Programmed death-1 expression on HIV-1-specific CD8+ T cells is shaped by epitope specificity, T-cell receptor clonotype usage and antigen load

PubMed Central

Kløverpris, Henrik N.; McGregor, Reuben; McLaren, James E.; Ladell, Kristin; Stryhn, Anette; Koofhethile, Catherine; Brener, Jacqui; Chen, Fabian; Riddell, Lynn; Graziano, Luzzi; Klenerman, Paul; Leslie, Alasdair; Buus, Søren; Price, David A.; Goulder, Philip

2014-01-01

Objectives: Although CD8+ T cells play a critical role in the control of HIV-1 infection, their antiviral efficacy can be limited by antigenic variation and immune exhaustion. The latter phenomenon is characterized by the upregulation of multiple inhibitory receptors, such as programmed death-1 (PD-1), CD244 and lymphocyte activation gene-3 (LAG-3), which modulate the functional capabilities of CD8+ T cells. Design and methods: Here, we used an array of different human leukocyte antigen (HLA)-B∗15 : 03 and HLA-B∗42 : 01 tetramers to characterize inhibitory receptor expression as a function of differentiation on HIV-1-specific CD8+ T-cell populations (n = 128) spanning 11 different epitope targets. Results: Expression levels of PD-1, but not CD244 or LAG-3, varied substantially across epitope specificities both within and between individuals. Differential expression of PD-1 on T-cell receptor (TCR) clonotypes within individual HIV-1-specific CD8+ T-cell populations was also apparent, independent of clonal dominance hierarchies. Positive correlations were detected between PD-1 expression and plasma viral load, which were reinforced by stratification for epitope sequence stability and dictated by effector memory CD8+ T cells. Conclusion: Collectively, these data suggest that PD-1 expression on HIV-1-specific CD8+ T cells tracks antigen load at the level of epitope specificity and TCR clonotype usage. These findings are important because they provide evidence that PD-1 expression levels are influenced by peptide/HLA class I antigen exposure. PMID:24906112

HIV infection impairs Th1 and Th17 Mycobacterium tuberculosis-specific T cell responses

PubMed Central

Murray, Lyle W; Satti, Iman; Meyerowitz, Jodi; Jones, Matthew; Willberg, Christian B; Ussher, James E; Goedhals, Dominique; Hurst, Jacob; Phillips, Rodney E; McShane, Helen

2018-01-01

Background HIV-infected individuals have a higher risk of developing active tuberculosis than HIV-uninfected individuals, but the mechanisms underpinning this are unclear. We hypothesized that depletion of specific components of Mycobacterium tuberculosis (M.tb)-specific CD4+ and CD8+ T cell responses contributed to this increased risk. Methods M.tb-specific T cell responses in 147 HIV-infected and 44 HIV-uninfected control subjects in a TB-endemic setting in Bloemfontein, South Africa were evaluated. Using a whole-blood flow cytometry assay, we measured expression of IFNγ, TNFα, IL-2 and IL-17 in CD4+ and CD8+ T cells in response to M.tb antigens (PPD, ESAT-6/CFP-10 (EC) and DosR regulon-encoded α-crystallin (Rv2031c)). Results Fewer HIV-infected individuals had detectable CD4+ and CD8+ T cell responses to PPD and Rv2031c than HIV-uninfected subjects. M.tb-specific T cells showed distinct patterns of cytokine expression comprising both Th1 (CD4 and CD8) and Th17 (CD4) cytokines, the latter at highest frequency for Rv2031c. Th17 antigen-specific responses to all antigens tested were specifically impaired in HIV-infected individuals. Conclusions HIV-associated impairment of CD4+ and CD8+ M.tb-specific T cell responses is antigen-specific, particularly impacting responses to PPD and Rv2031c. Preferential depletion of Th17 cytokine-expressing CD4+ T cells suggests this T cell subset may be key to TB susceptibility in HIV-infected individuals. PMID:29546381

Caspase-3 Is Transiently Activated without Cell Death during Early Antigen Driven Expansion of CD8+ T Cells In Vivo

PubMed Central

McComb, Scott; Mulligan, Rebecca; Sad, Subash

2010-01-01

Background CD8+ T cell responses develop rapidly during infection and are swiftly reduced during contraction, wherein >90% of primed CD8+ T cells are eliminated. The role of apoptotic mechanisms in controlling this rapid proliferation and contraction of CD8+ T cells remains unclear. Surprisingly, evidence has shown non-apoptotic activation of caspase-3 to occur during in vitro T-cell proliferation, but the relevance of these mechanisms to in vivo CD8+ T cell responses has yet to be examined. Methods and Findings We have evaluated the activity of caspase-3, a key downstream inducer of apoptosis, throughout the entirety of a CD8+ T cell response. We utilized two infection models that differ in the intensity, onset and duration of antigen-presentation and inflammation. Expression of cleaved caspase-3 in antigen specific CD8+ T cells was coupled to the timing and strength of antigen presentation in lymphoid organs. We also observed coordinated activation of additional canonical apoptotic markers, including phosphatidylserine exposure. Limiting dilution analysis directly showed that in the presence of IL7, very little cell death occurred in both caspase-3hi and caspase-3low CD8+ T cells. The expression of active caspase-3 peaked before effector phenotype (CD62Llow) CD8+ T cells emerged, and was undetectable in effector-phenotype cells. In addition, OVA-specific CD8+ cells remained active caspase-3low throughout the contraction phase. Conclusions Our results specifically implicate antigen and not inflammation in driving activation of apoptotic mechanisms without cell death in proliferating CD8+ T cells. Furthermore, the contraction of CD8+ T cell response following expansion is likely not mediated by the key downstream apoptosis inducer, caspase-3. PMID:21203525

Initial HIV-1 Antigen-Specific CD8+ T Cells in Acute HIV-1 Infection Inhibit Transmitted/Founder Virus Replication

PubMed Central

Freel, Stephanie A.; Picking, Ralph A.; Ferrari, Guido; Ding, Haitao; Ochsenbauer, Christina; Kappes, John C.; Kirchherr, Jennifer L.; Soderberg, Kelly A.; Weinhold, Kent J.; Cunningham, Coleen K.; Denny, Thomas N.; Crump, John A.; Cohen, Myron S.; McMichael, Andrew J.; Haynes, Barton F.

2012-01-01

CD8-mediated virus inhibition can be detected in HIV-1-positive subjects who naturally control virus replication. Characterizing the inhibitory function of CD8+ T cells during acute HIV-1 infection (AHI) can elucidate the nature of the CD8+ responses that can be rapidly elicited and that contribute to virus control. We examined the timing and HIV-1 antigen specificity of antiviral CD8+ T cells during AHI. Autologous and heterologous CD8+ T cell antiviral functions were assessed longitudinally during AHI in five donors from the CHAVI 001 cohort using a CD8+ T cell-mediated virus inhibition assay (CD8 VIA) and transmitted/founder (T/F) viruses. Potent CD8+ antiviral responses against heterologous T/F viruses appeared during AHI at the first time point sampled in each of the 5 donors (Fiebig stages 1/2 to 5). Inhibition of an autologous T/F virus was durable to 48 weeks; however, inhibition of heterologous responses declined concurrent with the resolution of viremia. HIV-1 viruses from 6 months postinfection were more resistant to CD8+-mediated virus inhibition than cognate T/F viruses, demonstrating that the virus escapes early from CD8+ T cell-mediated inhibition of virus replication. CD8+ T cell antigen-specific subsets mediated inhibition of T/F virus replication via soluble components, and these soluble responses were stimulated by peptide pools that include epitopes that were shown to drive HIV-1 escape during AHI. These data provide insights into the mechanisms of CD8-mediated virus inhibition and suggest that functional analyses will be important for determining whether similar antigen-specific virus inhibition can be induced by T cell-directed vaccine strategies. PMID:22514337

Retrogenic ICOS Expression Increases Differentiation of KLRG-1hi CD127lo CD8+ T Cells During Listeria Infection and Diminishes Recall Responses1

PubMed Central

Liu, Danya; Burd, Eileen M.; Coopersmith, Craig M.; Ford, Mandy L.

2016-01-01

Following T cell encounter with antigen, multiple signals are integrated to collectively induce distinct differentiation programs within antigen-specific CD8+ T cell populations. Several factors contribute to these cell fate decisions including the amount and duration of antigen, exposure to inflammatory cytokines, and degree of ligation of cosignaling molecules. The inducible costimulator (ICOS) is not expressed on resting T cells but is rapidly upregulated upon encounter with antigen. However, the impact of ICOS signaling on programmed differentiation is not well understood. In this study we therefore sought to determine the role of ICOS signaling on CD8+ T cell programmed differentiation. Through the creation of novel ICOS retrogenic antigen-specific TCR transgenic CD8+ T cells, we interrogated the phenotype, functionality, and recall potential of CD8+ T cells that receive early and sustained ICOS signaling during antigen exposure. Our results reveal that these ICOS signals critically impacted cell fate decisions of antigen-specific CD8+ T cells, resulting in increased frequencies of KLRG-1hiCD127lo cells, altered BLIMP-1, T-bet, and eomesodermin expression, and increased cytolytic capacity as compared to empty vector controls. Interestingly, however, ICOS retrogenic CD8+ T cells also preferentially homed to non-lymphoid organs, and exhibited reduced multi-cytokine functionality and reduced ability to mount secondary recall responses upon challenge in vivo. In sum, our results suggest that an altered differentiation program is induced following early and sustained ICOS expression, resulting in the generation of more cytolyticly potent, terminally differentiated effectors that possess limited capacity for recall response. PMID:26729800

Characterization of the Fine Specificity of Bovine CD8 T-Cell Responses to Defined Antigens from the Protozoan Parasite Theileria parva▿

PubMed Central

Graham, Simon P.; Pellé, Roger; Yamage, Mat; Mwangi, Duncan M.; Honda, Yoshikazu; Mwakubambanya, Ramadhan S.; de Villiers, Etienne P.; Abuya, Evelyne; Awino, Elias; Gachanja, James; Mbwika, Ferdinand; Muthiani, Anthony M.; Muriuki, Cecelia; Nyanjui, John K.; Onono, Fredrick O.; Osaso, Julius; Riitho, Victor; Saya, Rosemary M.; Ellis, Shirley A.; McKeever, Declan J.; MacHugh, Niall D.; Gilbert, Sarah C.; Audonnet, Jean-Christophe; Morrison, W. Ivan; van der Bruggen, Pierre; Taracha, Evans L. N.

2008-01-01

Immunity against the bovine intracellular protozoan parasite Theileria parva has been shown to be mediated by CD8 T cells. Six antigens targeted by CD8 T cells from T. parva-immune cattle of different major histocompatibility complex (MHC) genotypes have been identified, raising the prospect of developing a subunit vaccine. To facilitate further dissection of the specificity of protective CD8 T-cell responses and to assist in the assessment of responses to vaccination, we set out to identify the epitopes recognized in these T. parva antigens and their MHC restriction elements. Nine epitopes in six T. parva antigens, together with their respective MHC restriction elements, were successfully identified. Five of the cytotoxic-T-lymphocyte epitopes were found to be restricted by products of previously described alleles, and four were restricted by four novel restriction elements. Analyses of CD8 T-cell responses to five of the epitopes in groups of cattle carrying the defined restriction elements and immunized with live parasites demonstrated that, with one exception, the epitopes were consistently recognized by animals of the respective genotypes. The analysis of responses was extended to animals immunized with multiple antigens delivered in separate vaccine constructs. Specific CD8 T-cell responses were detected in 19 of 24 immunized cattle. All responder cattle mounted responses specific for antigens for which they carried an identified restriction element. By contrast, only 8 of 19 responder cattle displayed a response to antigens for which they did not carry an identified restriction element. These data demonstrate that the identified antigens are inherently dominant in animals with the corresponding MHC genotypes. PMID:18070892

Techniques to improve the direct ex vivo detection of low frequency antigen-specific CD8+ T cells with peptide-major histocompatibility complex class I tetramers

PubMed Central

Chattopadhyay, Pratip K.; Melenhorst, J. Joseph; Ladell, Kristin; Gostick, Emma; Scheinberg, Philip; Barrett, A. John; Wooldridge, Linda; Roederer, Mario; Sewell, Andrew K.; Price, David A.

2008-01-01

The ability to quantify and characterize antigen-specific CD8+ T cells irrespective of functional readouts using fluorochrome-conjugated tetrameric peptide-MHC class I (pMHCI) complexes in conjunction with flow cytometry has transformed our understanding of cellular immune responses over the past decade. In the case of prevalent CD8+ T cell populations that engage cognate pMHCI tetramers with high avidities, direct ex vivo identification and subsequent data interpretation is relatively straightforward. However, the accurate identification of low frequency antigen-specific CD8+ T cell populations can be complicated, especially in situations where TCR-mediated tetramer binding occurs at low avidities. Here, we highlight a few simple techniques that can be employed to improve the visual resolution, and hence the accurate quantification, of tetramer-binding CD8+ T cell populations by flow cytometry. These methodological modifications enhance signal intensity, especially in the case of specific CD8+ T cell populations that bind cognate antigen with low avidity, minimize background noise and enable improved discrimination of true pMHCI tetramer binding events from nonspecific uptake. PMID:18836993

Adoptive transfer of CD8+ T cells generated from induced pluripotent stem cells triggers regressions of large tumors along with immunological memory

PubMed Central

Saito, Hidehito; Okita, Keisuke; Chang, Alfred E.; Ito, Fumito

2016-01-01

Current approaches to adoptive T cell therapy are limited by the difficulty of obtaining sufficient numbers of T cells against targeted antigens with useful in vivo characteristics. Theoretically, this limitation could be overcome by using induced pluripotent stem cells (iPSCs) that could provide an unlimited source of autologous T cells. However, the therapeutic efficacy of iPSC-derived regenerated T cells remains to be demonstrated. Here we report the first successful reprogramming of T-cell receptor (TCR) transgenic CD8+ T cells into pluripotency. As part of the work, we established a syngeneic mouse model for evaluating in vitro and in vivo antitumor reactivity of regenerated T cells from iPSCs bearing a rearranged TCR of known antigen specificity. Stably TCR retained T cell-derived iPSCs differentiated into CD4+CD8+ T cells that expressed CD3 and the desired TCR in vitro. Stimulation of iPSC-derived CD4+CD8+ T cells with the cognate antigen in the presence of IL-7 and IL-15 followed by expansion with IL-2, IL-7 and IL-15 generated large numbers of less-differentiated CD8+ T cells with antigen-specific potent cytokine production and cytolytic capacity. Furthermore, adoptively transferred iPSC-derived CD8+ T cells escaped immune rejection, mediated effective regression of large tumors, improved survival, and established antigen-specific immunological memory. Our findings illustrate the translational potential of iPSCs to provide an unlimited number of phenotypically defined, functional, and expandable autologous antigen-specific T cells with the characteristics needed to enable in vivo effectiveness. PMID:27197199

Long-term in vivo provision of antigen-specific T cell immunity by programming hematopoietic stem cells

NASA Astrophysics Data System (ADS)

Yang, Lili; Baltimore, David

2005-03-01

A method to genetically program mouse hematopoietic stem cells to develop into functional CD8 or CD4 T cells of defined specificity in vivo is described. For this purpose, a bicistronic retroviral vector was engineered that efficiently delivers genes for both and chains of T cell receptor (TCR) to hematopoietic stem cells. When modified cell populations were used to reconstruct the hematopoietic lineages of recipient mice, significant percentages of antigen-specific CD8 or CD4 T cells were observed. These cells expressed normal surface markers and responded to peptide antigen stimulation by proliferation and cytokine production. Moreover, they could mature into memory cells after peptide stimulation. Using TCRs specific for a model tumor antigen, we found that the recipient mice were able to partially resist a challenge with tumor cells carrying the antigen. By combining cells modified with CD8- and CD4-specific TCRs, and boosting with dendritic cells pulsed with cognate peptides, complete suppression of tumor could be achieved and even tumors that had become established would regress and be eliminated after dendritic cell/peptide immunization. This methodology of "instructive immunotherapy" could be developed for controlling the growth of human tumors and attacking established pathogens.

Central Tolerance to Tissue-specific Antigens Mediated by Direct and Indirect Antigen Presentation

PubMed Central

Gallegos, Alena M.; Bevan, Michael J.

2004-01-01

Intrathymic expression of tissue-specific antigens (TSAs) by medullary thymic epithelial cells (Mtecs) leads to deletion of autoreactive T cells. However, because Mtecs are known to be poor antigen-presenting cells (APCs) for tolerance to ubiquitous antigens, and very few Mtecs express a given TSA, it was unclear if central tolerance to TSA was induced directly by Mtec antigen presentation or indirectly by thymic bone marrow (BM)-derived cells via cross-presentation. We show that professional BM-derived APCs acquire TSAs from Mtecs and delete autoreactive CD8 and CD4 T cells. Although direct antigen presentation by Mtecs did not delete the CD4 T cell population tested in this study, Mtec presentation efficiently deleted both monoclonal and polyclonal populations of CD8 T cells. For developing CD8 T cells, deletion by BM-derived APC and by Mtec presentation occurred abruptly at the transitional, CD4high CD8low TCRintermediate stage, presumably as the cells transit from the cortex to the medulla. These studies reveal a cooperative relationship between Mtecs and BM-derived cells in thymic elimination of autoreactive T cells. Although Mtecs synthesize TSAs and delete a subset of autoreactive T cells, BM-derived cells extend the range of clonal deletion by cross-presenting antigen captured from Mtecs. PMID:15492126

Specific Mutation of a Gammaherpesvirus-Expressed Antigen in Response to CD8 T Cell Selection In Vivo

PubMed Central

Loh, Joy; Popkin, Daniel L.; Droit, Lindsay; Braaten, Douglas C.; Zhao, Guoyan; Zhang, Xin; Vachharajani, Punit; Myers, Nancy; Hansen, Ted H.

2012-01-01

Herpesviruses are thought to be highly genetically stable, and their use as vaccine vectors has been proposed. However, studies of the human gammaherpesvirus, Epstein-Barr virus, have found viral isolates containing mutations in HLA class I-restricted epitopes. Using murine gammaherpesvirus 68 expressing ovalbumin (OVA), we examined the stability of a gammaherpesvirus antigenic locus under strong CD8 T cell selection in vivo. OVA-specific CD8 T cells selected viral isolates containing mutations in the OVA locus but minimal alterations in other genomic regions. Thus, a CD8 T cell response to a gammaherpesvirus-expressed antigen that is not essential for replication or pathogenesis can result in selective mutation of that antigen in vivo. This finding may have relevance for the use of herpesvirus vectors for chronic antigen expression in vivo. PMID:22171269

Antigen-specific tolerance inhibits autoimmune uveitis in pre-sensitized animals by deletion and CD4+CD25+ T-regulatory cells.

PubMed

Matta, Bharati; Jha, Purushottam; Bora, Puran S; Bora, Nalini S

2010-02-01

The objective of this study was to inhibit experimental autoimmune anterior uveitis (EAAU) by establishing antigen-specific immune tolerance in animals pre-sensitized with melanin-associated antigen (MAA). Intravenous administration of MAA on days 6, 7, 8 and 9 post-immunization induced tolerance and inhibited EAAU in all Lewis rats. The number of cells (total T cells, CD4(+) T cells and CD8(+) T cells) undergoing apoptosis dramatically increased in the popliteal lymph nodes (LNs) of the tolerized animals compared with non-tolerized animals. In addition, Fas ligand (FasL), TNF receptor 1 (TNFR1) and caspase-8 were upregulated in tolerized rats. Proliferation of total lymphocytes, CD4(+)T cells and CD8(+) T cells (harvested from the popliteal LNs) in response to antigenic stimulation was drastically reduced in the state of tolerance compared with the cells from non-tolerized animals. The level of interferon (IFN)-gamma and IL-2 decreased, whereas TGF-beta2 was elevated in the state of tolerance. Furthermore, the number of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) increased in the popliteal LNs of tolerized animals compared with non-tolerized animals. In conclusion, our results suggest that deletion of antigen-specific T cells by apoptosis and active suppression mediated by Tregs has an important role in the induction of antigen specific immune tolerance in animals with an established immune response against MAA.

Recognition of prostate-specific antigenic peptide determinants by human CD4 and CD8 T cells.

PubMed

Corman, J M; Sercarz, E E; Nanda, N K

1998-11-01

It is now becoming accepted that one is not tolerant to all the determinants of self proteins: the T cell repertoire directed to some sequences in self proteins is intact and can be activated. When a self protein is exclusively expressed by tumour cells, the T cell repertoire directed to the particular self antigen can potentially be activated to attack the tumour: this would amount to induction of a beneficial autoimmune response. Prostate cancer offers a unique opportunity for activation of a tumour-specific immune response owing to the exclusive synthesis of prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSM) by prostatic tissue and prostate tumour cells. In this study we examine the CD4 and CD8 T cell repertoires specific for peptides of PSA and PSM in normal human male individuals, using short-term, peptide antigen-driven CD4 and CD8 T cell lines. We show that short-term, CD4 T cell lines derived from six HLA-DR4 individuals showed strong proliferative responses to six of 10 tested peptides of PSA, selected as to contain a DR4 binding motif. Short-term, CD8 T cell lines from three HLA-A1 individuals showed specific cytolytic activity for autologous targets loaded with five of five tested peptides of PSA and PSM, selected to possess an HLA-A1 binding motif. One of the peptides chosen is termed a 'dual-motif' peptide, as it encodes determinants for both CD4 and CD8 T cells. These results, indicating the existence of CD4 and CD8 T cells against determinants of the self proteins, PSA and PSM, in healthy male individuals reveal the potential of the T cell repertoire from the typical prostate cancer patient to eradicate prostate tumours upon being appropriately activated.

Definition of epitopes and antigens recognized by vaccinia specific immune responses: their conservation in variola virus sequences, and use as a model system to study complex pathogens.

PubMed

Sette, Alessandro; Grey, Howard; Oseroff, Carla; Peters, Bjoern; Moutaftsi, Magdalini; Crotty, Shane; Assarsson, Erika; Greenbaum, Jay; Kim, Yohan; Kolla, Ravi; Tscharke, David; Koelle, David; Johnson, R Paul; Blum, Janice; Head, Steven; Sidney, John

2009-12-30

In the last few years, a wealth of information has become available relating to the targets of vaccinia virus (VACV)-specific CD4(+) T cell, CD8(+) T cell and antibody responses. Due to the large size of its genome, encoding more than 200 different proteins, VACV represents a useful model system to study immunity to complex pathogens. Our data demonstrate that both cellular and humoral responses target a large number of antigens and epitopes. This broad spectrum of targets is detected in both mice and humans. CD4(+) T cell responses target late and structural antigens, while CD8(+) T cells preferentially recognize early antigens. While both CD4(+) and CD8(+) T cell responses target different types of antigens, the antigens recognized by T(H) cells are highly correlated with those recognized by antibody responses. We further show that protein abundance and antibody recognition can be used to predict antigens recognized by CD4(+) T cell responses, while early expression at the mRNA level predicts antigens targeted by CD8(+) T cells. Finally, we find that the vast majority of VACV epitopes are conserved in variola virus (VARV), thus suggesting that the epitopes defined herein also have relevance for the efficacy of VACV as a smallpox vaccine.

Rapid generation of combined CMV-specific CD4+ and CD8+ T-cell lines for adoptive transfer into recipients of allogeneic stem cell transplants.

PubMed

Rauser, Georg; Einsele, Hermann; Sinzger, Christian; Wernet, Dorothee; Kuntz, Gabriele; Assenmacher, Mario; Campbell, John D M; Topp, Max S

2004-05-01

Adoptive transfer of cytomegalovirus (CMV)-specific T cells can restore long-lasting, virus-specific immunity and clear CMV viremia in recipients of allogeneic stem cell transplants if CD4(+) and CD8(+) CMV-specific T cells are detected in the recipient after transfer. Current protocols for generating virus-specific T cells use live virus, require leukapheresis of the donor, and are time consuming. To circumvent these limitations, a clinical-scale protocol was developed to generate CMV-specific T cells by using autologous cellular and serum components derived from a single 500-mL blood draw. CMV-specific T cells were stimulated simultaneously with CMV-specific major histocompatibility complex class I (MHC I)- restricted peptides and CMV antigen. Activated T cells were isolated with the interferon-gamma (IFN-gamma) secretion assay and expanded for 10 days. In 8 randomly selected, CMV-seropositive donors, 1.34 x 10(8) combined CD4(+) and CD8(+) CMV-specific T cells, on average, were generated, as determined by antigen-triggered IFN-gamma production. CMV-infected fibroblasts were efficiently lysed by the generated T cells, and CMV-specific CD4(+) and CD8(+) T cells expanded if they were stimulated with natural processed antigen. On the other hand, CD4(+) and CD8(+) T cell-mediated alloreactivity of generated CMV-specific T-cell lines was reduced compared with that of the starting population. In conclusion, the culture system developed allowed the rapid generation of allodepleted, highly enriched, combined CD4(+) and CD8(+) CMV-specific T cells under conditions mimicking good manufacturing practice.

Lawrence Transfer Factor: Transference of Specific Immune Memory by Dialyzable Leukocyte Extract from a CD8+ T Cell Line.

PubMed

Wang, Jason F; Park, Andrew J; Rendini, Tina; Levis, William R

2017-12-01

Lawrence transfer factor (TF) is defined as dialyzable leukocyte extract (DLE) that can transfer antigen-specific cell-mediated immunity from a person testing positive for the antigen in a delayed type hypersensitivity skin test manner to a person negative for the same antigen. A recent article by Myles et al1 has identified a DLE isolated from an established CD8+ T cell line capable of transferring antigen-specific immunity. The DLE contains a portion of the beta chain of the T cell receptor and additional nucleotide and protein factors that are being subjected to further modern biochemical analysis. After months of study that included interviews of TF physician-scientists, we conclude that an antigen-specific TF exists for most, if not all, antigens. By working from a CD8+ T cell line with modern biochemical technology, it should be possible to identify and patent products capable of treating infectious diseases, antigen-responsive cancers, and autoimmune disorders.

Development of Augmented Leukemia/Lymphoma-Specific T-Cell Immunotherapy for Deployment with Haploidentical, Hematompoietic Progenitor-Cell Transplant

DTIC Science & Technology

2008-05-01

adoptive therapy using CD19- specific chimeric antigen receptor re-directed T cells for recurrent/refractory follicular lymphoma. Mol Ther...T- cell therapies for B- cell malignancies we have developed a chimeric antigen receptor (CAR) which when expressed on the cell surface redirects T...that both CD4+ and CD8+ T cells expressing CD19-specific chimeric antigen receptor (CAR) can be generated usmg a novel non-viral gene

The link between CD8⁺ T-cell antigen-sensitivity and HIV-suppressive capacity depends on HLA restriction, target epitope and viral isolate.

PubMed

Lissina, Anna; Fastenackels, Solène; Inglesias, Maria C; Ladell, Kristin; McLaren, James E; Briceño, Olivia; Gostick, Emma; Papagno, Laura; Autran, Brigitte; Sauce, Delphine; Price, David A; Saez-Cirion, Asier; Appay, Victor

2014-02-20

Although it is established that CD8 T-cell immunity is critical for the control of HIV replication in vivo, the key factors that determine antiviral efficacy are yet to be fully elucidated. Antigen-sensitivity and T-cell receptor (TCR) avidity have been identified as potential determinants of CD8⁺ T-cell efficacy. However, there is no general consensus in this regard because the relationship between these parameters and the control of HIV infection has been established primarily in the context of immunodominant CD8⁺ T-cell responses against the Gag₂₆₃₋₂₇₂ KK10 epitope restricted by human leukocyte antigen (HLA)-B27. To investigate the relationship between antigen-sensitivity, TCR avidity and HIV-suppressive capacity in vitro across epitope specificities and HLA class I restriction elements, we used a variety of techniques to study CD8⁺ T-cell clones specific for Nef₇₃₋₈₂ QK10 and Gag₂₀₋₂₉ RY10, both restricted by HLA-A3, alongside CD8⁺ T-cell clones specific for Gag₂₆₃₋₂₇₂ KK10. For each targeted epitope, the linked parameters of antigen-sensitivity and TCR avidity correlated directly with antiviral efficacy. However, marked differences in HIV-suppressive capacity were observed between epitope specificities, HLA class I restriction elements and viral isolates. Collectively, these data emphasize the central role of the TCR as a determinant of CD8⁺ T-cell efficacy and demonstrate that the complexities of antigen recognition across epitope and HLA class I boundaries can confound simple relationships between TCR engagement and HIV suppression.

Split T Cell Tolerance against a Self/Tumor Antigen: Spontaneous CD4+ but Not CD8+ T Cell Responses against p53 in Cancer Patients and Healthy Donors

PubMed Central

Tsuji, Takemasa; Matsuzaki, Junko; Ritter, Erika; Miliotto, Anthony; Ritter, Gerd; Odunsi, Kunle; Old, Lloyd J.; Gnjatic, Sacha

2011-01-01

Analyses of NY-ESO-1-specific spontaneous immune responses in cancer patients revealed that antibody and both CD4+ and CD8+ T cell responses were induced together in cancer patients. To explore whether such integrated immune responses are also spontaneously induced for other tumor antigens, we have evaluated antibody and T cell responses against self/tumor antigen p53 in ovarian cancer patients and healthy individuals. We found that 21% (64/298) of ovarian cancer patients but no healthy donors showed specific IgG responses against wild-type p53 protein. While none of 12 patients with high titer p53 antibody showed spontaneous p53-specific CD8+ T cell responses following a single in vitro sensitization, significant p53-specific IFN-γ producing CD4+ T cells were detected in 6 patients. Surprisingly, similar levels of p53-specific CD4+ T cells but not CD8+ T cells were also detected in 5/10 seronegative cancer patients and 9/12 healthy donors. Importantly, p53-specific CD4+ T cells in healthy donors originated from a CD45RA− antigen-experienced T cell population and recognized naturally processed wild-type p53 protein. These results raise the possibility that p53-specific CD4+ T cells reflect abnormalities in p53 occurring in normal individuals and that they may play a role in processes of immunosurveillance or immunoregulation of p53-related neoplastic events. PMID:21858191

Engineering antigens for in situ erythrocyte binding induces T-cell deletion.

PubMed

Kontos, Stephan; Kourtis, Iraklis C; Dane, Karen Y; Hubbell, Jeffrey A

2013-01-02

Antigens derived from apoptotic cell debris can drive clonal T-cell deletion or anergy, and antigens chemically coupled ex vivo to apoptotic cell surfaces have been shown correspondingly to induce tolerance on infusion. Reasoning that a large number of erythrocytes become apoptotic (eryptotic) and are cleared each day, we engineered two different antigen constructs to target the antigen to erythrocyte cell surfaces after i.v. injection, one using a conjugate with an erythrocyte-binding peptide and another using a fusion with an antibody fragment, both targeting the erythrocyte-specific cell surface marker glycophorin A. Here, we show that erythrocyte-binding antigen is collected much more efficiently than free antigen by splenic and hepatic immune cell populations and hepatocytes, and that it induces antigen-specific deletional responses in CD4(+) and CD8(+) T cells. We further validated T-cell deletion driven by erythrocyte-binding antigens using a transgenic islet β cell-reactive CD4(+) T-cell adoptive transfer model of autoimmune type 1 diabetes: Treatment with the peptide antigen fused to an erythrocyte-binding antibody fragment completely prevented diabetes onset induced by the activated, autoreactive CD4(+) T cells. Thus, we report a translatable modular biomolecular approach with which to engineer antigens for targeted binding to erythrocyte cell surfaces to induce antigen-specific CD4(+) and CD8(+) T-cell deletion toward exogenous antigens and autoantigens.

Increased CD8+ T Cell Response to Epstein-Barr Virus Lytic Antigens in the Active Phase of Multiple Sclerosis

PubMed Central

Angelini, Daniela F.; Serafini, Barbara; Piras, Eleonora; Severa, Martina; Coccia, Eliana M.; Rosicarelli, Barbara; Ruggieri, Serena; Gasperini, Claudio; Buttari, Fabio; Centonze, Diego; Mechelli, Rosella; Salvetti, Marco; Borsellino, Giovanna; Aloisi, Francesca; Battistini, Luca

2013-01-01

It has long been known that multiple sclerosis (MS) is associated with an increased Epstein-Barr virus (EBV) seroprevalence and high immune reactivity to EBV and that infectious mononucleosis increases MS risk. This evidence led to postulate that EBV infection plays a role in MS etiopathogenesis, although the mechanisms are debated. This study was designed to assess the prevalence and magnitude of CD8+ T-cell responses to EBV latent (EBNA-3A, LMP-2A) and lytic (BZLF-1, BMLF-1) antigens in relapsing-remitting MS patients (n = 113) and healthy donors (HD) (n = 43) and to investigate whether the EBV-specific CD8+ T cell response correlates with disease activity, as defined by clinical evaluation and gadolinium-enhanced magnetic resonance imaging. Using HLA class I pentamers, lytic antigen-specific CD8+ T cell responses were detected in fewer untreated inactive MS patients than in active MS patients and HD while the frequency of CD8+ T cells specific for EBV lytic and latent antigens was higher in active and inactive MS patients, respectively. In contrast, the CD8+ T cell response to cytomegalovirus did not differ between HD and MS patients, irrespective of the disease phase. Marked differences in the prevalence of EBV-specific CD8+ T cell responses were observed in patients treated with interferon-β and natalizumab, two licensed drugs for relapsing-remitting MS. Longitudinal studies revealed expansion of CD8+ T cells specific for EBV lytic antigens during active disease in untreated MS patients but not in relapse-free, natalizumab-treated patients. Analysis of post-mortem MS brain samples showed expression of the EBV lytic protein BZLF-1 and interactions between cytotoxic CD8+ T cells and EBV lytically infected plasma cells in inflammatory white matter lesions and meninges. We therefore propose that inability to control EBV infection during inactive MS could set the stage for intracerebral viral reactivation and disease relapse. PMID:23592979

CD4 and CD8 T-Cell Responses to Mycobacterial Antigens in African Children

PubMed Central

Tena-Coki, Nontobeko G.; Scriba, Thomas J.; Peteni, Nomathemba; Eley, Brian; Wilkinson, Robert J.; Andersen, Peter; Hanekom, Willem A.; Kampmann, Beate

2010-01-01

Rationale: The current tuberculosis (TB) vaccine, bacille Calmette-Guérin (BCG), does not provide adequate protection against TB disease in children. Furthermore, more efficacious TB vaccines are needed for children with immunodeficiencies such as HIV infection, who are at highest risk of disease. Objectives: To characterize mycobacteria-specific T cells in children who might benefit from vaccination against TB, focusing on responses to antigens contained in novel TB vaccines. Methods: Whole blood was collected from three groups of BCG-vaccinated children: HIV-seronegative children receiving TB treatment (n = 30), HIV-infected children (n = 30), and HIV-unexposed healthy children (n = 30). Blood was stimulated with Ag85B and TB10.4, or purified protein derivative, and T-cell cytokine production by CD4 and CD8 was determined by flow cytometry. The memory phenotype of antigen-specific CD4 and CD8 T cells was also determined. Measurements and Main Results: Mycobacteria-specific CD4 and CD8 T-cell responses were detectable in all three groups of children. Children receiving TB treatment had significantly higher frequencies of antigen-specific CD4 T cells compared with HIV-infected children (P = 0.0176). No significant differences in magnitude, function, or phenotype of specific T cells were observed in HIV-infected children compared with healthy control subjects. CD4 T cells expressing IFN-γ, IL-2, or both expressed a CD45RA−CCR7−CD27+/− effector memory phenotype. Mycobacteria-specific CD8 T cells expressed mostly IFN-γ in all groups of children; these cells expressed CD45RA−CCR7−CD27+/− or CD45RA+CCR7−CD27+/− effector memory phenotypes. Conclusions: Mycobacteria-specific T-cell responses could be demonstrated in all groups of children, suggesting that the responses could be boosted by new TB vaccines currently in clinical trials. PMID:20224065

CD8α+ DC trans-presentation of IL-15 to naïve CD8+ T cells produces antigen inexperienced T cells in the periphery with memory phenotype and function

PubMed Central

Sosinowski, Tomasz; White, Jason T.; Cross, Eric; Haluszczak, Catherine; Marrack, Philippa; Gapin, Laurent; Kedl, Ross M.

2013-01-01

Various populations of memory phenotype CD8+ T cells have been described over the last 15–20 years, all of which possess elevated effector functions relative to naïve phenotype cells. Using a technique for isolating antigen specific cells from unprimed hosts, we recently identified a new subset of cells, specific for nominal antigen, but phenotypically and functionally similar to memory cells arising as a result of homeostatic proliferation (HP). We show here that these “Virtual Memory” cells are independent of previously identified “innate memory” cells, arising as a result of their response to IL-15 trans-presentation by lymphoid tissue-resident CD8α+ DCs in the periphery. The absence of IL-15, CD8+ T cell expression of either CD122 or Eomes, or of CD8a+ DCs all lead to the loss of Virtual Memory cells in the host. Our results show that CD8+ T cell homeostatic expansion is an active process within the non-lymphopenic environment, is mediated by IL-15, and produces antigen inexperienced memory cells which retain the capacity to respond to nominal antigen with memory-like function. Preferential engagement of these “Virtual Memory” T cells into a vaccine response could dramatically enhance the rate by which immune protection develops. PMID:23355737

Recombinant yellow fever vaccine virus 17D expressing simian immunodeficiency virus SIVmac239 gag induces SIV-specific CD8+ T-cell responses in rhesus macaques.

PubMed

Bonaldo, Myrna C; Martins, Mauricio A; Rudersdorf, Richard; Mudd, Philip A; Sacha, Jonah B; Piaskowski, Shari M; Costa Neves, Patrícia C; Veloso de Santana, Marlon G; Vojnov, Lara; Capuano, Saverio; Rakasz, Eva G; Wilson, Nancy A; Fulkerson, John; Sadoff, Jerald C; Watkins, David I; Galler, Ricardo

2010-04-01

Here we describe a novel vaccine vector for expressing human immunodeficiency virus (HIV) antigens. We show that recombinant attenuated yellow fever vaccine virus 17D expressing simian immunodeficiency virus SIVmac239 Gag sequences can be used as a vector to generate SIV-specific CD8(+) T-cell responses in the rhesus macaque. Priming with recombinant BCG expressing SIV antigens increased the frequency of these SIV-specific CD8(+) T-cell responses after recombinant YF17D boosting. These recombinant YF17D-induced SIV-specific CD8(+) T cells secreted several cytokines, were largely effector memory T cells, and suppressed viral replication in CD4(+) T cells.

High frequencies of circulating IFN-gamma-secreting CD8 cytotoxic T cells specific for a novel MHC class I-restricted Mycobacterium tuberculosis epitope in M. tuberculosis-infected subjects without disease.

PubMed

Pathan, A A; Wilkinson, K A; Wilkinson, R J; Latif, M; McShane, H; Pasvol, G; Hill, A V; Lalvani, A

2000-09-01

MHC class I-restricted CD8 cytotoxic T lymphocytes (CTL) are essential for protective immunity to Mycobacterium tuberculosis in animal models but their role in humans remains unclear. We therefore studied subjects who had successfully contained M. tuberculosis infection in vivo, i.e. exposed healthy household contacts and individuals with inactive self-healed pulmonary tuberculosis. Using the ELISPOT assay for IFN-gamma, we screened peptides from ESAT-6, a secreted antigen that is highly specific for M. tuberculosis. We identified a novel nonamer epitope: unstimulated peripheral blood-derived CD8 T cells displayed peptide-specific IFN-gamma release ex vivo while CD8 T cell lines and clones exhibited HLA-A68.02-restricted cytolytic activity and recognized endogenously processed antigen. The frequency of CD8 CTL specific for this single M. tuberculosis epitope, 1/2500 peripheral blood lymphocytes, was equivalent to the combined frequency of all IFN-gamma-secreting purified protein derivative-reactive T cells ex vivo. This highly focused CTL response was maintained in an asymptomatic contact over 2 years and is the most potent antigen-specific antimycobacterial CD8 CTL response hitherto described. Thus, human M. tuberculosis-specific CD8 CTL are not necessarily associated with active disease per se. Rather, our results are consistent with a protective role for these ESAT-6-specific CD8 T cells in the long-term control of M. tuberculosis in vivo in humans.

Efficacy Against Human Prostate Cancer by Prostate-specific Membrane Antigen-specific, Transforming Growth Factor-β Insensitive Genetically Targeted CD8+ T-cells Derived from Patients with Metastatic Castrate-resistant Disease.

PubMed

Zhang, Qiang; Helfand, Brian T; Carneiro, Benedito A; Qin, Weijun; Yang, Ximing J; Lee, Chung; Zhang, Weipeng; Giles, Francis J; Cristofanilli, Massimo; Kuzel, Timothy M

2018-05-01

Current immunotherapy has limited efficacy on metastatic castrate-resistant prostate cancer (mCRPC). We therefore sought to improve the antitumor ability of mCRPC patient-derived CD8 + T-cells by the endowment of specificity to prostate-specific membrane antigen (PSMA) and insensitivity to immunosuppressant molecule transforming growth factor-β (TGF-ß) under the control of herpes simplex virus-1 thymidine kinase. CD8 + T-cells were collected by leukapheresis and cultured in a Food and Drug Administration-approved Cell Processing Work Station. We developed a chimeric antigen receptor retroviral construct using an anti-PSMA chimeric immunoglobulin-T-cell receptor(ζ) gene (PZ1) and dominant negative TGF-ß type II receptor (TßRIIDN), that could induce CD8 + T-cells to be PSMA reactive and insensitive to TGF-ß. Cr 51 release assay was performed on PC-3 and PC-3-PSMA. The further antitumor functions of PSMA-specific, TGF-ß insensitive CD8 + T-cells was evaluated using an immunodeficient RAG-1 -/- mouse model. We found PSMA-specific, TGF-ß insensitive CD8 + T-cells from mCRPC were expanded with strong expression of PZ1 and thymidine kinase genes, and their growth was not suppressed by TGF-ß. The survival of these cells decreased sharply after treatment with ganciclovir. Treatment of PSMA-specific TGF-ß, insensitive CD8 + T-cells was associated with 61.58% specific lysis on PC-3-PSMA, and significantly suppressed PC3-PSMA tumor compared with the PC3 tumor. A large amount of tumor apoptosis and CD8 + T-cell infiltration were found only in the PC3-PSMA tumor. This study verified that PSMA-specific, TGF-ß insensitive CD8 + T-cells derived from mCRPC patients could be successfully expanded and used to overcome the immunosuppressive effects of the tumor microenvironment to control PSMA-expressing PC in vitro and in vivo. This may provide a promising approach for men with mCRPC who fail androgen deprivation therapy. We investigated the role of a novel chimeric antigen receptor T-immunotherapy based on autologous metastatic castrate-resistant prostate cancer patient-derived prostate-specific membrane antigen (PSMA)-specific, transforming growth factor-ß insensitive CD8 + T-cells on PSMA-positive prostate cancer. We found that this chimeric antigen receptor T-cells could kill PSMA-positive prostate cancer specifically. The results suggest that this novel immunotherapy treatment is a potential new approach for men with metastatic castrate-resistant prostate cancer. Copyright © 2017 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Quantitative and qualitative features of heterologous virus-vector-induced antigen-specific CD8+ T cells against Trypanosoma cruzi infection.

PubMed

Takayama, Eiji; Ono, Takeshi; Carnero, Elena; Umemoto, Saori; Yamaguchi, Yoko; Kanayama, Atsuhiro; Oguma, Takemi; Takashima, Yasuhiro; Tadakuma, Takushi; García-Sastre, Adolfo; Miyahira, Yasushi

2010-11-01

We studied some aspects of the quantitative and qualitative features of heterologous recombinant (re) virus-vector-induced, antigen-specific CD8(+) T cells against Trypanosoma cruzi. We used three different, highly attenuated re-viruses, i.e., influenza virus, adenovirus and vaccinia virus, which all expressed a single, T. cruzi antigen-derived CD8(+) T-cell epitope. The use of two out of three vectors or the triple virus-vector vaccination regimen not only confirmed that the re-vaccinia virus, which was placed last in order for sequential immunisation, was an effective booster for the CD8(+) T-cell immunity in terms of the number of antigen-specific CD8(+) T cells, but also demonstrated that (i) the majority of cells exhibit the effector memory (T(EM)) phenotype, (ii) robustly secrete IFN-γ, (iii) express higher intensity of the CD122 molecule and (iv) present protective activity against T. cruzi infection. In contrast, placing the re-influenza virus last in sequential immunisation had a detrimental effect on the quantitative and qualitative features of CD8(+) T cells. The triple virus-vector vaccination was more effective at inducing a stronger CD8(+) T-cell immunity than using two re-viruses. The different quantitative and qualitative features of CD8(+) T cells induced by different immunisation regimens support the notion that the refinement of the best choice of multiple virus-vector combinations is indispensable for the induction of a maximum number of CD8(+) T cells of high quality. Copyright © 2010 Australian Society for Parasitology Inc. All rights reserved.

Roles of lymphatic endothelial cells expressing peripheral tissue antigens in CD4 T-cell tolerance induction.

PubMed

Rouhani, Sherin J; Eccles, Jacob D; Riccardi, Priscila; Peske, J David; Tewalt, Eric F; Cohen, Jarish N; Liblau, Roland; Mäkinen, Taija; Engelhard, Victor H

2015-04-10

Lymphatic endothelial cells (LECs) directly express peripheral tissue antigens and induce CD8 T-cell deletional tolerance. LECs express MHC-II molecules, suggesting they might also tolerize CD4 T cells. We demonstrate that when β-galactosidase (β-gal) is expressed in LECs, β-gal-specific CD8 T cells undergo deletion via the PD-1/PD-L1 and LAG-3/MHC-II pathways. In contrast, LECs do not present endogenous β-gal in the context of MHC-II molecules to β-gal-specific CD4 T cells. Lack of presentation is independent of antigen localization, as membrane-bound haemagglutinin and I-Eα are also not presented by MHC-II molecules. LECs express invariant chain and cathepsin L, but not H2-M, suggesting that they cannot load endogenous antigenic peptides onto MHC-II molecules. Importantly, LECs transfer β-gal to dendritic cells, which subsequently present it to induce CD4 T-cell anergy. Therefore, LECs serve as an antigen reservoir for CD4 T-cell tolerance, and MHC-II molecules on LECs are used to induce CD8 T-cell tolerance via LAG-3.

Roles of lymphatic endothelial cells expressing peripheral tissue antigens in CD4 T-cell tolerance induction

PubMed Central

Rouhani, Sherin J.; Eccles, Jacob D.; Riccardi, Priscila; Peske, J. David; Tewalt, Eric F.; Cohen, Jarish N.; Liblau, Roland; Mäkinen, Taija; Engelhard, Victor H.

2015-01-01

Lymphatic endothelial cells (LECs) directly express peripheral tissue antigens and induce CD8 T-cell deletional tolerance. LECs express MHC-II molecules, suggesting they might also tolerize CD4 T cells. We demonstrate that when β-galactosidase (β-gal) is expressed in LECs, β-gal-specific CD8 T cells undergo deletion via the PD-1/PD-L1 and LAG-3/MHC-II pathways. In contrast, LECs do not present endogenous β-gal in the context of MHC-II molecules to β-gal-specific CD4 T cells. Lack of presentation is independent of antigen localization, as membrane-bound haemagglutinin and I-Eα are also not presented by MHC-II molecules. LECs express invariant chain and cathepsin L, but not H2-M, suggesting that they cannot load endogenous antigenic peptides onto MHC-II molecules. Importantly, LECs transfer β-gal to dendritic cells, which subsequently present it to induce CD4 T-cell anergy. Therefore, LECs serve as an antigen reservoir for CD4 T-cell tolerance, and MHC-II molecules on LECs are used to induce CD8 T-cell tolerance via LAG-3. PMID:25857745

Memory vs memory-like: The different facets of CD8+ T-cell memory in HCV infection.

PubMed

Hofmann, Maike; Wieland, Dominik; Pircher, Hanspeter; Thimme, Robert

2018-05-01

Memory CD8 + T cells are essential in orchestrating protection from re-infection. Hallmarks of virus-specific memory CD8 + T cells are the capacity to mount recall responses with rapid induction of effector cell function and antigen-independent survival. Growing evidence reveals that even chronic infection does not preclude virus-specific CD8 + T-cell memory formation. However, whether this kind of CD8 + T-cell memory that is established during chronic infection is indeed functional and provides protection from re-infection is still unclear. Human chronic hepatitis C virus infection represents a unique model system to study virus-specific CD8 + T-cell memory formation during and after cessation of persisting antigen stimulation. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

IL-15 regulates memory CD8+ T cell O-glycan synthesis and affects trafficking

PubMed Central

Nolz, Jeffrey C.; Harty, John T.

2014-01-01

Memory and naive CD8+ T cells exhibit distinct trafficking patterns. Specifically, memory but not naive CD8+ T cells are recruited to inflamed tissues in an antigen-independent manner. However, the molecular mechanisms that regulate memory CD8+ T cell trafficking are largely unknown. Here, using murine models of infection and T cell transfer, we found that memory but not naive CD8+ T cells dynamically regulate expression of core 2 O-glycans, which interact with P- and E-selectins to modulate trafficking to inflamed tissues. Following infection, antigen-specific effector CD8+ T cells strongly expressed core 2 O-glycans, but this glycosylation pattern was lost by most memory CD8+ T cells. After unrelated infection or inflammatory challenge, memory CD8+ T cells synthesized core 2 O-glycans independently of antigen restimulation. The presence of core 2 O-glycans subsequently directed these cells to inflamed tissue. Memory and naive CD8+ T cells exhibited the opposite pattern of epigenetic modifications at the Gcnt1 locus, which encodes the enzyme that initiates core 2 O-glycan synthesis. The open chromatin configuration in memory CD8+ T cells permitted de novo generation of core 2 O-glycans in a TCR-independent, but IL-15–dependent, manner. Thus, IL-15 stimulation promotes antigen-experienced memory CD8+ T cells to generate core 2 O-glycans, which subsequently localize them to inflamed tissues. These findings suggest that CD8+ memory T cell trafficking potentially can be manipulated to improve host defense and immunotherapy. PMID:24509081

IL-1 enhances expansion, effector function, tissue localization, and memory response of antigen-specific CD8 T cells

PubMed Central

Ben-Sasson, Shlomo Z.; Hogg, Alison; Hu-Li, Jane; Wingfield, Paul; Chen, Xi; Crank, Michelle; Caucheteux, Stephane; Ratner-Hurevich, Maya; Berzofsky, Jay A.; Nir-Paz, Ran

2013-01-01

Here, we show that interleukin-1 (IL-1) enhances antigen-driven CD8 T cell responses. When administered to recipients of OT-I T cell receptor transgenic CD8 T cells specific for an ovalbumin (OVA) peptide, IL-1 results in an increase in the numbers of wild-type but not IL1R1−/− OT-I cells, particularly in spleen, liver, and lung, upon immunization with OVA and lipopolysaccharide. IL-1 administration also results in an enhancement in the frequency of antigen-specific cells that are granzyme B+, have cytotoxic activity, and/ or produce interferon γ (IFN-γ). Cells primed in the presence of IL-1 display enhanced expression of granzyme B and increased capacity to produce IFN-γ when rechallenged 2 mo after priming. In three in vivo models, IL-1 enhances the protective value of weak immunogens. Thus, IL-1 has a marked enhancing effect on antigen-specific CD8 T cell expansion, differentiation, migration to the periphery, and memory. PMID:23460726

Protective CD8 Memory T Cell Responses to Mouse Melanoma Are Generated in the Absence of CD4 T Cell Help

PubMed Central

Steinberg, Shannon M.; Zhang, Peisheng; Turk, Mary Jo

2011-01-01

Background We have previously demonstrated that temporary depletion of CD4 T cells in mice with progressive B16 melanoma, followed by surgical tumor excision, induces protective memory CD8 T cell responses to melanoma/melanocyte antigens. We also showed that persistence of these CD8 T cells is supported, in an antigen-dependent fashion, by concurrent autoimmune melanocyte destruction. Herein we explore the requirement of CD4 T cell help in priming and maintaining this protective CD8 T cell response to melanoma. Methodology and Principal Findings To induce melanoma/melanocyte antigen-specific CD8 T cells, B16 tumor bearing mice were depleted of regulatory T cells (Treg) by either temporary, or long-term continuous treatment with anti-CD4 (mAb clone GK1.5). Total depletion of CD4 T cells led to significant priming of IFN-γ-producing CD8 T cell responses to TRP-2 and gp100. Surprisingly, treatment with anti-CD25 (mAb clone PC61), to specifically deplete Treg cells while leaving help intact, was ineffective at priming CD8 T cells. Thirty to sixty days after primary tumors were surgically excised, mice completely lacking CD4 T cell help developed autoimmune vitiligo, and maintained antigen-specific memory CD8 T cell responses that were highly effective at producing cytokines (IFN-γ, TNF-α, and IL-2). Mice lacking total CD4 T cell help also mounted protection against re-challenge with B16 melanoma sixty days after primary tumor excision. Conclusions and Significance This work establishes that CD4 T cell help is dispensable for the generation of protective memory T cell responses to melanoma. Our findings support further use of CD4 T cell depletion therapy for inducing long-lived immunity to cancer. PMID:22046294

Heterologous prime-boost regimens with a recombinant chimpanzee adenoviral vector and adjuvanted F4 protein elicit polyfunctional HIV-1-specific T-Cell responses in macaques.

PubMed

Lorin, Clarisse; Vanloubbeeck, Yannick; Baudart, Sébastien; Ska, Michaël; Bayat, Babak; Brauers, Geoffroy; Clarinval, Géraldine; Donner, Marie-Noëlle; Marchand, Martine; Koutsoukos, Marguerite; Mettens, Pascal; Cohen, Joe; Voss, Gerald

2015-01-01

HIV-1-specific CD4+ and CD8+ T lymphocytes are important for HIV-1 replication control. F4/AS01 consists of F4 recombinant fusion protein (containing clade B Gag/p24, Pol/RT, Nef and Gag/p17) formulated in AS01 Adjuvant System, and was shown to induce F4-specific polyfunctional CD4+ T-cell responses in humans. While replication-incompetent recombinant HIV-1/SIV antigen-expressing human adenoviral vectors can elicit high-frequency antigen-specific CD8+ T-cell responses, their use is hampered by widespread pre-existing immunity to human serotypes. Non-human adenovirus serotypes associated with lower prevalence may offer an alternative strategy. We evaluated the immunogenicity of AdC7-GRN ('A'), a recombinant chimpanzee adenovirus type 7 vector expressing clade B Gag, RT and Nef, and F4/AS01 ('P'), when delivered intramuscularly in homologous (PP or AA) and heterologous (AAPP or PPAA) prime-boost regimens, in macaques and mice. Vaccine-induced HIV-1-antigen-specific T cells in peripheral blood (macaques), liver, spleen, and intestinal and genital mucosa (mice) were characterized by intracellular cytokine staining. Vaccine-specific IgG antibodies (macaques) were detected using ELISA. In macaques, only the heterologous prime-boost regimens induced polyfunctional, persistent and balanced CD4+ and CD8+ T-cell responses specific to each HIV-1 vaccine antigen. AdC7-GRN priming increased the polyfunctionality of F4/AS01-induced CD4+ T cells. Approximately 50% of AdC7-GRN-induced memory CD8+ T cells exhibited an effector-memory phenotype. HIV-1-specific antibodies were detected with each regimen. In mice, antigen-specific CD4+ and CD8+ T-cell responses were detected in the mucosal and systemic anatomical compartments assessed. When administered in heterologous prime-boost regimens, AdC7-GRN and F4/AS01 candidate vaccines acted complementarily in inducing potent and persistent peripheral blood HIV-1-specific CD4+ and CD8+ T-cell responses and antibodies in macaques. Besides, adenoviral vector priming modulated the cytokine-expression profile of the protein-induced CD4+ T cells. Each regimen induced HIV-1-specific T-cell responses in systemic/local tissues in mice. This suggests that prime-boost regimens combining adjuvanted protein and low-seroprevalent chimpanzee adenoviral vectors represent an attractive vaccination strategy for clinical evaluation.

Heterologous Prime-Boost Regimens with a Recombinant Chimpanzee Adenoviral Vector and Adjuvanted F4 Protein Elicit Polyfunctional HIV-1-Specific T-Cell Responses in Macaques

PubMed Central

Lorin, Clarisse; Vanloubbeeck, Yannick; Baudart, Sébastien; Ska, Michaël; Bayat, Babak; Brauers, Geoffroy; Clarinval, Géraldine; Donner, Marie-Noëlle; Marchand, Martine; Koutsoukos, Marguerite; Mettens, Pascal; Cohen, Joe; Voss, Gerald

2015-01-01

HIV-1-specific CD4+ and CD8+ T lymphocytes are important for HIV-1 replication control. F4/AS01 consists of F4 recombinant fusion protein (containing clade B Gag/p24, Pol/RT, Nef and Gag/p17) formulated in AS01 Adjuvant System, and was shown to induce F4-specific polyfunctional CD4+ T-cell responses in humans. While replication-incompetent recombinant HIV-1/SIV antigen-expressing human adenoviral vectors can elicit high-frequency antigen-specific CD8+ T-cell responses, their use is hampered by widespread pre-existing immunity to human serotypes. Non-human adenovirus serotypes associated with lower prevalence may offer an alternative strategy. We evaluated the immunogenicity of AdC7-GRN (‘A’), a recombinant chimpanzee adenovirus type 7 vector expressing clade B Gag, RT and Nef, and F4/AS01 (‘P’), when delivered intramuscularly in homologous (PP or AA) and heterologous (AAPP or PPAA) prime-boost regimens, in macaques and mice. Vaccine-induced HIV-1-antigen-specific T cells in peripheral blood (macaques), liver, spleen, and intestinal and genital mucosa (mice) were characterized by intracellular cytokine staining. Vaccine-specific IgG antibodies (macaques) were detected using ELISA. In macaques, only the heterologous prime-boost regimens induced polyfunctional, persistent and balanced CD4+ and CD8+ T-cell responses specific to each HIV-1 vaccine antigen. AdC7-GRN priming increased the polyfunctionality of F4/AS01-induced CD4+ T cells. Approximately 50% of AdC7-GRN-induced memory CD8+ T cells exhibited an effector-memory phenotype. HIV-1-specific antibodies were detected with each regimen. In mice, antigen-specific CD4+ and CD8+ T-cell responses were detected in the mucosal and systemic anatomical compartments assessed. When administered in heterologous prime-boost regimens, AdC7-GRN and F4/AS01 candidate vaccines acted complementarily in inducing potent and persistent peripheral blood HIV-1-specific CD4+ and CD8+ T-cell responses and antibodies in macaques. Besides, adenoviral vector priming modulated the cytokine-expression profile of the protein-induced CD4+ T cells. Each regimen induced HIV-1-specific T-cell responses in systemic/local tissues in mice. This suggests that prime-boost regimens combining adjuvanted protein and low-seroprevalent chimpanzee adenoviral vectors represent an attractive vaccination strategy for clinical evaluation. PMID:25856308

Protective Capacity of Memory CD8+ T Cells is Dictated by Antigen Exposure History and Nature of the Infection

PubMed Central

Nolz, Jeffrey C.; Harty, John T.

2011-01-01

SUMMARY Infection or vaccination confers heightened resistance to pathogen re-challenge due to quantitative and qualitative differences between naïve and primary memory T cells. Herein, we show that secondary (boosted) memory CD8+ T cells were better than primary memory CD8+ T cells in controlling some, but not all acute infections with diverse pathogens. However, secondary memory CD8+ T cells were less efficient than an equal number of primary memory cells at preventing chronic LCMV infection and are more susceptible to functional exhaustion. Importantly, localization of memory CD8+ T cells within lymph nodes, which is reduced by antigen re-stimulation, was critical for both viral control in lymph nodes and for the sustained CD8+ T cell response required to prevent chronic LCMV infection. Thus, repeated antigen-stimulation shapes memory CD8+ T cell populations to either enhance or decrease per cell protective immunity in a pathogen-specific manner, a concept of importance in vaccine design against specific diseases. PMID:21549619

Induction of hapten-specific tolerance of human CD8+ urushiol (poison ivy)-reactive T lymphocytes.

PubMed

Kalish, R S; Wood, J A

1997-03-01

The interaction of CD28 with B7 molecules (CD80 or CD86) is an essential second signal for both the activation of CD4+ T cells through the T-cell receptor and the prevention of anergy. We studied the requirement of hapten-specific human CD8+ cells for CD28 co-stimulation in recognition of hapten, and anergy induction. Urushiol, the immunogenic hapten of poison ivy (Toxicodendron radicans), elicits a predominantly CD8+ T-cell response. Autologous PBMC were pre-incubated with urushiol prior to fixation by paraformaldehyde. Fixed antigen-presenting cells were unable to present urushiol to human CD8+ urushiol-specific T cells. Addition of anti-CD28, however, overcame this antigen-presenting defect, enabling CD8+ cells to proliferate. Fixation of antigen-presenting cells prevents upregulation of B7, and addition of anti-CD28 substitutes for this signal. Proliferation of CD8+ T cells in response to urushiol was blocked by CTLA4Ig, a recombinant fusion protein that blocks CD28/B7 interactions. Preincubation of urushiol-specific CD8+ cells with fixed PBMC + urushiol for 7 d induced anergy. Anergic CD8+ cells were viable and able to proliferate in response to IL-2, but not in response to urushiol. Induction of anergy required the presence of urushiol, and pre-incubation with irradiated PBMC + urushiol did not have this effect. It is proposed that anergy was induced by presentation of urushiol by fixed PBMC, in the absence of adequate co-stimulation signals. Induction of anergy by blocking of co-stimulation could potentially induce clinical hyposensitization to haptens.

Polyfunctional analysis of Gag and Nef specific CD8+ T-cell responses in HIV-1 infected Indian individuals.

PubMed

Mendiratta, Sanjay; Vajpayee, Madhu; Mojumdar, Kamalika; Chauhan, Neeraj K; Sreenivas, Vishnubhatla

2011-02-01

Polyfunctional CD8+ T-cells have been described as most competent in controlling viral replication. We studied the impact of antigen persistence on the polyfunctional immune responses of CD8+ T-lymphocytes to HIV Gag and Nef peptides and polyclonal stimuli in 40 ART naïve HIV infected individuals and analyzed the alterations in T-cell functionality in early and late stages of infection. Significantly elevated level of global response and polyfunctional profile of CD8+ T-cells were observed to polyclonal stimulation, than HIV specific antigens in chronically infected individuals. However no key differences were observed in CD8+ T-cell functional profile in any of the 15 unique subsets for Gag and Nef specific antigens. The subjects in early stage of infection (defined as a gap of 6 months or less between seroconversion and enrolment and with no apparent clinical symptoms) had a higher degree of response functionality (4+ or 3+ different functions simultaneously) than in the late stage infection (defined as time duration since seroconversion greater than 6 months). The data suggest that persistence of antigen during chronic infection leads to functional impairment of HIV specific responses. Copyright © 2010 Elsevier Ltd. All rights reserved.

SLAP deficiency increases TCR avidity leading to altered repertoire and negative selection of cognate antigen-specific CD8+ T cells

PubMed Central

Friend, Samantha F.; Peterson, Lisa K.; Kedl, Ross M.; Dragone, Leonard L.

2014-01-01

How T cell receptor (TCR) avidity influences CD8+ T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP−/− mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP−/− Vβ5 mice, expressing only the β-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8+ T cell development and repertoire selection. In comparing SLAP−/− OT-1 and Vβ5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP−/− Vβ5 mice. We have found that SLAP−/− OT-1 mice have fewer CD8+ thymocytes but have increased CD5 expression. SLAP−/− OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8+ splenocytes upon tetramer staining. Our data demonstrate that SLAP−/− Vβ5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vβ5 CD8+ T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8+ T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8+ T cell development influences repertoire selection. PMID:22956467

SLAP deficiency increases TCR avidity leading to altered repertoire and negative selection of cognate antigen-specific CD8+ T cells.

PubMed

Friend, Samantha F; Peterson, Lisa K; Kedl, Ross M; Dragone, Leonard L

2013-03-01

How T cell receptor (TCR) avidity influences CD8(+) T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP(-/-) mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP(-/-) Vβ5 mice, expressing only the β-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8(+) T cell development and repertoire selection. In comparing SLAP(-/-) OT-1 and Vβ5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP(-/-) Vβ5 mice. We have found that SLAP(-/-) OT-1 mice have fewer CD8(+) thymocytes but have increased CD5 expression. SLAP(-/-) OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8(+) splenocytes upon tetramer staining. Our data demonstrate that SLAP(-/-) Vβ5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vβ5 CD8(+) T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8(+) T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8(+) T cell development influences repertoire selection.

β-cell-specific CD8 T cell phenotype in type 1 diabetes reflects chronic autoantigen exposure

PubMed Central

McLaren, James E.; Dolton, Garry; Matthews, Katherine K.; Gostick, Emma; Kronenberg-Versteeg, Deborah; Eichmann, Martin; Knight, Robin R.; Heck, Susanne; Powrie, Jake; Bingley, Polly J.; Dayan, Colin M.; Miles, John J.; Sewell, Andrew K.

2015-01-01

Autoreactive CD8 T cells play a central role in the destruction of pancreatic islet β-cells that leads to type 1 diabetes, yet the key features of this immune-mediated process remain poorly defined. In this study, we combined high definition polychromatic flow cytometry with ultrasensitive peptide-human leukocyte antigen class I (pHLAI) tetramer staining to quantify and characterize β-cell-specific CD8 T cell populations in patients with recent onset type 1 diabetes and healthy controls. Remarkably, we found that β-cell-specific CD8 T cell frequencies in peripheral blood were similar between subject groups. In contrast to healthy controls, however, patients with newly diagnosed type 1 diabetes displayed hallmarks of antigen-driven expansion uniquely within the β-cell-specific CD8 T cell compartment. Molecular analysis of selected β-cell-specific CD8 T cell populations further revealed highly skewed oligoclonal T cell receptor (TCR) repertoires comprising exclusively private clonotypes. Collectively, these data identify novel and distinctive features of disease-relevant CD8 T cells that inform the immunopathogenesis of type 1 diabetes. PMID:25249579

Impact of benznidazole treatment on the functional response of Trypanosoma cruzi antigen-specific CD4+CD8+ T cells in chronic Chagas disease patients.

PubMed

Pérez-Antón, Elena; Egui, Adriana; Thomas, M Carmen; Puerta, Concepción J; González, John Mario; Cuéllar, Adriana; Segovia, Manuel; López, Manuel Carlos

2018-05-11

Chagas disease is caused by Trypanosoma cruzi. The persistence of the parasite is associated with the disease chronicity and the impairment of the cellular immune response. It has been reported that the CD4+CD8+ T cell population expands in chronic Chagas disease patients. Few studies have focused on this subset of cells, and very little is known about the impact of antiparasitic treatment on this population. Thirty-eight chronic Chagas disease patients (20 asymptomatic and 18 symptomatic) and twelve healthy controls were enrolled in this study. Peripheral blood mononuclear cells were stimulated with soluble T. cruzi antigens to analyze the production of cytokines and cytotoxic molecules by CD4+CD8+ T cells before and after benznidazole treatment. Additionally, expression and co-expression of five inhibitory receptors in these patients after treatment were studied using a multiparameter flow cytometry technique. The frequency of CD4+CD8+ T cells was higher in chronic Chagas disease patients compared with healthy donors. Furthermore, a higher ratio of CD4+CD8low/CD4+CD8high subpopulations was observed in chronic Chagas disease patients than in healthy donors. Additionally, CD4+CD8+ T cells from these patients expressed and co-expressed higher levels of inhibitory receptors in direct proportion to the severity of the pathology. Benznidazole treatment reduced the frequency of CD4+CD8+ T cells and decreased the ratio of CD4+CD8low/CD4+CD8high subpopulations. The co-expression level of the inhibitory receptor was reduced after treatment simultaneously with the enhancement of the multifunctional capacity of CD4+CD8+ T cells. After treatment, an increase in the frequency of T. cruzi antigen-specific CD4+CD8+ T cells expressing IL-2 and TNF-α was also observed. CD4+CD8+ T cells could play an important role in the control of T. cruzi infection since they were able to produce effector molecules for parasite control. Benznidazole treatment partially reversed the exhaustion process caused by T. cruzi infection in these cells with an improvement in the functional response of the T. cruzi antigen-specific CD4+CD8+ T cells.

Impact of benznidazole treatment on the functional response of Trypanosoma cruzi antigen-specific CD4+CD8+ T cells in chronic Chagas disease patients

PubMed Central

Pérez-Antón, Elena; Egui, Adriana; Thomas, M. Carmen; Puerta, Concepción J.; González, John Mario; Cuéllar, Adriana; Segovia, Manuel

2018-01-01

Background Chagas disease is caused by Trypanosoma cruzi. The persistence of the parasite is associated with the disease chronicity and the impairment of the cellular immune response. It has been reported that the CD4+CD8+ T cell population expands in chronic Chagas disease patients. Few studies have focused on this subset of cells, and very little is known about the impact of antiparasitic treatment on this population. Methodology Thirty-eight chronic Chagas disease patients (20 asymptomatic and 18 symptomatic) and twelve healthy controls were enrolled in this study. Peripheral blood mononuclear cells were stimulated with soluble T. cruzi antigens to analyze the production of cytokines and cytotoxic molecules by CD4+CD8+ T cells before and after benznidazole treatment. Additionally, expression and co-expression of five inhibitory receptors in these patients after treatment were studied using a multiparameter flow cytometry technique. Principal findings The frequency of CD4+CD8+ T cells was higher in chronic Chagas disease patients compared with healthy donors. Furthermore, a higher ratio of CD4+CD8low/CD4+CD8high subpopulations was observed in chronic Chagas disease patients than in healthy donors. Additionally, CD4+CD8+ T cells from these patients expressed and co-expressed higher levels of inhibitory receptors in direct proportion to the severity of the pathology. Benznidazole treatment reduced the frequency of CD4+CD8+ T cells and decreased the ratio of CD4+CD8low/CD4+CD8high subpopulations. The co-expression level of the inhibitory receptor was reduced after treatment simultaneously with the enhancement of the multifunctional capacity of CD4+CD8+ T cells. After treatment, an increase in the frequency of T. cruzi antigen-specific CD4+CD8+ T cells expressing IL-2 and TNF-α was also observed. Conclusions CD4+CD8+ T cells could play an important role in the control of T. cruzi infection since they were able to produce effector molecules for parasite control. Benznidazole treatment partially reversed the exhaustion process caused by T. cruzi infection in these cells with an improvement in the functional response of the T. cruzi antigen-specific CD4+CD8+ T cells. PMID:29750791

Recruitment of bone marrow CD11b+Gr-1+ cells by polymeric nanoparticles for antigen cross-presentation

NASA Astrophysics Data System (ADS)

Yang, Ya-Wun; Luo, Wen-Hui

2017-03-01

The objective of this study was to investigate the function of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) on the activation of antigen-specific CD8+ T cell responses via the CD11b+Gr-1+ myeloid subpopulations in murine bone marrow (BM). PLGA NPs containing ovalbumin (OVA) were fabricated by the double-emulsion method. The CD11b+Gr-1lowLy-6Chigh and CD11b+Gr-1highLy-6Clow subsets from mice bone marrow were sorted and treated with the PLGA/OVA NPs, followed by co-culture with the carboxyfluorescein succinimidyl ester (CFSE)-labelled OT-I CD8+ cells. Co-culture of OT-I CD8+ T cells with PLGA/OVA NPs-primed CD11b+Gr-1+ subsets upregulated the expression of IL-2, TNF-α, INF-γ, granzyme B, and perforin, resulting in proliferation of CD8+ T cells and differentiation into effector cytotoxic T lymphocytes (CTLs). In vivo proliferation of CFSE-labelled OT-I CD8+ cells in response to OVA was also obtained in the animals immunized with PLGA/OVA NPs. The results presented in this study demonstrate the ability of polymeric NPs to recruit two CD11b+Gr-1+ myeloid subsets for effective presentation of exogenous antigen to OT-I CD8+ T cells in the context of major histocompatibility complex (MHC) class I, leading to an induction of antigen-specific cell proliferation and differentiation into effector cells.

Human leucocyte antigen class I-redirected anti-tumour CD4+ T cells require a higher T cell receptor binding affinity for optimal activity than CD8+ T cells.

PubMed

Tan, M P; Dolton, G M; Gerry, A B; Brewer, J E; Bennett, A D; Pumphrey, N J; Jakobsen, B K; Sewell, A K

2017-01-01

CD4 + T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour-specific CD4 + T cells occur in low frequency, express relatively low-affinity T cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leucocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4 + T cells with tumour-specific HLA class I-restricted TCRs prior to adoptive transfer. The lack of help from the co-receptor CD8 glycoprotein in CD4 + cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4 + and CD8 + T cells expressing wild-type and a range of affinity-enhanced TCRs specific for the HLA A*0201-restricted NY-ESO-1- and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4 + T cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and (ii) optimal TCR binding affinity is higher in CD4 + T cells than CD8 + T cells. These results indicate that the CD4 + T cell component of current adoptive therapies using TCRs optimized for CD8 + T cells is below par and that there is room for substantial improvement. © 2016 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.

Naive CD8 T-Cells Initiate Spontaneous Autoimmunity to a Sequestered Model Antigen of the Central Nervous System

ERIC Educational Resources Information Center

Na, Shin-Young; Cao, Yi; Toben, Catherine; Nitschke, Lars; Stadelmann, Christine; Gold, Ralf; Schimpl, Anneliese; Hunig, Thomas

2008-01-01

In multiple sclerosis, CD8 T-cells are thought play a key pathogenetic role, but mechanistic evidence from rodent models is limited. Here, we have tested the encephalitogenic potential of CD8 T-cells specific for the model antigen ovalbumin (OVA) sequestered in oligodendrocytes as a cytosolic molecule. We show that in these "ODC-OVA" mice, the…

Foxp3+ regulatory T cells impede the priming of protective CD8+ T cells

PubMed Central

Ertelt, James M.; Rowe, Jared H.; Mysz, Margaret A.; Singh, Charanjeet; Roychowdhury, Monika; Aguilera, Marijo N.; Way, Sing Sing

2011-01-01

T cell activation is controlled by incompletely defined opposing stimulation and suppression signals that together sustain the balance between optimal host defense against infection and peripheral tolerance. Herein, we explored the impacts of Foxp3+ regulatory T cell (Treg) suppression in priming antigen-specific T cell activation under non-infection and infection conditions. We find the transient ablation of Foxp3+ Tregs unleashes the robust expansion and activation of peptide stimulated CD8+ T cells that provide protection against Listeria monocytogenes (Lm) infection in an antigen-specific fashion. By contrast, Treg-ablation had non-significant impacts on the CD8+ T cell response primed by infection with recombinant Lm. Similarly, non-recombinant Lm administered with peptide stimulated the expansion and activation of CD8+ T cells that paralleled the response primed by Treg-ablation. Interestingly, these adjuvant properties of Lm did not require CD8+ T cell stimulation by IL-12 produced in response to infection, but instead were associated with sharp reductions in Foxp3+ Treg suppressive potency. Therefore, Foxp3+ Tregs impose critical barriers that when overcome naturally during infection or artificially with ablation allows the priming of protective antigen-specific CD8+ T cells. PMID:21810602

The molecular determinants of CD8 co-receptor function.

PubMed

Cole, David K; Laugel, Bruno; Clement, Mathew; Price, David A; Wooldridge, Linda; Sewell, Andrew K

2012-10-01

CD8(+) T cells respond to signals mediated through a specific interaction between the T-cell receptor (TCR) and a composite antigen in the form of an epitopic peptide bound between the polymorphic α1 and α2 helices of an MHC class I (MHCI) molecule. The CD8 glycoprotein 'co-receives' antigen by binding to an invariant region of the MHCI molecule and can enhance ligand recognition by up to 1 million-fold. In recent years, a number of structural and biophysical investigations have shed light on the role of the CD8 co-receptor during T-cell antigen recognition. Here, we provide a collated resource for these data, and discuss how the structural and biophysical parameters governing CD8 co-receptor function further our understanding of T-cell cross-reactivity and the productive engagement of low-affinity antigenic ligands. © 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.

Antigen sensitivity is a major determinant of CD8+ T-cell polyfunctionality and HIV-suppressive activity

PubMed Central

Almeida, Jorge R.; Sauce, Delphine; Price, David A.; Papagno, Laura; Shin, So Youn; Moris, Arnaud; Larsen, Martin; Pancino, Gianfranco; Douek, Daniel C.; Autran, Brigitte; Sáez-Cirión, Asier

2009-01-01

CD8+ T cells are major players in the immune response against HIV. However, recent failures in the development of T cell–based vaccines against HIV-1 have emphasized the need to reassess our basic knowledge of T cell–mediated efficacy. CD8+ T cells from HIV-1–infected patients with slow disease progression exhibit potent polyfunctionality and HIV-suppressive activity, yet the factors that unify these properties are incompletely understood. We performed a detailed study of the interplay between T-cell functional attributes using a bank of HIV-specific CD8+ T-cell clones isolated in vitro; this approach enabled us to overcome inherent difficulties related to the in vivo heterogeneity of T-cell populations and address the underlying determinants that synthesize the qualities required for antiviral efficacy. Conclusions were supported by ex vivo analysis of HIV-specific CD8+ T cells from infected donors. We report that attributes of CD8+ T-cell efficacy against HIV are linked at the level of antigen sensitivity. Highly sensitive CD8+ T cells display polyfunctional profiles and potent HIV-suppressive activity. These data provide new insights into the mechanisms underlying CD8+ T-cell efficacy against HIV, and indicate that vaccine strategies should focus on the induction of HIV-specific T cells with high levels of antigen sensitivity to elicit potent antiviral efficacy. PMID:19389882

Antigen sensitivity is a major determinant of CD8+ T-cell polyfunctionality and HIV-suppressive activity.

PubMed

Almeida, Jorge R; Sauce, Delphine; Price, David A; Papagno, Laura; Shin, So Youn; Moris, Arnaud; Larsen, Martin; Pancino, Gianfranco; Douek, Daniel C; Autran, Brigitte; Sáez-Cirión, Asier; Appay, Victor

2009-06-18

CD8(+) T cells are major players in the immune response against HIV. However, recent failures in the development of T cell-based vaccines against HIV-1 have emphasized the need to reassess our basic knowledge of T cell-mediated efficacy. CD8(+) T cells from HIV-1-infected patients with slow disease progression exhibit potent polyfunctionality and HIV-suppressive activity, yet the factors that unify these properties are incompletely understood. We performed a detailed study of the interplay between T-cell functional attributes using a bank of HIV-specific CD8(+) T-cell clones isolated in vitro; this approach enabled us to overcome inherent difficulties related to the in vivo heterogeneity of T-cell populations and address the underlying determinants that synthesize the qualities required for antiviral efficacy. Conclusions were supported by ex vivo analysis of HIV-specific CD8(+) T cells from infected donors. We report that attributes of CD8(+) T-cell efficacy against HIV are linked at the level of antigen sensitivity. Highly sensitive CD8(+) T cells display polyfunctional profiles and potent HIV-suppressive activity. These data provide new insights into the mechanisms underlying CD8(+) T-cell efficacy against HIV, and indicate that vaccine strategies should focus on the induction of HIV-specific T cells with high levels of antigen sensitivity to elicit potent antiviral efficacy.

Photon manipulation in silicon nanophotonic circuits

NASA Astrophysics Data System (ADS)

Elshaari, Ali Wanis

2011-12-01

CD8+ T cells are the branch of the adaptive immune system responsible for recognizing and killing tumor cells or cells infected with intracellular pathogens, such as Listeria monocytogenes (LM). However, when CD8+ T cells target our own tissues, they can cause autoimmune diseases, such as type I diabetes, rheumatoid arthritis. For CD8+ T cells to fulfill these functions, the T cell receptors (TCRs) on CD8+ T cells must recognize pathogens or antigens presented on the surface of target cells. TCR ligation triggers multiple signaling pathways that lead to the activation and proliferation of CD8+ T cells. The goal of our research is to define the TCR-proximal signaling events that regulate CD8+ T cell-mediated immunity. To accomplish this goal, we are focusing on an adaptor protein Gads, which is critical for optimal TCR-mediated calcium mobilization. We reported the first analysis of the function of Gads in peripheral naive CD8+ T cells. To examine the function of Gads in CD8+ T cell mediated immune responses, we crossed Gads-/- mice with mice expressing an MHC class I-restricted transgenic TCR recognizing ovalbumin (OVA). The transgenic mice are called ovalbumin-specific T cell receptor-major histocompatibility complex class I restricted (OT-I) mice. We investigated the effect of Gads on the proliferation of CD8+ T cells following stimulation with peptide antigen in vivo and in vitro. We stimulated splenocytes from Gads+/+ OT-I and Gads -/- OT-I mice with the peptide agonist. The experiments revealed that Gads is required for optimal proliferation of CD8+ T cells. The regulation of Gads is most evident at the early time points of proliferation. Then we demonstrated that Gads-/- CD8+ T cells have impaired TCR-mediated exit from G0 phase of the cell cycle. In addition, Gads-/- CD8+ T cells have delayed expression of c-myc and the activation markers CD69 and CD25, upon stimulation with peptide antigen. Next, we investigated how Gads affects CD8+ T cell-mediated immunity in the context of infection with LM. We adoptively transferred naive CD8+ T cells from Gads+/+ OT-I mice and/or Gads -/- OT-I mice into congenic wild-type hosts. Then the recipient mice were infected with recombinant LM expressing ovalbumin (rLM-OVA). The CD8 + T cells from OT-I mice recognize and respond to the ovalbumin provided by this strain of LM. By using this system, we investigated how Gads regulates the activation of antigen-specific CD8+ T cells as well as the expansion and memory phases of CD8+ T cell-mediated immune responses following infection with rLM-OVA. We also examined the recall response of CD8+ T cells after the secondary encounter with the same pathogen. Our data demonstrated that Gads regulates the expression of activation markers CD69 and CD25 of antigen-specific CD8+ T cells but Gads is not required for the onset of accumulation of antigen-specific CD8 + T cells following infection. However, Gads is critical to sustain the expansion of CD8+ T cell-mediated immune response following infection. Although the differentiation of naive CD8+ T cells into memory cells is independent of Gads, Gads is required for an optimal recall response. Our data indicating that Gads regulates the initiation of proliferation of CD8+ T cells upon TCR ligation by peptide antigen seemed to contradict with our in vivo infection data showing that Gads is not required for the initiation of expansion of CD8+ T cell population. In order to explain the "discrepancy", we hypothesized that the homotypic interactions among CD8+ T cells compensate for Gads deficiency at the initial stage of accumulation of antigen-specific CD8+ T cells upon infection. Our data indicated that the need for Gads in cell cycle progression of CD8+ T cells when total splenocytes were stimulated could be overcome by stimulating purified CD8 + T cells. These data suggested that the homotypic interactions among CD8+ T cells facilitate the TCR signaling so as to compensate for Gads deficiency in promoting cell cycle entry and proliferation. To conclude, the role of Gads in TCR-mediated activation and proliferation of CD8+ T cells is dependent on the interactions of CD8 + T cells and their partners. Interestingly, if CD8+ T cells interact with non-CD8+ T cells, Gads regulates the kinetics of cell cycle entry; however, if CD8+ T cells interact with other CD8+ T cells, Gads is dispensable for cell cycle entry of CD8+ T cells. Overall, these studies will help us better understand how TCR-proximal signaling regulates the activation of CD8 + T cells.

CD8+ T cells produce a dialyzable antigen-specific activator of dendritic cells

PubMed Central

Myles, Ian A.; Zhao, Ming; Nardone, Glenn; Olano, Lisa R.; Reckhow, Jensen D.; Saleem, Danial; Break, Timothy J.; Lionakis, Michail S.; Myers, Timothy G.; Gardina, Paul J.; Kirkpatrick, Charles H.; Holland, Steven M.; Datta, Sandip K.

2017-01-01

Cellular lysates from PPD+ donors have been reported to transfer tuberculin reactivity to naïve recipients, but not diphtheria reactivity, and vice versa. A historically controversial topic, the terms "transfer factor" and "DLE" were used to characterize the reactivity-transferring properties of lysates. Intrigued by these reported phenomena, we found that the cellular extract derived from antigen-specific memory CD8+ T cells induces IL-6 from antigen-matched APCs. This ultimately elicits IL-17 from bystander memory CD8+ T cells. We have identified that dialyzable peptide sequences, S100a9, and the TCR β chain from CD8+ T cells contribute to the molecular nature of this activity. We further show that extracts from antigen-targeted T cells enhance immunity to Staphylococcus aureus and Candida albicans. These effects are sensitive to immunization protocols and extraction methodology in ways that may explain past discrepancies in the reproducibility of passive cellular immunity. PMID:27515950

Impact of regulated secretion on anti-parasitic CD8 T cell responses

PubMed Central

Grover, Harshita Satija; Chu, H. Hamlet; Kelly, Felice D.; Yang, Soo Jung; Reese, Michael L.; Blanchard, Nicolas; Gonzalez, Federico; Chan, Shiao Wei; Boothroyd, John C.; Shastri, Nilabh; Robey, Ellen A.

2014-01-01

Summary CD8 T cells play a key role in defense against the intracellular parasite Toxoplasma but why certain CD8 responses are more potent than others is not well understood. Here, we describe a parasite antigen ROP5 that elicits a modest CD8 T cell response in genetically susceptible mice. ROP5 is secreted via parasite organelles termed rhoptries that are injected directly into host cells during invasion, whereas the protective, dense granule antigen, GRA6, is constitutively secreted into the parasitophorous vacuole. Transgenic parasites in which the ROP5 antigenic epitope was targeted for secretion through dense granules led to enhanced CD8 T cell responses, whereas targeting the GRA6 epitope to rhoptries led to reduced CD8 responses. CD8 T cell responses to the dense granule-targeted ROP5 epitope resulted in reduced parasite load in the brain. These data suggest that the mode of secretion impacts the efficacy of parasite-specific CD8 T cell responses. PMID:24857659

Trypanosoma cruzi Subverts Host Cell Sialylation and May Compromise Antigen-specific CD8+ T Cell Responses*

PubMed Central

Freire-de-Lima, Leonardo; Alisson-Silva, Frederico; Carvalho, Sebastião T.; Takiya, Christina M.; Rodrigues, Maurício M.; DosReis, George A.; Mendonça-Previato, Lucia; Previato, José O.; Todeschini, Adriane R.

2010-01-01

Upon activation, cytotoxic CD8+ T lymphocytes are desialylated exposing β-galactose residues in a physiological change that enhances their effector activity and that can be monitored on the basis of increased binding of the lectin peanut agglutinin. Herein, we investigated the impact of sialylation mediated by trans-sialidase, a specific and unique Trypanosoma transglycosylase for sialic acid, on CD8+ T cell response of mice infected with T. cruzi. Our data demonstrate that T. cruzi uses its trans-sialidase enzyme to resialylate the CD8+ T cell surface, thereby dampening antigen-specific CD8+ T cell response that might favor its own persistence in the mammalian host. Binding of the monoclonal antibody S7, which recognizes sialic acid-containing epitopes on the 115-kDa isoform of CD43, was augmented on CD8+ T cells from ST3Gal-I-deficient infected mice, indicating that CD43 is one sialic acid acceptor for trans-sialidase activity on the CD8+ T cell surface. The cytotoxic activity of antigen-experienced CD8+ T cells against the immunodominant trans-sialidase synthetic peptide IYNVGQVSI was decreased following active trans-sialidase- mediated resialylation in vitro and in vivo. Inhibition of the parasite's native trans-sialidase activity during infection strongly decreased CD8+ T cell sialylation, reverting it to the glycosylation status expected in the absence of parasite manipulation increasing mouse survival. Taken together, these results demonstrate, for the first time, that T. cruzi subverts sialylation to attenuate CD8+ T cell interactions with peptide-major histocompatibility complex class I complexes. CD8+ T cell resialylation may represent a sophisticated strategy to ensure lifetime host parasitism. PMID:20106975

Enhancement of antigen-specific CD4 + and CD8 + T cell responses using a self-assembled biologic nanolipoprotein particle vaccine

DOE PAGES

Weilhammer, Dina; Dunkle, Alexis D.; Blanchette, Craig D.; ...

2017-02-14

To address the need for vaccine platforms that induce robust cell-mediated immunity, we investigated the potential of utilizing self-assembling biologic nanolipoprotein particles (NLPs) as an antigen and adjuvant delivery system to induce antigen-specific murine T cell responses. Here, we utilized OT-I and OT-II TCR-transgenic mice to investigate the effects of NLP-mediated delivery of the model antigen ovalbumin (OVA) on T cell activation. Delivery of OVA with the TLR4 agonist monophosphoryl lipid A (MPLA) in the context of NLPs significantly enhanced the activation of both CD4 + and CD8 + T cells in vitro compared to co-administration of free OVA andmore » MPLA. Upon intranasal immunization of mice harboring TCR-transgenic cells, NLPs enhanced the adjuvant effects of MPLA and the in vivo delivery of OVA, leading to significantly increased expansion of CD4 + and CD8 + T cells in lung-draining lymph nodes. Therefore, NLPs are a promising vaccine platform for inducing T cell responses following intranasal administration.« less

Enhancement of antigen-specific CD4 + and CD8 + T cell responses using a self-assembled biologic nanolipoprotein particle vaccine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weilhammer, Dina; Dunkle, Alexis D.; Blanchette, Craig D.

To address the need for vaccine platforms that induce robust cell-mediated immunity, we investigated the potential of utilizing self-assembling biologic nanolipoprotein particles (NLPs) as an antigen and adjuvant delivery system to induce antigen-specific murine T cell responses. Here, we utilized OT-I and OT-II TCR-transgenic mice to investigate the effects of NLP-mediated delivery of the model antigen ovalbumin (OVA) on T cell activation. Delivery of OVA with the TLR4 agonist monophosphoryl lipid A (MPLA) in the context of NLPs significantly enhanced the activation of both CD4 + and CD8 + T cells in vitro compared to co-administration of free OVA andmore » MPLA. Upon intranasal immunization of mice harboring TCR-transgenic cells, NLPs enhanced the adjuvant effects of MPLA and the in vivo delivery of OVA, leading to significantly increased expansion of CD4 + and CD8 + T cells in lung-draining lymph nodes. Therefore, NLPs are a promising vaccine platform for inducing T cell responses following intranasal administration.« less

A pathway of costimulation that prevents anergy in CD28- T cells: B7- independent costimulation of CD1-restricted T cells

PubMed Central

1995-01-01

A class of molecules that is expressed on antigen presenting cells, exemplified by CD80 (B7), has been found to provide a necessary costimulatory signal for T cell activation and proliferation. CD28 and CTLA4 are the B7 counterreceptors and are expressed on the majority of human CD4+ T cells and many CD8+ T cells. The signal these molecules mediate is distinguished from other costimulatory signals by the finding that T cell recognition of antigen results in a prolonged state of T cell unresponsiveness or anergy, unless these costimulatory molecules are engaged. However, nearly half of the CD8+ and CD4-CD8- T cells lack CD28, and the costimulatory signals required for the activation of such cells are unknown. To understand the pathways of activation used by CD28- T cells, we have examined the costimulatory requirements of antigen-specific CD4-CD8- TCR(+)-alpha/beta circulating T cells that lack the expression of CD28. We have characterized two T cell lines, DN1 and DN6, that recognize a mycobacterial antigen, and are restricted not by major histocompatibility complex class I or II, but by CD1b or CD1c, two members of a family of major histocompatibility complex-related molecules that have been recently implicated in a distinct pathway for antigen presentation. Comparison of antigen-specific cytolytic responses of the DN1 and DN6 T cell lines against antigen-pulsed CD1+ monocytes or CD1+ B lymphoblastoid cell lines (B-LCL) demonstrated that these T cells recognized antigen presented by both types of cells. However, T cell proliferation occurred only when antigen was presented by CD1+ monocytes, indicating that the CD1+ monocytes expressed a costimulatory molecule that the B- LCL transfectants lacked. This hypothesis was confirmed by demonstrating that the T cells became anergic when incubated with the CD1(+)-transfected B-LCL in the presence of antigen, but not in the absence of antigen. The required costimulatory signal occurred by a CD28-independent mechanism since both the CD1+ monocytes and CD1+ B-LCL transfectants expressed B7-1 and B7-2, and DN1 and DN6 lacked surface expression of CD28. We propose that these data define a previously unrecognized pathway of costimulation for T cells distinct from that involving CD28 and its counterreceptors. We suggest that this B7- independent pathway plays a crucial role in the activation and maintenance of tolerance of at least a subset of CD28- T cells. PMID:7500046

Normalized Synergy Predicts That CD8 Co-Receptor Contribution to T Cell Receptor (TCR) and pMHC Binding Decreases As TCR Affinity Increases in Human Viral-Specific T Cells

PubMed Central

Williams, Chad M.; Schonnesen, Alexandra A.; Zhang, Shu-Qi; Ma, Ke-Yue; He, Chenfeng; Yamamoto, Tori; Eckhardt, S. Gail; Klebanoff, Christopher A.; Jiang, Ning

2017-01-01

The discovery of naturally occurring T cell receptors (TCRs) that confer specific, high-affinity recognition of pathogen and cancer-associated antigens remains a major goal in cellular immunotherapies. The contribution of the CD8 co-receptor to the interaction between the TCR and peptide-bound major histocompatibility complex (pMHC) has previously been correlated with the activation and responsiveness of CD8+ T cells. However, these studies have been limited to model systems of genetically engineered hybridoma TCRs or transgenic mouse TCRs against either a single epitope or an array of altered peptide ligands. CD8 contribution in a native human antigen-specific T cell response remains elusive. Here, using Hepatitis C Virus-specific precursor CTLs spanning a large range of TCR affinities, we discovered that the functional responsiveness of any given TCR correlated with the contribution of CD8 to TCR/pMHC binding. Furthermore, we found that CD8 contribution to TCR/pMHC binding in the two-dimensional (2D) system was more accurately reflected by normalized synergy (CD8 cooperation normalized by total TCR/pMHC bonds) rather than synergy (total CD8 cooperation) alone. While synergy showed an increasing trend with TCR affinity, normalized synergy was demonstrated to decrease with the increase of TCR affinity. Critically, normalized synergy was shown to correlate with CTL functionality and peptide sensitivity, corroborating three-dimensional (3D) analysis of CD8 contribution with respect to TCR affinity. In addition, we identified TCRs that were independent of CD8 for TCR/pMHC binding. Our results resolve the current discrepancy between 2D and 3D analysis on CD8 contribution to TCR/pMHC binding, and demonstrate that naturally occurring high-affinity TCRs are more capable of CD8-independent interactions that yield greater functional responsiveness even with CD8 blocking. Taken together, our data suggest that addition of the normalized synergy parameter to our previously established TCR discovery platform using 2D TCR affinity and sequence test would allow for selection of TCRs specific to any given antigen with the desirable attributes of high TCR affinity, CD8 co-receptor independence and functional superiority. Utilizing TCRs with less CD8 contribution could be beneficial for adoptive cell transfer immunotherapies using naturally occurring or genetically engineered T cells against viral or cancer-associated antigens. PMID:28804489

Minimally Activated CD8 Autoreactive T Cells Specific for IRBP Express a High Level of Foxp3 and Are Functionally Suppressive

PubMed Central

Peng, Yong; Shao, Hui; Ke, Yan; Zhang, Ping; Han, Gencheng; Kaplan, Henry J.; Sun, Deming

2008-01-01

Purpose Results in previous reports have demonstrated that immunization of the EAU-prone B6 mouse activates both CD4 and CD8 IRBP-specific T cells. The purpose of this study was to investigate structural and functional differences between CD4 and CD8 autoreactive T cells activated by the uveitogenic peptide. Methods Purified CD4 and CD8 isolated from B6 mice immunized with an uveitogenic peptide, interphotoreceptor retin-oid-binding protein (IRBP)1-20, were stimulated in vitro with various doses of immunizing peptide. The activated T cells were determined for cytokine production, expression of Foxp3, and suppressor activity. Results CD4 autoreactive T cells underwent full activation when stimulated with high or medium concentrations of immunizing peptide, whereas a high dose of antigenic peptide resulted in only modest activation of CD8 autoreactive T cells. When stimulated by a low dose (<0.1 μg/mL) of antigen or by of a high dose of antigen and a small amount of TGF-β1, the minimally activated CD8 T cells expressed a high level of Foxp3 and gained suppressor function. Conclusions Minimally activated CD8 autoreactive T cells can be functionally suppressive and may neutralize the tissue-damaging effect of the CD4 autoreactive T cells. PMID:17460277

Monitoring of antigen-specific CD8 T cells in patients with type 1 diabetes treated with antiCD3 monoclonal antibodies.

PubMed

Cernea, Simona; Herold, Kevan C

2010-02-01

The way in which anti-CD3 monoclonal antibodies (mAbs) modify human immune responses in type 1 diabetes (T1DM) is not known. We prepared a panel of Class I HLA-A2.1 tetramers with peptides from diabetes-associated antigens and studied the frequency and phenotype of the cells in patients with T1DM and blood donors and in patients treated with anti-CD3 mAb (Teplizumab). More patients with T1DM showed positive staining for at least 1 tetramer using frozen and fresh samples (p<0.05). Three months following treatment with anti-CD3 mAb, the proportion of GAD65- and InsB-peptide reactive CD8+ T cells increased (p<0.05). The phenotype of these cells was modulated from naïve to effector memoryRA+. We concludethat Class I MHC tetramers can identify antigen specific CD8+ T cells in patients with T1DM. The frequency of certain specificities increases after treatment with anti-CD3 mAb. Their modulated phenotype may have functional consequences for their pathogenicity. Copyright 2009 Elsevier Inc. All rights reserved.

Helminth Infections Coincident with Active Pulmonary Tuberculosis Inhibit Mono- and Multifunctional CD4+ and CD8+ T Cell Responses in a Process Dependent on IL-10

PubMed Central

George, Parakkal Jovvian; Anuradha, Rajamanickam; Kumar, Nathella Pavan; Sridhar, Rathinam; Banurekha, Vaithilingam V.; Nutman, Thomas B.; Babu, Subash

2014-01-01

Tissue invasive helminth infections and tuberculosis (TB) are co-endemic in many parts of the world and can trigger immune responses that might antagonize each other. We have previously shown that helminth infections modulate the Th1 and Th17 responses to mycobacterial-antigens in latent TB. To determine whether helminth infections modulate antigen-specific and non-specific immune responses in active pulmonary TB, we examined CD4+ and CD8+ T cell responses as well as the systemic (plasma) cytokine levels in individuals with pulmonary TB with or without two distinct helminth infections—Wuchereria bancrofti and Strongyloides stercoralis infection. By analyzing the frequencies of Th1 and Th17 CD4+ and CD8+ T cells and their component subsets (including multifunctional cells), we report a significant diminution in the mycobacterial–specific frequencies of mono- and multi–functional CD4+ Th1 and (to a lesser extent) Th17 cells when concomitant filarial or Strongyloides infection occurs. The impairment in CD4+ and CD8+ T cell cytokine responses was antigen-specific as polyclonal activated T cell frequencies were equivalent irrespective of helminth infection status. This diminution in T cell responses was also reflected in diminished circulating levels of Th1 (IFN-γ, TNF-α and IL-2)- and Th17 (IL-17A and IL-17F)-associated cytokines. Finally, we demonstrate that for the filarial co-infections at least, this diminished frequency of multifunctional CD4+ T cell responses was partially dependent on IL-10 as IL-10 blockade significantly increased the frequencies of CD4+ Th1 cells. Thus, co-existent helminth infection is associated with an IL-10 mediated (for filarial infection) profound inhibition of antigen-specific CD4+ T cell responses as well as protective systemic cytokine responses in active pulmonary TB. PMID:25211342

HIV-specific cytotoxic T lymphocyte precursors exist in a CD28-CD8+ T cell subset and increase with loss of CD4 T cells.

PubMed

Lewis, D E; Yang, L; Luo, W; Wang, X; Rodgers, J R

1999-06-18

To determine whether the CD28-CD8+ T cells that develop during HIV infection contain HIV-specific cytotoxic precursor cells. CD8 subpopulations from six asymptomatic HIV-positive adults, with varying degrees of CD4 T cell loss, were sorted by flow cytometry and HIV-specific precursor cytotoxic T lymphocyte frequencies were measured. Three populations of CD8 T cells were tested: CD28+CD5-- T cells, CD28-CD57+ T cells (thought to be memory cells) and CD28-CD57- T cells (function unknown). Sorted CD8 subsets were stimulated with antigen presenting cells expressing HIV-1 Gag/Pol molecules. Cytotoxic T cell assays on Gag/Pol expressing 51Cr-labeled Epstein-Barr virus transformed autologous B cells lines or control targets were performed after 2 weeks. Specific lysis and precursor frequencies were calculated. Both CD28 positive and CD28-CD57+ populations contained appreciable numbers of precursors (9-1720 per 10(6) CD8+ T cells). However, the CD28-CD57- population had fewer precursors in five out of six people studied. More CD28 positive HIV-specific cytotoxic T lymphocyte precursors were found in patients with CD4:CD8 ratios > 1, whereas more CD28-CD57+ precursors were found in patients whose CD4:CD8 ratios were < 1 (r2, 0.68). Memory HIV-specific precursor cytotoxic T lymphocytes are found in both CD28 positive and CD28-CD8+ cells, however, a CD28-CD57- subpopulation had fewer. Because CD28-CD57+ cells are antigen-driven with limited diversity, the loss of CD28 on CD8 T cells during disease progression may reduce the response to new HIV mutations; this requires further testing.

Involvement of CD8+ T cell-mediated immune responses in LcrV DNA vaccine induced protection against lethal Yersinia pestis challenge.

PubMed

Wang, Shixia; Goguen, Jon D; Li, Fusheng; Lu, Shan

2011-09-09

Yersinia pestis (Y. pestis) is the causative pathogen of plague, a highly fatal disease for which an effective vaccine, especially against mucosal transmission, is still not available. Like many bacterial infections, antigen-specific antibody responses have been traditionally considered critical, if not solely responsible, for vaccine-induced protection against Y. pestis. Studies in recent years have suggested the importance of T cell immune responses against Y. pestis infection but information is still limited about the details of Y. pestis antigen-specific T cell immune responses. In current report, studies are conducted to identify the presence of CD8+ T cell epitopes in LcrV protein, the leading antigen of plague vaccine development. Furthermore, depletion of CD8+ T cells in LcrV DNA vaccinated Balb/C mice led to reduced protection against lethal intranasal challenge of Y. pestis. These findings establish that an LcrV DNA vaccine is able to elicit CD8+ T cell immune responses against specific epitopes of this key plague antigen and that a CD8+ T cell immune response is involved in LcrV DNA vaccine-elicited protection. Future studies in plague vaccine development will need to examine if the presence of detectable T cell immune responses, in particular CD8+ T-cell immune responses, will enhance the protection against Y. pestis in higher animal species or humans. Copyright © 2010 Elsevier Ltd. All rights reserved.

Spontaneous CD4+ and CD8+ T‐cell responses directed against cancer testis antigens are present in the peripheral blood of testicular cancer patients

PubMed Central

Pearce, Hayden; Hutton, Paul; Chaudhri, Shalini; Porfiri, Emilio; Patel, Prashant; Viney, Richard

2017-01-01

Cancer/testis antigen (CTAg) expression is restricted to spermatogenic cells in an immune‐privileged site within the testis. However, these proteins are expressed aberrantly by malignant cells and T‐cell responses against CTAgs develop in many cancer patients. We investigated the prevalence, magnitude and phenotype of CTAg‐specific T cells in the blood of patients with testicular germ cell tumors (TGCTs). CD8+ and CD4+ T‐cell responses against MAGE‐A family antigens were present in 44% (20/45) of patients’ samples assayed by ex vivo IFN‐γ ELISPOT. The presence of MAGE‐specific CD8+ T cells was further determined following short‐term in vitro expansion through the use of pMHC‐I multimers containing known immunogenic peptides. Longitudinal analysis revealed that the frequency of MAGE‐specific T cells decreased by 89% following orchidectomy suggesting that persistence of tumor antigen is required to sustain CTAg‐specific T‐cell immunity. Notably, this decrease correlated with a decline in the global effector/memory T‐cell pool following treatment. Spontaneous T‐cell immunity against CTAg proteins therefore develops in many patients with testicular cancer and may play an important role in the excellent clinical outcome of patients with this tumor subtype. PMID:28555838

Development of tumor-reactive T cells after nonmyeloablative allogeneic hematopoietic stem cell transplant for chronic lymphocytic leukemia.

PubMed

Nishida, Tetsuya; Hudecek, Michael; Kostic, Ana; Bleakley, Marie; Warren, Edus H; Maloney, David; Storb, Rainer; Riddell, Stanley R

2009-07-15

Allogeneic nonmyeloablative hematopoietic stem cell transplant (NM-HSCT) can result in durable remission of chronic lymphocytic leukemia (CLL). It is thought that the efficacy of NM-HSCT is mediated by recognition of tumor cells by T cells in the donor stem cell graft. We evaluated the development of CTLs specific for CLL after NM-HSCT to determine if their presence correlated with antitumor efficacy. Peripheral blood mononuclear cells obtained from 12 transplant recipients at intervals after NM-HSCT were stimulated in vitro with CLL cells. Polyclonal T-cell lines and CD8(+) T-cell clones were derived from these cultures and evaluated for lysis of donor and recipient target cells including CLL. The presence and specificity of responses was correlated with clinical outcomes. Eight of the 12 patients achieved remission or a major antitumor response and all 8 developed CD8(+) and CD4(+) T cells specific for antigens expressed by CLL. A clonal analysis of the CD8(+) T-cell response identified T cells specific for multiple minor histocompatibility (H) antigens expressed on CLL in six of the responding patients. A significant fraction of the CD8(+) T-cell response in some patients was also directed against nonshared tumor-specific antigens. By contrast, CLL-reactive T cells were not detected in the four patients who had persistent CLL after NM-HSCT, despite the development of graft-versus-host disease. The development of a diverse T-cell response specific for minor H and tumor-associated antigens expressed by CLL predicts an effective graft-versus-leukemia response after NM-HSCT.

In Situ Activation of Antigen-Specific CD8+ T Cells in the Presence of Antigen in Organotypic Brain Slices1

PubMed Central

Ling, Changying; Verbny, Yakov I.; Banks, Matthew I.; Sandor, Matyas; Fabry, Zsuzsanna

2012-01-01

The activation of Ag-specific T cells locally in the CNS could potentially contribute to the development of immune-mediated brain diseases. We addressed whether Ag-specific T cells could be stimulated in the CNS in the absence of peripheral lymphoid tissues by analyzing Ag-specific T cell responses in organotypic brain slice cultures. Organotypic brain slice cultures were established 1 h after intracerebral OVA Ag microinjection. We showed that when OVA-specific CD8+ T cells were added to Ag-containing brain slices, these cells became activated and migrated into the brain to the sites of their specific Ags. This activation of OVA-specific T cells was abrogated by the deletion of CD11c+ cells from the brain slices of the donor mice. These data suggest that brain-resident CD11c+ cells stimulate Ag-specific naive CD8+ T cells locally in the CNS and may contribute to immune responses in the brain. PMID:18523307

Antibodies attenuate the capacity of dendritic cells to stimulate HIV-specific cytotoxic T lymphocytes

PubMed Central

Posch, Wilfried; Cardinaud, Sylvain; Hamimi, Chiraz; Fletcher, Adam; Mühlbacher, Annelies; Loacker, Klaus; Eichberger, Paul; Dierich, Manfred P.; Pancino, Gianfranco; Lass-Flörl, Cornelia; Moris, Arnaud; Saez-Cirion, Asier; Wilflingseder, Doris

2014-01-01

Background Control of HIV is suggested to depend on potent effector functions of the virus-specific CD8+ T-cell response. Antigen opsonization can modulate the capture of antigen, its presentation, and the priming of specific CD8+ T-cell responses. Objective We have previously shown that opsonization of retroviruses acts as an endogenous adjuvant for dendritic cell (DC)–mediated induction of specific cytotoxic T lymphocytes (CTLs). However, in some HIV-positive subjects, high levels of antibodies and low levels of complement fragments coat the HIV surface. Methods Therefore we analyzed the effect of IgG opsonization on the antigen-presenting capacity of DCs by using CD8+ T-cell proliferation assays after repeated prime boosting, by measuring the antiviral activity against HIV-infected autologous CD4+ T cells, and by determining IFN-γ secretion from HIV-specific CTL clones. Results We find that DCs exposed to IgG-opsonized HIV significantly decreased the HIV-specific CD8+ T-cell response compared with the earlier described efficient CD8+ T-cell activation induced by DCs loaded with complement-opsonized HIV. DCs exposed to HIV bearing high surface IgG levels after incubation in plasma from HIV-infected subjects acted as weak stimulators for HIV-specific CTL clones. In contrast, HIV opsonized with plasma from patients exhibiting high complement and low IgG deposition on the viral surface favored significantly higher activation of HIV-specific CD8+ T-cell clones. Conclusion Our ex vivo and in vitro observations provide the first evidence that IgG opsonization of HIV is associated with a decreased CTL-stimulatory capacity of DCs. PMID:23063584

Human melanoma immunotherapy using tumor antigen-specific T cells generated in humanized mice

PubMed Central

Hu, Zheng; Xia, Jinxing; Fan, Wei; Wargo, Jennifer; Yang, Yong-Guang

2016-01-01

A major factor hindering the exploration of adoptive immunotherapy in preclinical settings is the limited availability of tumor-reactive human T cells. Here we developed a humanized mouse model that permits large-scale production of human T cells expressing the engineered melanoma antigen MART-1-specific TCR. Humanized mice, made by transplantation of human fetal thymic tissue and CD34+ cells virally-transduced with HLA class I-restricted melanoma antigen (MART-1)-specific TCR gene, showed efficient development of MART-1-TCR+ human T cells with predominantly CD8+ cells. Importantly, MART-1-TCR+CD8+ T cells developing in these mice were capable of mounting antigen-specific responses in vivo, as evidenced by their proliferation, phenotypic conversion and IFN-γ production following MART-1 peptide immunization. Moreover, these MART-1-TCR+CD8+ T cells mediated efficient killing of melanoma cells in an HLA/antigen-dependent manner. Adoptive transfer of in vitro expanded MART-1-TCR+CD8+ T cells induced potent antitumor responses that were further enhanced by IL-15 treatment in melanoma-bearing recipients. Finally, a short incubation of MART-1-specific T cells with rapamycin acted synergistically with IL-15, leading to significantly improved tumor-free survival in recipients with metastatic melanoma. These data demonstrate the practicality of using humanized mice to produce potentially unlimited source of tumor-specific human T cells for experimental and preclinical exploration of cancer immunotherapy. This study also suggests that pretreatment of tumor-reactive T cells with rapamycin in combination with IL-15 administration may be a novel strategy to improve the efficacy of adoptive T cell therapy. PMID:26824989

Tomatine adjuvantation of protective immunity to a major pre-erythrocytic vaccine candidate of malaria is mediated via CD8+ T cell release of IFN-gamma.

PubMed

Heal, Karen G; Taylor-Robinson, Andrew W

2010-01-01

The glycoalkaloid tomatine, derived from the wild tomato, can act as a powerful adjuvant to elicit an antigen-specific cell-mediated immune response to the circumsporozoite (CS) protein, a major pre-erythrocytic stage malaria vaccine candidate antigen. Using a defined MHC-class-I-restricted CS epitope in a Plasmodium berghei rodent model, antigen-specific cytotoxic T lymphocyte activity and IFN-gamma secretion ex vivo were both significantly enhanced compared to responses detected from similarly stimulated splenocytes from naive and tomatine-saline-immunized mice. Further, through lymphocyte depletion it is demonstrated that antigen-specific IFN-gamma is produced exclusively by the CD8(+) T cell subset. We conclude that the processing of the P. berghei CS peptide as an exogenous antigen and its presentation via MHC class I molecules to CD8(+) T cells leads to an immune response that is an in vitro correlate of protection against pre-erythrocytic malaria. Further characterization of tomatine as an adjuvant in malaria vaccine development is indicated.

Treatment of malignant pleural mesothelioma by fibroblast activation protein-specific re-directed T cells

PubMed Central

2013-01-01

Introduction Malignant pleural mesothelioma (MPM) is an incurable malignant disease, which results from chronic exposition to asbestos in at least 70% of the cases. Fibroblast activation protein (FAP) is predominantly expressed on the surface of reactive tumor-associated fibroblasts as well as on particular cancer types. Because of its expression on the cell surface, FAP is an attractive target for adoptive T cell therapy. T cells can be re-directed by retroviral transfer of chimeric antigen receptors (CAR) against tumor-associated antigens (TAA) and therefore represent a therapeutic strategy of adoptive immunotherapy. Methods To evaluate FAP expression immunohistochemistry was performed in tumor tissue from MPM patients. CD8+ human T cells were retrovirally transduced with an anti-FAP-F19-∆CD28/CD3ζ-CAR. T cell function was evaluated in vitro by cytokine release and cytotoxicity assays. In vivo function was tested with an intraperitoneal xenograft tumor model in immunodeficient mice. Results FAP was found to be expressed in all subtypes of MPM. Additionally, FAP expression was evaluated in healthy adult tissue samples and was only detected in specific areas in the pancreas, the placenta and very weakly for cervix and uterus. Expression of the anti-FAP-F19-∆CD28/CD3ζ-CAR in CD8+ T cells resulted in antigen-specific IFNγ release. Additionally, FAP-specific re-directed T cells lysed FAP positive mesothelioma cells and inflammatory fibroblasts in an antigen-specific manner in vitro. Furthermore, FAP-specific re-directed T cells inhibited the growth of FAP positive human tumor cells in the peritoneal cavity of mice and significantly prolonged survival of mice. Conclusion FAP re-directed CD8+ T cells showed antigen-specific functionality in vitro and in vivo. Furthermore, FAP expression was verified in all MPM histotypes. Therefore, our data support performing a phase I clinical trial in which MPM patients are treated with adoptively transferred FAP-specific re-directed T cells. PMID:23937772

Podocytes Are Nonhematopoietic Professional Antigen-Presenting Cells

PubMed Central

Burkard, Miriam; Ölke, Martha; Daniel, Christoph; Amann, Kerstin; Hugo, Christian; Kurts, Christian; Steinkasserer, Alexander; Gessner, André

2013-01-01

Podocytes are essential to the structure and function of the glomerular filtration barrier; however, they also exhibit increased expression of MHC class II molecules under inflammatory conditions, and they remove Ig and immune complexes from the glomerular basement membrane (GBM). This finding suggests that podocytes may act as antigen-presenting cells, taking up and processing antigens to initiate specific T cell responses, similar to professional hematopoietic cells such as dendritic cells or macrophages. Here, MHC–antigen complexes expressed exclusively on podocytes of transgenic mice were sufficient to activate CD8+ T cells in vivo. In addition, deleting MHC class II exclusively on podocytes prevented the induction of experimental anti-GBM nephritis. Podocytes ingested soluble and particulate antigens, activated CD4+ T cells, and crosspresented exogenous antigen on MHC class I molecules to CD8+ T cells. In conclusion, podocytes participate in the antigen-specific activation of adaptive immune responses, providing a potential target for immunotherapies of inflammatory kidney diseases and transplant rejection. PMID:23539760

The WT hemochromatosis protein HFE inhibits CD8⁺ T-lymphocyte activation.

PubMed

Reuben, Alexandre; Phénix, Mikaël; Santos, Manuela M; Lapointe, Réjean

2014-06-01

MHC class I (MHC I) antigen presentation is a ubiquitous process by which cells present endogenous proteins to CD8(+) T lymphocytes during immune surveillance and response. Hereditary hemochromatosis protein, HFE, is involved in cellular iron uptake but, while structurally homologous to MHC I, is unable to bind peptides. However, increasing evidence suggests a role for HFE in the immune system. Here, we investigated the impact of HFE on CD8(+) T-lymphocyte activation. Using transient HFE transfection assays in a model of APCs, we show that WT HFE (HFEWT ), but not C282Y-mutated HFE, inhibits secretion of MIP-1β from antigen-specific CD8(+) T lymphocytes. HFEWT expression also resulted in major decreases in CD8(+) T-lymphocyte activation as measured by 4-1BB expression. We further demonstrate that inhibition of CD8(+) T-lymphocyte activation was independent of MHC I surface levels, β2-m competition, HFE interaction with transferrin receptor, antigen origin, or epitope affinity. Finally, we identified the α1-2 domains of HFEWT as being responsible for inhibiting CD8(+) T-lymphocyte activation. Our data imply a new role for HFEWT in altering CD8(+) T-lymphocyte reactivity, which could modulate antigen immunogenicity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

CD4+CD25+ regulatory T cells suppress allograft rejection mediated by memory CD8+ T cells via a CD30-dependent mechanism.

PubMed

Dai, Zhenhua; Li, Qi; Wang, Yinong; Gao, Ge; Diggs, Lonnette S; Tellides, George; Lakkis, Fadi G

2004-01-01

CD4(+)CD25(+) regulatory T (Treg) cells suppress naive T cell responses, prevent autoimmunity, and delay allograft rejection. It is not known, however, whether Treg cells suppress allograft rejection mediated by memory T cells, as the latter mount faster and stronger immune responses than their naive counterparts. Here we show that antigen-induced, but not naive, Treg cells suppress allograft rejection mediated by memory CD8(+) T cells. Suppression was allospecific, as Treg cells induced by third-party antigens did not delay allograft rejection. In vivo and in vitro analyses revealed that the apoptosis of allospecific memory CD8(+) T cells is significantly increased in the presence of antigen-induced Treg cells, while their proliferation remains unaffected. Importantly, neither suppression of allograft rejection nor enhanced apoptosis of memory CD8(+) T cells was observed when Treg cells lacked CD30 or when CD30 ligand-CD30 interaction was blocked with anti-CD30 ligand Ab. This study therefore provides direct evidence that pathogenic memory T cells are amenable to suppression in an antigen-specific manner and identifies CD30 as a molecule that is critical for the regulation of memory T cell responses.

CD4+CD25+ regulatory T cells suppress allograft rejection mediated by memory CD8+ T cells via a CD30-dependent mechanism

PubMed Central

Dai, Zhenhua; Li, Qi; Wang, Yinong; Gao, Ge; Diggs, Lonnette S.; Tellides, George; Lakkis, Fadi G.

2004-01-01

CD4+CD25+ regulatory T (Treg) cells suppress naive T cell responses, prevent autoimmunity, and delay allograft rejection. It is not known, however, whether Treg cells suppress allograft rejection mediated by memory T cells, as the latter mount faster and stronger immune responses than their naive counterparts. Here we show that antigen-induced, but not naive, Treg cells suppress allograft rejection mediated by memory CD8+ T cells. Suppression was allospecific, as Treg cells induced by third-party antigens did not delay allograft rejection. In vivo and in vitro analyses revealed that the apoptosis of allospecific memory CD8+ T cells is significantly increased in the presence of antigen-induced Treg cells, while their proliferation remains unaffected. Importantly, neither suppression of allograft rejection nor enhanced apoptosis of memory CD8+ T cells was observed when Treg cells lacked CD30 or when CD30 ligand–CD30 interaction was blocked with anti–CD30 ligand Ab. This study therefore provides direct evidence that pathogenic memory T cells are amenable to suppression in an antigen-specific manner and identifies CD30 as a molecule that is critical for the regulation of memory T cell responses. PMID:14722622

Dynamic Imaging of CD8(+) T cells and dendritic cells during infection with Toxoplasma gondii.

PubMed

John, Beena; Harris, Tajie H; Tait, Elia D; Wilson, Emma H; Gregg, Beth; Ng, Lai Guan; Mrass, Paulus; Roos, David S; Dzierszinski, Florence; Weninger, Wolfgang; Hunter, Christopher A

2009-07-01

To better understand the initiation of CD8(+) T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8(+) T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8(+) T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8(+) T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8(+) T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis.

Dynamic Imaging of CD8+ T Cells and Dendritic Cells during Infection with Toxoplasma gondii

PubMed Central

John, Beena; Harris, Tajie H.; Tait, Elia D.; Wilson, Emma H.; Gregg, Beth; Ng, Lai Guan; Mrass, Paulus; Roos, David S.; Dzierszinski, Florence; Weninger, Wolfgang; Hunter, Christopher A.

2009-01-01

To better understand the initiation of CD8+ T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8+ T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8+ T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8+ T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8+ T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis. PMID:19578440

Lentiviral Protein Transfer Vectors Are an Efficient Vaccine Platform and Induce a Strong Antigen-Specific Cytotoxic T Cell Response

PubMed Central

Uhlig, Katharina M.; Schülke, Stefan; Scheuplein, Vivian A. M.; Malczyk, Anna H.; Reusch, Johannes; Kugelmann, Stefanie; Muth, Anke; Koch, Vivian; Hutzler, Stefan; Bodmer, Bianca S.; Schambach, Axel; Buchholz, Christian J.; Waibler, Zoe; Scheurer, Stephan

2015-01-01

ABSTRACT To induce and trigger innate and adaptive immune responses, antigen-presenting cells (APCs) take up and process antigens. Retroviral particles are capable of transferring not only genetic information but also foreign cargo proteins when they are genetically fused to viral structural proteins. Here, we demonstrate the capacity of lentiviral protein transfer vectors (PTVs) for targeted antigen transfer directly into APCs and thereby induction of cytotoxic T cell responses. Targeting of lentiviral PTVs to APCs can be achieved analogously to gene transfer vectors by pseudotyping the particles with truncated wild-type measles virus (MV) glycoproteins (GPs), which use human SLAM (signaling lymphocyte activation molecule) as a main entry receptor. SLAM is expressed on stimulated lymphocytes and APCs, including dendritic cells. SLAM-targeted PTVs transferred the reporter protein green fluorescent protein (GFP) or Cre recombinase with strict receptor specificity into SLAM-expressing CHO and B cell lines, in contrast to broadly transducing vesicular stomatitis virus G protein (VSV-G) pseudotyped PTVs. Primary myeloid dendritic cells (mDCs) incubated with targeted or nontargeted ovalbumin (Ova)-transferring PTVs stimulated Ova-specific T lymphocytes, especially CD8+ T cells. Administration of Ova-PTVs into SLAM-transgenic and control mice confirmed the observed predominant induction of antigen-specific CD8+ T cells and demonstrated the capacity of protein transfer vectors as suitable vaccines for the induction of antigen-specific immune responses. IMPORTANCE This study demonstrates the specificity and efficacy of antigen transfer by SLAM-targeted and nontargeted lentiviral protein transfer vectors into antigen-presenting cells to trigger antigen-specific immune responses in vitro and in vivo. The observed predominant activation of antigen-specific CD8+ T cells indicates the suitability of SLAM-targeted and also nontargeted PTVs as a vaccine for the induction of cytotoxic immune responses. Since cytotoxic CD8+ T lymphocytes are a mainstay of antitumoral immune responses, PTVs could be engineered for the transfer of specific tumor antigens provoking tailored antitumoral immunity. Therefore, PTVs can be used as safe and efficient alternatives to gene transfer vectors or live attenuated replicating vector platforms, avoiding genotoxicity or general toxicity in highly immunocompromised patients, respectively. Thereby, the potential for easy envelope exchange allows the circumventing of neutralizing antibodies, e.g., during repeated boost immunizations. PMID:26085166

Cytotoxicity of Tumor Antigen Specific Human T Cells Is Unimpaired by Arginine Depletion

PubMed Central

Knies, Diana; Medenhoff, Sergej; Wabnitz, Guido; Luckner-Minden, Claudia; Feldmeyer, Nadja; Voss, Ralf-Holger; Kropf, Pascale; Müller, Ingrid; Conradi, Roland; Samstag, Yvonne; Theobald, Matthias; Ho, Anthony D.; Goldschmidt, Hartmut; Hundemer, Michael

2013-01-01

Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8+ T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i) CD8+ T cells with specificity against the MART-1aa26–35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii) clonal CMV pp65aa495–503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495–503 specific T cell receptor were analyzed. Our data demonstrate that human CD8+ T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency. PMID:23717444

Performance Assessment of a Trypanosoma cruzi Chimeric Antigen in Multiplex Liquid Microarray Assays.

PubMed

Santos, Fred Luciano Neves; Celedon, Paola Alejandra Fiorani; Zanchin, Nilson Ivo Tonin; Leitolis, Amanda; Crestani, Sandra; Foti, Leonardo; de Souza, Wayner Vieira; Gomes, Yara de Miranda; Krieger, Marco Aurélio

2017-10-01

Diagnosing chronic Chagas disease (CD) requires antibody-antigen detection methods, which are traditionally based on enzymatic assay techniques whose performance depend on the type and quality of antigen used. Previously, 4 recombinant chimeric proteins from the Instituto de Biologia Molecular do Paraná (IBMP-8.1 to 8.4) comprising immuno-dominant regions of diverse Trypanosoma cruzi antigens showed excellent diagnostic performance in enzyme-linked immunosorbent assays. Considering that next-generation platforms offer improved CD diagnostic accuracy with different T. cruzi -specific recombinant antigens, we assessed the performance of these chimeras in liquid microarrays (LMAs). The chimeric proteins were expressed in Escherichia coli and purified by chromatography. Sera from 653 chagasic and 680 healthy individuals were used to assess the performance of these chimeras in detecting specific anti- T. cruzi antibodies. Accuracies ranged from 98.1 to 99.3%, and diagnostic odds ratio values were 3,548 for IBMP-8.3, 4,826 for IBMP-8.1, 7,882 for IBMP-8.2, and 25,000 for IBMP-8.4. A separate sera bank (851 samples) was employed to assess cross-reactivity with other tropical diseases. Leishmania , a pathogen with high similarity to T. cruzi , showed cross-reactivity rates ranging from 0 to 2.17%. Inconclusive results were negligible (0 to 0.71%). Bland-Altman and Deming regression analysis based on 200 randomly selected CD-positive and negative samples demonstrated interchangeability with respect to CD diagnostic performance in both singleplex and multiplex assays. Our results suggested that these chimeras can potentially replace antigens currently used in commercially available assay kits. Moreover, the use of multiplex platforms, such as LMA assays employing 2 or more IBMP antigens, would abrogate the need for 2 different testing techniques when diagnosing CD. Copyright © 2017 American Society for Microbiology.

Secreted HSP Vaccine for Malaria Prophylaxis

DTIC Science & Technology

2017-10-01

thereby stimulating an avid, antigen specific, cytotoxic CD8 T cell response. Here we developed malaria vaccine that relies on secreted gp96-Ig...vaccine is expected to provide prophylactic immunity for malaria by removing infected liver cells before sporozoites can replicate and spread to the...vaccine to the immunogenicity of NMRC-M3V-D/Ad-PfCA vaccine. We found that gp96-Ig vaccination provided stronger antigen specific CD8 T cell

Topical CpG Adjuvantation of a Protein-Based Vaccine Induces Protective Immunity to Listeria monocytogenes

PubMed Central

Cheng, Wing Ki; Wee, Kathleen; Kollmann, Tobias R.

2014-01-01

Robust CD8+ T cell responses are essential for immune protection against intracellular pathogens. Using parenteral administration of ovalbumin (OVA) protein as a model antigen, the effect of the Toll-like receptor 9 (TLR9) agonist, CpG oligodeoxynucleotide (ODN) 1826, as an adjuvant delivered either topically, subcutaneously, or intramuscularly on antigen-specific CD8+ T cell responses in a mouse model was evaluated. Topical CpG adjuvant increased the frequency of OVA-specific CD8+ T cells in the peripheral blood and in the spleen. The more effective strategy to administer topical CpG adjuvant to enhance CD8+ T cell responses was single-dose administration at the time of antigen injection with a prime-boost regimen. Topical CpG adjuvant conferred both rapid and long-lasting protection against systemic challenge with recombinant Listeria monocytogenes expressing the cytotoxic T lymphocyte (CTL) epitope of OVA257–264 (strain Lm-OVA) in a TLR9-dependent manner. Topical CpG adjuvant induced a higher proportion of CD8+ effector memory T cells than parenteral administration of the adjuvant. Although traditional vaccination strategies involve coformulation of antigen and adjuvant, split administration using topical adjuvant is effective and has advantages of safety and flexibility. Split administration of topical CpG ODN 1826 with parenteral protein antigen is superior to other administration strategies in enhancing both acute and memory protective CD8+ T cell immune responses to subcutaneous protein vaccines. This vaccination strategy induces rapid and persistent protective immune responses against the intracellular organism L. monocytogenes. PMID:24391136

Zoledronic acid induces dose-dependent increase of antigen-specific CD8 T-cell responses in combination with peptide/poly-IC vaccine.

PubMed

Park, Hye-Mi; Cho, Hyun-Il; Shin, Chang-Ae; Shon, Hyun-Jung; Kim, Tai-Gyu

2016-03-04

Zoledronic acid (ZA) is used for treating osteoporosis and for preventing skeletal fractures in cancer patients suffering from myeloma and prostate cancer. It is also reported to directly induce cancer cell apoptosis and indirectly modulate T-cell immune response as an antitumor agent. In this study, the effect of ZA following peptide/polyinosinic-polycytidylic acid (poly-IC) vaccination was investigated in a murine tumor model. The combination of ZA with peptide/poly-IC vaccine showed a synergistic effect on the induction of antigen-specific CD8 T-cell response. Three consecutive intravenous administrations of ZA was defined to induce the highest CD8 T-cell response. Further, total splenocyte counts and antigen-specific CD8 T-cell response gradually increased depending on the dose of ZA. In tumor-bearing mice, ZA showed a dose-dependent decrease of growth and prolonged survival. Treatment with ZA only decreased the number of CD11b(+)Gr1(+) myeloid cells in blood. Our results demonstrate that the use of ZA could improve antitumor immune responses induced by the peptide/poly-IC vaccine. Copyright © 2016 Elsevier Ltd. All rights reserved.

Antigen-specific CD8{sup +} T cells induced by the ubiquitin fusion degradation pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Imai, Takashi; Duan Xuefeng; Hisaeda, Hajime

We have developed a DNA vaccine encoding a fusion protein of ubiquitin (Ub) and target proteins at the N-terminus for effective induction of antigen-specific CD8{sup +} T cells. A series of expression plasmids encoding a model antigen, ovalbumin (OVA), fused with mutated Ub, was constructed. Western blotting analyses using COS7 cells transfected with these plasmids revealed that there were three types of amino acid causing different binding capacities between Ub and OVA. Natural Ub with a C-terminal glycine readily dissociated from OVA; on the other hand, artificially mutated Ub, the C-terminal amino acid of which had been exchanged to valinemore » or arginine, stably united with the polypeptide, while Ub with a C-terminal alanine partially dissociated. The ability of DNA vaccination to induce OVA-specific CD8{sup +} T cells closely correlated with the stability of Ub fusion to OVA. Our strategy could be used to optimize the effect of genetic vaccines on the induction of CD8{sup +} T cells.« less

Transfer of allogeneic CD4+ T cells rescues CD8+ T cells in anti-PD-L1–resistant tumors leading to tumor eradication

PubMed Central

Arina, Ainhoa; Karrison, Theodore; Galka, Eva; Schreiber, Karin; Weichselbaum, Ralph R.; Schreiber, Hans

2017-01-01

Adoptively transferred CD8+ T cells can stabilize the size of solid tumors over long periods of time by exclusively recognizing antigen cross-presented on tumor stroma. However, these tumors eventually escape T cell–mediated growth control. The aim of this study was to eradicate such persistent cancers. In our model, the SIYRYYGL antigen is expressed by cancer cells that lack the MHC-I molecule Kb needed for direct presentation, but the antigen is picked up and cross-presented by tumor stroma. A single injection of antigen-specific 2C CD8+ T cells caused long-term inhibition of tumor growth, but without further intervention, tumors started to progress after approximately 3 months. Escape was associated with reduced numbers of circulating 2C cells. Tumor-infiltrating 2C cells produced significantly less TNFα and expressed more of the “exhaustion” markers PD-1 and Tim-3 than T cells from lymphoid organs. High-dose local ionizing radiation, depletion of myeloid-derived suppressor cells, infusions of additional 2C cells, and antibodies blocking PD-L1 did not prevent tumor escape. In contrast, adoptive transfer of allogeneic CD4+ T cells restored the numbers of circulating Ag-specific CD8+ T cells and their intratumoral function, resulting in tumor eradication. These CD4+ T cells had no antitumor effects in the absence of CD8+ T cells and recognized the alloantigen cross-presented on tumor stroma. CD4+ T cells might also be effective in cancer patients when PD1/PD-L1 blockade does not rescue intratumoral CD8+ T-cell function and tumors persist. PMID:28077434

Evaluation of hydrophobic chitosan-based particulate formulations of porcine reproductive and respiratory syndrome virus vaccine candidate T cell antigens.

PubMed

Mokhtar, Helen; Biffar, Lucia; Somavarapu, Satyanarayana; Frossard, Jean-Pierre; McGowan, Sarah; Pedrera, Miriam; Strong, Rebecca; Edwards, Jane C; Garcia-Durán, Margarita; Rodriguez, Maria Jose; Stewart, Graham R; Steinbach, Falko; Graham, Simon P

2017-09-01

PRRS control is hampered by the inadequacies of existing vaccines to combat the extreme diversity of circulating viruses. Since immune clearance of PRRSV infection may not be dependent on the development of neutralising antibodies and the identification of broadly-neutralising antibody epitopes have proven elusive, we hypothesised that conserved T cell antigens represent potential candidates for development of a novel PRRS vaccine. Previously we had identified the M and NSP5 proteins as well-conserved targets of polyfunctional CD8 and CD4 T cells. To assess their vaccine potential, peptides representing M and NSP5 were encapsulated in hydrophobically-modified chitosan particles adjuvanted by incorporation of a synthetic multi-TLR2/TLR7 agonist and coated with a model B cell PRRSV antigen. For comparison, empty particles and adjuvanted particles encapsulating inactivated PRRSV-1 were prepared. Vaccination with the particulate formulations induced antigen-specific antibody responses, which were most pronounced following booster immunisation. M and NSP5-specific CD4, but not CD8, T cell IFN-γ reactivity was measurable following the booster immunisation in a proportion of animals vaccinated with peptide-loaded particles. Upon challenge, CD4 and CD8 T cell reactivity was detected in all groups, with the greatest responses being detected in the peptide vaccinated group but with limited evidence of an enhanced control of viraemia. Analysis of the lungs during the resolution of infection showed significant M/NSP5 specific IFN-γ responses from CD8 rather than CD4 T cells. Vaccine primed CD8 T cell responses may therefore be required for protection and future work should focus on enhancing the cross-presentation of M/NSP5 to CD8 T cells. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Enhanced Vaccine-Induced CD8+ T Cell Responses to Malaria Antigen ME-TRAP by Fusion to MHC Class II Invariant Chain

PubMed Central

Spencer, Alexandra J.; Cottingham, Matthew G.; Jenks, Jennifer A.; Longley, Rhea J.; Capone, Stefania; Colloca, Stefano; Folgori, Antonella; Cortese, Riccardo; Nicosia, Alfredo; Bregu, Migena; Hill, Adrian V. S.

2014-01-01

The orthodox role of the invariant chain (CD74; Ii) is in antigen presentation to CD4+ T cells, but enhanced CD8+ T cells responses have been reported after vaccination with vectored viral vaccines encoding a fusion of Ii to the antigen of interest. In this study we assessed whether fusion of the malarial antigen, ME-TRAP, to Ii could increase the vaccine-induced CD8+ T cell response. Following single or heterologous prime-boost vaccination of mice with a recombinant chimpanzee adenovirus vector, ChAd63, or recombinant modified vaccinia virus Ankara (MVA), higher frequencies of antigen-specific CD4+ and CD8+ T cells were observed, with the largest increases observed following a ChAd63-MVA heterologous prime-boost regimen. Studies in non-human primates confirmed the ability of Ii-fusion to augment the T cell response, where a 4-fold increase was maintained up to 11 weeks after the MVA boost. Of the numerous different approaches explored to increase vectored vaccine induced immunogenicity over the years, fusion to the invariant chain showed a consistent enhancement in CD8+ T cell responses across different animal species and may therefore find application in the development of vaccines against human malaria and other diseases where high levels of cell-mediated immunity are required. PMID:24945248

Spontaneous CD4+ and CD8+ T-cell responses directed against cancer testis antigens are present in the peripheral blood of testicular cancer patients.

PubMed

Pearce, Hayden; Hutton, Paul; Chaudhri, Shalini; Porfiri, Emilio; Patel, Prashant; Viney, Richard; Moss, Paul

2017-07-01

Cancer/testis antigen (CTAg) expression is restricted to spermatogenic cells in an immune-privileged site within the testis. However, these proteins are expressed aberrantly by malignant cells and T-cell responses against CTAgs develop in many cancer patients. We investigated the prevalence, magnitude and phenotype of CTAg-specific T cells in the blood of patients with testicular germ cell tumors (TGCTs). CD8 + and CD4 + T-cell responses against MAGE-A family antigens were present in 44% (20/45) of patients' samples assayed by ex vivo IFN-γ ELISPOT. The presence of MAGE-specific CD8 + T cells was further determined following short-term in vitro expansion through the use of pMHC-I multimers containing known immunogenic peptides. Longitudinal analysis revealed that the frequency of MAGE-specific T cells decreased by 89% following orchidectomy suggesting that persistence of tumor antigen is required to sustain CTAg-specific T-cell immunity. Notably, this decrease correlated with a decline in the global effector/memory T-cell pool following treatment. Spontaneous T-cell immunity against CTAg proteins therefore develops in many patients with testicular cancer and may play an important role in the excellent clinical outcome of patients with this tumor subtype. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Missing Link in Epstein-Barr Virus Immune Evasion: the BDLF3 Gene Induces Ubiquitination and Downregulation of Major Histocompatibility Complex Class I (MHC-I) and MHC-II

PubMed Central

Quinn, Laura L.; Williams, Luke R.; White, Claire; Forrest, Calum; Rowe, Martin

2015-01-01

ABSTRACT The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8+ cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8+ cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8+ cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4+ cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8+ and CD4+ T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner. IMPORTANCE Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8+ T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8+ T cells specific for early lytic cycle antigens. The present work identifies an additional immune evasion protein, BDLF3, that is expressed late in the lytic cycle and impairs CD8+ T cell recognition by targeting cell surface MHC class I molecules for ubiquitination and proteasome-dependent downregulation. Interestingly, BDLF3 also targets MHC class II molecules to impair CD4+ T cell recognition. BDLF3 is therefore a rare example of a viral protein that impairs both the MHC class I and class II antigen-presenting pathways. PMID:26468525

The Missing Link in Epstein-Barr Virus Immune Evasion: the BDLF3 Gene Induces Ubiquitination and Downregulation of Major Histocompatibility Complex Class I (MHC-I) and MHC-II.

PubMed

Quinn, Laura L; Williams, Luke R; White, Claire; Forrest, Calum; Zuo, Jianmin; Rowe, Martin

2016-01-01

The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8(+) cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8(+) cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8(+) cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4(+) cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8(+) and CD4(+) T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner. Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8(+) T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8(+) T cells specific for early lytic cycle antigens. The present work identifies an additional immune evasion protein, BDLF3, that is expressed late in the lytic cycle and impairs CD8(+) T cell recognition by targeting cell surface MHC class I molecules for ubiquitination and proteasome-dependent downregulation. Interestingly, BDLF3 also targets MHC class II molecules to impair CD4(+) T cell recognition. BDLF3 is therefore a rare example of a viral protein that impairs both the MHC class I and class II antigen-presenting pathways. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

CD8+ T-cell responses rapidly select for antigen-negative tumor cells in the prostate.

PubMed

Bak, S Peter; Barnkob, Mike Stein; Wittrup, K Dane; Chen, Jianzhu

2013-12-01

Stimulation of patients' immune systems for the treatment of solid tumors is an emerging therapeutic paradigm. The use of enriched autologous T cells for adoptive cell therapy or vaccination with antigen-loaded dendritic cells have shown clinical efficacy in melanoma and prostate cancer, respectively. However, the long-term effects of immune responses on selection and outgrowth of antigen-negative tumor cells in specific tumor types must be determined to understand and achieve long-term therapeutic effects. In this study, we have investigated the expression of a tumor-specific antigen in situ after treatment with tumor-specific CD8(+) T cells in an autochthonous mouse model of prostate cancer. After T-cell treatment, aggregates of dead antigen-positive tumor cells were concentrated in the lumen of the prostate gland and were eventually eliminated from the prostate tissue. Despite the elimination of antigen-positive tumor cells, prostate tumor continued to grow in T-cell-treated mice. Interestingly, the remaining tumor cells were antigen negative and downregulated MHC class I expression. These results show that CD8(+) T cells are effective in eliminating antigen-bearing prostate tumor cells but they also can select for the outgrowth of antigen-negative tumor cells. These findings provide insights into the requirements for an effective cancer immunotherapy within the prostate that not only induces potent immune responses but also avoids selection and outgrowth of antigen-negative tumor cells. ©2013 AACR.

Cooperation between Epstein-Barr Virus Immune Evasion Proteins Spreads Protection from CD8+ T Cell Recognition across All Three Phases of the Lytic Cycle

PubMed Central

Quinn, Laura L.; Zuo, Jianmin; Abbott, Rachel J. M.; Shannon-Lowe, Claire; Tierney, Rosemary J.; Hislop, Andrew D.; Rowe, Martin

2014-01-01

CD8+ T cell responses to Epstein-Barr virus (EBV) lytic cycle expressed antigens display a hierarchy of immunodominance, in which responses to epitopes of immediate-early (IE) and some early (E) antigens are more frequently observed than responses to epitopes of late (L) expressed antigens. It has been proposed that this hierarchy, which correlates with the phase-specific efficiency of antigen presentation, may be due to the influence of viral immune-evasion genes. At least three EBV-encoded genes, BNLF2a, BGLF5 and BILF1, have the potential to inhibit processing and presentation of CD8+ T cell epitopes. Here we examined the relative contribution of these genes to modulation of CD8+ T cell recognition of EBV lytic antigens expressed at different phases of the replication cycle in EBV-transformed B-cells (LCLs) which spontaneously reactivate lytic cycle. Selective shRNA-mediated knockdown of BNLF2a expression led to more efficient recognition of immediate-early (IE)- and early (E)-derived epitopes by CD8+ T cells, while knock down of BILF1 increased recognition of epitopes from E and late (L)-expressed antigens. Contrary to what might have been predicted from previous ectopic expression studies in EBV-negative model cell lines, the shRNA-mediated inhibition of BGLF5 expression in LCLs showed only modest, if any, increase in recognition of epitopes expressed in any phase of lytic cycle. These data indicate that whilst BNLF2a interferes with antigen presentation with diminishing efficiency as lytic cycle progresses (IE>E>>L), interference by BILF1 increases with progression through lytic cycle (IE

Immunotherapy expands and maintains the function of high affinity tumor infiltrating CD8 T cells in situ

PubMed Central

Moran, Amy E.; Polesso, Fanny; Weinberg, Andrew D.

2016-01-01

Cancer cells harbor high affinity tumor-associated antigens capable of eliciting potent anti-tumor T cell responses yet detecting these polyclonal T cells is challenging. Therefore, surrogate markers of T cell activation such as CD69, CD44, and PD-1 have been used. We report here that in mice, expression of activation markers including PD-1 is insufficient in the tumor microenvironment to identify tumor-antigen specific T cells. Using the Nur77GFP T cell affinity reporter mouse, we highlight that PD-1 expression can be induced independent of TCR ligation within the tumor. Given this, we characterized the utility of the Nur77GFP model system in elucidating mechanisms of action of immunotherapies independent of PD-1 expression. Co-expression of Nur77GFP and OX40 identifies a polyclonal population of high affinity tumor-associated antigen-specific CD8+ T cells, which produce more IFNγ in situ than OX40 negative and doubles in quantity with anti-OX40 and anti-CTLA4 mAb therapy but not with anti-PD-1 or PD-L1. Moreover, expansion of these high affinity CD8 T cells prolongs survival of tumor bearing animals. Upon chronic stimulation in tumors and after adoptive cell therapy, CD8 TCR signaling and Nur77GFP induction is impaired and tumors progress. However, this can be reversed and overall survival significantly enhanced after adoptive cell therapy with agonist OX40 immunotherapy. Therefore, we propose that OX40 agonist immunotherapy can maintain functional TCR signaling of chronically stimulated tumor resident CD8 T cells thereby increasing the frequency of cytolytic, high affinity, tumor-associated antigen-specific cells. PMID:27503208

CHEMOKINE RECEPTOR 7 (CCR7)-EXPRESSION AND IFNγ PRODUCTION DEFINE VACCINE-SPECIFIC CANINE T CELL SUBSETS

PubMed Central

Hartley, Ashley N.; Tarleton, Rick L.

2015-01-01

Canines suffer from and serve as strong translational animals models for many immunological disorders and infectious diseases. Routine vaccination has been a mainstay of protecting dogs through the stimulation of robust antibody responses and expansion of memory T cell populations. Commercially available reagents and described techniques are limited for identifying and characterizing canine T cell subsets and evaluating T cell-specific effector function. To define reagents for delineating naïve versus activated T cells and identify antigen-specific T cells, we tested anti-human and anti-bovine T-cell specific cell surface marker reagents for cross-reactivity with canine peripheral blood mononuclear cells (PBMCs. Both CD4+ and CD8+ T cells from healthy canine donors showed reactivity to CCL19-Ig, a CCR7 ligand, and coexpression with CD62L. An in vitro stimulation with concanavalin A validated downregulation of CCR7 and CD62L expression on stimulated healthy control PBMCs, consistent with an activated T cell phenotype. Anti-IFNγ antibodies identified antigen-specific IFNγ-producing CD4+ and CD8+ T cells upon in vitro vaccine antigen PBMC stimulation. PBMC isolation within 24 hours of sample collection allowed for efficient cell recovery and accurate T cell effector function characterization. These data provide a reagent and techniques platform via flow cytometry for identifying canine T cell subsets and characterizing circulating antigen-specific canine T cells for potential use in diagnostic and field settings. PMID:25758065

A CD22-reactive TCR from the T-cell allorepertoire for the treatment of acute lymphoblastic leukemia by TCR gene transfer

PubMed Central

Jahn, Lorenz; Hagedoorn, Renate S.; van der Steen, Dirk M.; Hombrink, Pleun; Kester, Michel G.D.; Schoonakker, Marjolein P.; de Ridder, Daniëlle; van Veelen, Peter A.; Falkenburg, J.H. Frederik; Heemskerk, Mirjam H.M.

2016-01-01

CD22 is currently evaluated as a target-antigen for the treatment of B-cell malignancies using chimeric antigen receptor (CAR)-engineered T-cells or monoclonal antibodies (mAbs). CAR- and mAbs-based immunotherapies have been successfully applied targeting other antigens, however, occurrence of refractory disease to these interventions urges the identification of additional strategies. Here, we identified a TCR recognizing the CD22-derived peptide RPFPPHIQL (CD22RPF) presented in human leukocyte antigen (HLA)-B*07:02. To overcome tolerance to self-antigens such as CD22, we exploited the immunogenicity of allogeneic HLA. CD22RPF-specific T-cell clone 9D4 was isolated from a healthy HLA-B*07:02neg individual, efficiently produced cytokines upon stimulation with primary acute lymphoblastic leukemia and healthy B-cells, but did not react towards healthy hematopoietic and nonhematopoietic cell subsets, including dendritic cells (DCs) and macrophages expressing low levels of CD22. Gene transfer of TCR-9D4 installed potent CD22-specificity onto recipient CD8+ T-cells that recognized and lysed primary B-cell leukemia. TCR-transduced T-cells spared healthy CD22neg hematopoietic cell subsets but weakly lysed CD22low-expressing DCs and macrophages. CD22-specific TCR-engineered T-cells could form an additional immunotherapeutic strategy with a complementary role to CAR- and antibody-based interventions in the treatment of B-cell malignancies. However, CD22 expression on non-B-cells may limit the attractiveness of CD22 as target-antigen in cellular immunotherapy. PMID:27689397

A CD22-reactive TCR from the T-cell allorepertoire for the treatment of acute lymphoblastic leukemia by TCR gene transfer.

PubMed

Jahn, Lorenz; Hagedoorn, Renate S; van der Steen, Dirk M; Hombrink, Pleun; Kester, Michel G D; Schoonakker, Marjolein P; de Ridder, Daniëlle; van Veelen, Peter A; Falkenburg, J H Frederik; Heemskerk, Mirjam H M

2016-11-01

CD22 is currently evaluated as a target-antigen for the treatment of B-cell malignancies using chimeric antigen receptor (CAR)-engineered T-cells or monoclonal antibodies (mAbs). CAR- and mAbs-based immunotherapies have been successfully applied targeting other antigens, however, occurrence of refractory disease to these interventions urges the identification of additional strategies. Here, we identified a TCR recognizing the CD22-derived peptide RPFPPHIQL (CD22RPF) presented in human leukocyte antigen (HLA)-B*07:02. To overcome tolerance to self-antigens such as CD22, we exploited the immunogenicity of allogeneic HLA. CD22RPF-specific T-cell clone 9D4 was isolated from a healthy HLA-B*07:02neg individual, efficiently produced cytokines upon stimulation with primary acute lymphoblastic leukemia and healthy B-cells, but did not react towards healthy hematopoietic and nonhematopoietic cell subsets, including dendritic cells (DCs) and macrophages expressing low levels of CD22. Gene transfer of TCR-9D4 installed potent CD22-specificity onto recipient CD8+ T-cells that recognized and lysed primary B-cell leukemia. TCR-transduced T-cells spared healthy CD22neg hematopoietic cell subsets but weakly lysed CD22low-expressing DCs and macrophages. CD22-specific TCR-engineered T-cells could form an additional immunotherapeutic strategy with a complementary role to CAR- and antibody-based interventions in the treatment of B-cell malignancies. However, CD22 expression on non-B-cells may limit the attractiveness of CD22 as target-antigen in cellular immunotherapy.

CD8+ T Cells Induce Fatal Brainstem Pathology during Cerebral Malaria via Luminal Antigen-Specific Engagement of Brain Vasculature

PubMed Central

Swanson, Phillip A.; Hart, Geoffrey T.; Russo, Matthew V.; Nayak, Debasis; Yazew, Takele; Peña, Mirna; Khan, Shahid M.; Pierce, Susan K.; McGavern, Dorian B.

2016-01-01

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection that results in thousands of deaths each year, mostly in African children. The in vivo mechanisms underlying this fatal condition are not entirely understood. Using the animal model of experimental cerebral malaria (ECM), we sought mechanistic insights into the pathogenesis of CM. Fatal disease was associated with alterations in tight junction proteins, vascular breakdown in the meninges / parenchyma, edema, and ultimately neuronal cell death in the brainstem, which is consistent with cerebral herniation as a cause of death. At the peak of ECM, we revealed using intravital two-photon microscopy that myelomonocytic cells and parasite-specific CD8+ T cells associated primarily with the luminal surface of CNS blood vessels. Myelomonocytic cells participated in the removal of parasitized red blood cells (pRBCs) from cerebral blood vessels, but were not required for the disease. Interestingly, the majority of disease-inducing parasite-specific CD8+ T cells interacted with the lumen of brain vascular endothelial cells (ECs), where they were observed surveying, dividing, and arresting in a cognate peptide-MHC I dependent manner. These activities were critically dependent on IFN-γ, which was responsible for activating cerebrovascular ECs to upregulate adhesion and antigen-presenting molecules. Importantly, parasite-specific CD8+ T cell interactions with cerebral vessels were impaired in chimeric mice rendered unable to present EC antigens on MHC I, and these mice were in turn resistant to fatal brainstem pathology. Moreover, anti-adhesion molecule (LFA-1 / VLA-4) therapy prevented fatal disease by rapidly displacing luminal CD8+ T cells from cerebrovascular ECs without affecting extravascular T cells. These in vivo data demonstrate that parasite-specific CD8+ T cell-induced fatal vascular breakdown and subsequent neuronal death during ECM is associated with luminal, antigen-dependent interactions with cerebrovasculature. PMID:27907215

Nonclassical MHC Ib-restricted CD8+ T Cells Recognize Mycobacterium tuberculosis-Derived Protein Antigens and Contribute to Protection Against Infection

PubMed Central

Shang, Shaobin; Siddiqui, Sarah; Bian, Yao; Zhao, Jie; Wang, Chyung-Ru

2016-01-01

MHC Ib-restricted CD8+ T cells have been implicated in host defense against Mycobacterium tuberculosis (Mtb) infection. However, the relative contribution of various MHC Ib-restricted T cell populations to anti-mycobacterial immunity remains elusive. In this study, we used mice that lack MHC Ia (Kb-/-Db-/-), MHC Ia/H2-M3 (Kb-/-Db-/-M3-/-), or β2m (β2m-/-) to study the role of M3-restricted and other MHC Ib-restricted T cells in immunity against Mtb. Unlike their dominant role in Listeria infection, we found that M3-restricted CD8+ T cells only represented a small proportion of the CD8+ T cells responding to Mtb infection. Non-M3, MHC Ib-restricted CD8+ T cells expanded preferentially in the lungs of Mtb-infected Kb-/-Db-/-M3-/- mice, exhibited polyfunctional capacities and conferred protection against Mtb. These MHC Ib-restricted CD8+ T cells recognized several Mtb-derived protein antigens at a higher frequency than MHC Ia-restricted CD8+ T cells. The presentation of Mtb antigens to MHC Ib-restricted CD8+ T cells was mostly β2m-dependent but TAP-independent. Interestingly, a large proportion of Mtb-specific MHC Ib-restricted CD8+ T cells in Kb-/-Db-/-M3-/- mice were Qa-2-restricted while no considerable numbers of MR1 or CD1-restricted Mtb-specific CD8+ T cells were detected. Our findings indicate that nonclassical CD8+ T cells other than the known M3, CD1, and MR1-restricted CD8+ T cells contribute to host immune responses against Mtb infection. Targeting these MHC Ib-restricted CD8+ T cells would facilitate the design of better Mtb vaccines with broader coverage across MHC haplotypes due to the limited polymorphism of MHC class Ib molecules. PMID:27272249

CD4 T cell-mediated masking effects of the immunogenicity of tumor-associated antigens are qualitatively and quantitatively different depending on the individual antigens.

PubMed

Okano, Shinji; Matsumoto, Yoshihiro; Yoshiya, Shohei; Yamashita, Yo-ichi; Harimoto, Norifumi; Ikegami, Toru; Shirabe, Ken; Harada, Mamoru; Yoshikai, Yasunobu; Maehara, Yoshihiko

2013-01-01

The use of cancer immunotherapy as part of multidisciplinary therapies for cancer is a promising strategy for the cure of advanced cancer patients. In cancer immunotherapy, the effective priming of tumor-associated antigen (TAA)-specific CD8+ T cells is essential, and therefore, the appropriate selection of the best peptide for targeting the cancer is a most important concern. One criticism in the selection of a TAA is the immunogenicity of the TAA, the vaccination of which effectively elicits clinical responses. However, the critical basic immunological factors that affect the differences in the immunogenicity of TAAs remain to be elucidated. Here we found that CD4 T-cell responses suppressed the immunogenicity of the concomitant TAA in a murine melanoma model in which intratumoral activated dendritic therapy (ITADT) was used for treatment of the established cancer, and we observed that the antitumor effects were largely dependent on the CD8 T-cell response. CD4 T-cell depletion simply enhanced the tyrosinase-related protein (TRP)-2(180-188) peptide-specific cytotoxic T-cell (CTL) responses, and CD4 T-cell depletion provided immunogenicity for mgp100(25-33) peptide, to which a CTL response could not be detected at all in CD4 T-cell-intact mice in the early therapeutic phase. Further, the mgp100(25-33) peptide-specific CTL response again became undetectable after the recovery of CD4 T cells in previously CD4-depleted, tumor-eradicated mice, whereas the TRP-2(180-188) peptide-specific CTL response was still much stronger in CD4-depleted mice than in CD4-intact mice. These findings suggest that the CD4 T cell-mediated masking effects of the immunogenicity of tumor-associated antigens are qualitatively and quantitatively different depending on the individual antigens.

Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens into the cytosol to induce T cell immunity

NASA Astrophysics Data System (ADS)

Lu, Yichen; Friedman, Rachel; Kushner, Nicholas; Doling, Amy; Thomas, Lawrence; Touzjian, Neal; Starnbach, Michael; Lieberman, Judy

2000-07-01

Bacillus anthrax lethal toxin can be engineered to deliver foreign proteins to the cytosol for antigen presentation to CD8 T cells. Vaccination with modified toxins carrying 8-9 amino acid peptide epitopes induces protective immunity in mice. To evaluate whether large protein antigens can be used with this system, recombinant constructs encoding several HIV antigens up to 500 amino acids were produced. These candidate HIV vaccines are safe in animals and induce CD8 T cells in mice. Constructs encoding gag p24 and nef stimulate gag-specific CD4 proliferation and a secondary cytotoxic T lymphocyte response in HIV-infected donor peripheral blood mononuclear cells in vitro. These results lay the foundation for future clinical vaccine studies.

Efficient Culture of Human Naïve and Memory B cells for Use as Antigen-presenting Cells

PubMed Central

Su, Kuei-Ying; Watanabe, Akiko; Yeh, Chen-Hao; Kelsoe, Garnett; Kuraoka, Masayuki

2016-01-01

The ability to culture and expand B cells in vitro has become a useful tool for studying human immunity. A limitation of current methods for human B-cell culture is the capacity to support mature B-cell proliferation. We have developed a culture method to support the efficient activation and proliferation of both naïve and memory human B cells. This culture supports extensive B-cell proliferation, with approximately 103-fold increases following 8 days in culture, and 106-fold increases when cultures are split and cultured for 8 more days. In culture, a significant fraction of naïve B cells undergo isotype switching and differentiate into plasmacytes. Culture-derived (CD) B cells are readily cryopreserved, and when recovered, retain their ability to proliferate and differentiate. Significantly, proliferating CD B cells express high levels of MHCII, CD80, and CD86. CD B cells act as APCs and present both alloantigens and microbial antigens to T cells. We are able to activate and expand antigen-specific memory B cells; these cultured cells are highly effective in presenting antigen to T cells. We have characterized the TCR repertoire of rare antigen-specific CD4+ T cells that proliferated in response to tetanus toxoid (TT) presented by autologous CD B cells. TCR Vβ usage by TT-activated CD4+ T cells differs from both resting and unspecifically activated CD4+ T cells. Moreover, we found that TT-specific TCR Vβ usage by CD4+ T cells was substantially different between donors. This culture method provides a platform for studying the BCR and TCR repertoires within a single individual. PMID:27815447

Plasmacytoid Dendritic Cells Require Direct Infection To Sustain the Pulmonary Influenza A Virus-Specific CD8 T Cell Response

PubMed Central

Hemann, Emily A.; Sjaastad, Louisa E.; Langlois, Ryan A.

2015-01-01

ABSTRACT Following influenza A virus (IAV) infection, development of a robust IAV-specific CD8 T cell response is required for clearance of primary infection and enhances memory protection. Following IAV infection, plasmacytoid dendritic cells (pDC) or CD8α+ DC regulate pulmonary effector CD8 T cell responses within the lung. Without this DC-T cell interaction, insufficient effector CD8 T cells are maintained in the lungs, leading to enhanced morbidity and mortality. Previous studies have demonstrated that pDC are capable of classical presentation or cross-presentation of IAV antigens and could potentially regulate IAV-specific CD8 T cell responses through either mechanism. Our results demonstrate that pDC from the lungs of donor mice infected with an IAV that is not able to replicate in hematopoietic cells (142t-IAV), unlike donor pDC isolated from the lungs of control infected mice, are not able to rescue the host IAV-specific CD8 T cell response from apoptosis. This indicates that pDC must utilize the direct presentation pathway for this rescue. This inability of pDC from 142t-IAV donors to rescue the IAV-specific CD8 T cell response is not due to differences in the overall ability of 142t-IAV to replicate within the lungs or generate defective viral genomes or to differences in levels of costimulatory molecules required for this interaction. We further demonstrate that bypassing the antigen presentation pathway by coating the 142t-IAV pDC with IAV peptide epitopes restores their ability to rescue the IAV-specific CD8 T cell response. IMPORTANCE IAV continues to be a global health burden, infecting 5 to 20% of the global population annually. Continued investigation into the mechanisms that mediate protective immune responses against IAV is important to improving current vaccination and antiviral strategies antagonistic toward IAV. Our findings presented herein demonstrate a key requirement for pDC promotion of effector CD8 T cell survival: that rather than utilizing cross-presentation, pDC must be infected and utilize the endogenous pathway for presentation of antigens to CD8 T cells during in vivo IAV infections. This suggests that targeting presentation via the endogenous pathway in pDC could be important for the development of unique antiviral cellular therapies. PMID:26719269

Specific recognition and inhibition of Ewing tumour growth by antigen-specific allo-restricted cytotoxic T cells

PubMed Central

Thiel, U; Pirson, S; Müller-Spahn, C; Conrad, H; Busch, D H; Bernhard, H; Burdach, S; Richter, G H S

2011-01-01

Background: The development of a successful immunotherapy is hampered by an ineffective T-cell repertoire against tumour antigens and the inability of the patient's immune system to overcome tolerance-inducing mechanisms. Here, we test the specific recognition and lytical potential of allo-restricted CD8+ T cells against Ewing tumour (ET) associated antigens Enhancer of Zeste, Drosophila Homolog 2 (EZH2), and Chondromodulin-I (CHM1) identified through previous microarray analysis. Methods: Following repetitive CHM1319 (VIMPCSWWV) and EZH2666 (YMCSFLFNL) peptide-driven stimulations with HLA-A*0201+ dendritic cells (DC), allo-restricted HLA-A*0201− CD8+ T cells were stained with HLA-A*0201/peptide multimers, sorted and expanded by limiting dilution. Results: Expanded T cells specifically recognised peptide-pulsed target cells or antigen-transfected cells in the context of HLA-A*0201 and killed HLA-A*0201+ ET lines expressing the antigen while HLA-A*0201– ET lines were not affected. Furthermore, adoptively transferred T cells caused significant ET growth delay in Rag2−/−γC−/− mice. Within this context, we identified the CHM1319 peptide as a new candidate target antigen for ET immunotherapy. Conclusion: These results clearly identify the ET-derived antigens, EZH2666 and CHM1319, as suitable targets for protective allo-restricted human CD8+ T-cell responses against non-immunogenic ET and may benefit new therapeutic strategies in ET patients treated with allogeneic stem cell transplantation. PMID:21407224

Myristoylation increases the CD8+T-cell response to a GFP prototype antigen delivered by modified vaccinia virus Ankara.

PubMed

Marr, Lisa; Lülf, Anna-Theresa; Freudenstein, Astrid; Sutter, Gerd; Volz, Asisa

2016-04-01

Activation of CD8(+)T-cells is an essential part of immune responses elicited by recombinant modified vaccinia virus Ankara (MVA). Strategies to enhance T-cell responses to antigens may be particularly necessary for broadly protective immunization against influenza A virus infections or for candidate vaccines targeting chronic infections and cancer. Here, we tested recombinant MVAs that targeted a model antigen, GFP, to different localizations in infected cells. In vitro characterization demonstrated that GFP accumulated in the nucleus (MVA-nls-GFP), associated with cellular membranes (MVA-myr-GFP) or was equally distributed throughout the cell (MVA-GFP). On vaccination, we found significantly higher levels of GFP-specific CD8(+)T-cells in MVA-myr-GFP-vaccinated BALB/c mice than in those immunized with MVA-GFP or MVA-nls-GFP. Thus, myristoyl modification may be a useful strategy to enhance CD8(+)T-cell responses to MVA-delivered target antigens.

CD8+ T Lymphocyte Epitopes From The Herpes Simplex Virus Type 2 ICP27, VP22 and VP13/14 Proteins To Facilitate Vaccine Design And Characterization

PubMed Central

Platt, Rebecca J.; Khodai, Tansi; Townend, Tim J.; Bright, Helen H.; Cockle, Paul; Perez-Tosar, Luis; Webster, Rob; Champion, Brian; Hickling, Timothy P.; Mirza, Fareed

2013-01-01

CD8+ T cells have the potential to control HSV-2 infection. However, limited information has been available on CD8+ T cell epitopes or the functionality of antigen specific T cells during infection or following immunization with experimental vaccines. Peptide panels from HSV-2 proteins ICP27, VP22 and VP13/14 were selected from in silico predictions of binding to human HLA-A*0201 and mouse H-2Kd, Ld and Dd molecules. Nine previously uncharacterized CD8+ T cell epitopes were identified from HSV-2 infected BALB/c mice. HSV-2 specific peptide sequences stabilized HLA-A*02 surface expression with intermediate or high affinity binding. Peptide specific CD8+ human T cell lines from peripheral blood lymphocytes were generated from a HLA-A*02+ donor. High frequencies of peptide specific CD8+ T cell responses were elicited in mice by DNA vaccination with ICP27, VP22 and VP13/14, as demonstrated by CD107a mobilization. Vaccine driven T cell responses displayed a more focused immune response than those induced by viral infection. Furthermore, vaccination with ICP27 reduced viral shedding and reduced the clinical impact of disease. In conclusion, this study describes novel HSV-2 epitopes eliciting strong CD8+ T cell responses that may facilitate epitope based vaccine design and aid immunomonitoring of antigen specific T cell frequencies in preclinical and clinical settings. PMID:24709642

Characteristics of Prevotella intermedia-specific CD4+ T cell clones from peripheral blood of a chronic adult periodontitis patient

PubMed Central

Wassenaar, A; Reinhardus, C; Abraham-Inpijn, L; Snijders, A; Kievits, F

1998-01-01

Periodontitis is a chronic destructive inflammatory disease associated with periodontopathic bacteria. In addition, autoantigens such as collagen and heat shock proteins (hsp) have been suggested to play a role. Established periodontal lesions are characterized by dense infiltrations of immune cells such as cytokine-producing CD4+ and CD8+ T cells. CD4+ T cells specific for Prevotella intermedia can be isolated from lesional gingiva, suggesting an active role for CD4+ T cells in the response to this bacterium. We therefore investigated the characteristics of a panel of 13 P. intermedia-specific CD4+ T cells generated from the peripheral blood of a patient with chronic adult periodontitis. All 13 P. intermedia-specific CD4+ T cells recognized the antigens in the context of HLA-DR. The T cell clones were mainly classified as Th0, producing comparable amounts of interferon-gamma (IFN-γ) and IL-4, and Th2, producing high amounts of IL-4 and almost no IFN-γ. None of the P. intermedia-specific T cell clones recognized antigens of the periodontopathic bacteria Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis and of the autoantigens collagen and hsp. The reactivity profile of the T cell clones to size-fractionated cell envelope antigens of P. intermedia indicated that P. intermedia-specific CD4+ T cell clones recognize probably five different antigen specificities in the context of the MHC class II molecules, DR7 or DR15. These results suggest that a broad panel of cell-associated protein antigens play a role in the induction of P. intermedia-specific CD4+ T cell response. PMID:9697992

Peripheral self-reactivity regulates antigen-specific CD8 T-cell responses and cell division under physiological conditions.

PubMed

Swee, Lee Kim; Tan, Zhen Wei; Sanecka, Anna; Yoshida, Nagisa; Patel, Harshil; Grotenbreg, Gijsbert; Frickel, Eva-Maria; Ploegh, Hidde L

2016-11-01

T-cell identity is established by the expression of a clonotypic T-cell receptor (TCR), generated by somatic rearrangement of TCRα and β genes. The properties of the TCR determine both the degree of self-reactivity and the repertoire of antigens that can be recognized. For CD8 T cells, the relationship between TCR identity-hence reactivity to self-and effector function(s) remains to be fully understood and has rarely been explored outside of the H-2 b haplotype. We measured the affinity of three structurally distinct CD8 T-cell-derived TCRs that recognize the identical H-2 L d -restricted epitope, derived from the Rop7 protein of Toxoplasma gondii We used CD8 T cells obtained from mice generated by somatic cell nuclear transfer as the closest approximation of primary T cells with physiological TCR rearrangements and TCR expression levels. First, we demonstrate the common occurrence of secondary rearrangements in endogenously rearranged loci. Furthermore, we characterized and compared the response of Rop7-specific CD8 T-cell clones upon Toxoplasma gondii infection as well as effector function and TCR signalling upon antigenic stimulation in vitro Antigen-independent TCR cross-linking in vitro uncovered profound intrinsic differences in the effector functions between T-cell clones. Finally, by assessing the degree of self-reactivity and comparing the transcriptomes of naive Rop7 CD8 T cells, we show that lower self-reactivity correlates with lower effector capacity, whereas higher self-reactivity is associated with enhanced effector function as well as cell cycle entry under physiological conditions. Altogether, our data show that potential effector functions and basal proliferation of CD8 T cells are set by self-reactivity thresholds. © 2016 The Authors.

Detailed analysis of Epstein–Barr virus-specific CD4+ and CD8+ T cell responses during infectious mononucleosis

PubMed Central

Scherrenburg, J; Piriou, E R W A N; Nanlohy, N M; van Baarle, D

2008-01-01

We studied simultaneously Epstein–Barr virus (EBV)-specific CD4+ and CD8+ T cell responses during and after infectious mononucleosis (IM), using a previously described 12-day stimulation protocol with EBNA1 or BZLF1 peptide pools. Effector function of EBV-specific T cells was determined after restimulation by measuring intracellular interferon-γ production. During IM, BZLF1-specifc CD4+ T cell responses were dominant compared with CD8+ T cell responses. EBNA1-specific CD4+ and CD8+ T cell responses were low and remained similar for 6 months. However, 6 months after IM, BZLF1-specific CD4+ T cell responses had declined, but CD8+ T cell responses had increased. At diagnosis, EBV-specific CD8+ T cells as studied by human leucocyte antigen class I tetramer staining comprised a tetramerbrightCD8bright population consisting mainly of CD27+ memory T cells and a tetramerdimCD8dim population consisting primarily of CD27- effector T cells. The remaining EBV-specific CD8+ T cell population 6 months after the diagnosis of IM consisted mainly of tetramerbrightCD8bright CD27+ T cells, suggesting preferential preservation of memory T cells after contraction of the EBV-specific T cell pool. PMID:18549439

Connective tissue growth factor linked to the E7 tumor antigen generates potent antitumor immune responses mediated by an antiapoptotic mechanism.

PubMed

Cheng, W-F; Chang, M-C; Sun, W-Z; Lee, C-N; Lin, H-W; Su, Y-N; Hsieh, C-Y; Chen, C-A

2008-07-01

A novel method for generating an antigen-specific cancer vaccine and immunotherapy has emerged using a DNA vaccine. However, antigen-presenting cells (APCs) have a limited life span, which hinders their long-term ability to prime antigen-specific T cells. Connective tissue growth factor (CTGF) has a role in cell survival. This study explored the intradermal administration of DNA encoding CTGF with a model tumor antigen, human papilloma virus type 16 E7. Mice vaccinated with CTGF/E7 DNA exhibited a dramatic increase in E7-specific CD4(+) and CD8(+) T-cell precursors. They also showed an impressive antitumor effect against E7-expressing tumors compared with mice vaccinated with the wild-type E7 DNA. The delivery of DNA encoding CTGF and E7 or CTGF alone could prolong the survival of transduced dendritic cells (DCs) in vivo. In addition, CTGF/E7-transduced DCs could enhance a higher number of E7-specific CD8(+) T cells than E7-transduced DCs. By prolonging the survival of APCs, DNA vaccine encoding CTGF linked to a tumor antigen represents an innovative approach to enhance DNA vaccine potency and holds promise for cancer prophylaxis and immunotherapy.

A novel T cell receptor single-chain signaling complex mediates antigen-specific T cell activity and tumor control

PubMed Central

Stone, Jennifer D.; Harris, Daniel T.; Soto, Carolina M.; Chervin, Adam S.; Aggen, David H.; Roy, Edward J.; Kranz, David M.

2014-01-01

Adoptive transfer of genetically modified T cells to treat cancer has shown promise in several clinical trials. Two main strategies have been applied to redirect T cells against cancer: 1) introduction of a full-length T cell receptor (TCR) specific for a tumor-associated peptide-MHC, or 2) introduction of a chimeric antigen receptor (CAR), including an antibody fragment specific for a tumor cell surface antigen, linked intracellularly to T cell signaling domains. Each strategy has advantages and disadvantages for clinical applications. Here, we present data on the in vitro and in vivo effectiveness of a single-chain signaling receptor incorporating a TCR variable fragment as the targeting element (referred to as TCR-SCS). This receptor contained a single-chain TCR (Vβ-linker-Vα) from a high-affinity TCR called m33, linked to the intracellular signaling domains of CD28 and CD3ζ. This format avoided mispairing with endogenous TCR chains, and mediated specific T cell activity when expressed in either CD4 or CD8 T cells. TCR-SCS-transduced CD8-negative cells showed an intriguing sensitivity, compared to full-length TCRs, to higher densities of less stable pepMHC targets. T cells that expressed this peptide-specific receptor persisted in vivo, and exhibited polyfunctional responses. Growth of metastatic antigen-positive tumors was significantly inhibited by T cells that expressed this receptor, and tumor cells that escaped were antigen loss variants. TCR-SCS receptors represent an alternative targeting receptor strategy that combines the advantages of single-chain expression, avoidance of TCR chain mispairing, and targeting of intracellular antigens presented in complex with MHC proteins. PMID:25082071

Comprehensive definition of human immunodominant CD8 antigens in tuberculosis.

PubMed

Lewinsohn, Deborah A; Swarbrick, Gwendolyn M; Park, Byung; Cansler, Meghan E; Null, Megan D; Toren, Katelynne G; Baseke, Joy; Zalwango, Sarah; Mayanja-Kizza, Harriet; Malone, LaShaunda L; Nyendak, Melissa; Wu, Guanming; Guinn, Kristi; McWeeney, Shannon; Mori, Tomi; Chervenak, Keith A; Sherman, David R; Boom, W Henry; Lewinsohn, David M

2017-01-01

Despite widespread use of the Bacillus Calmette-Guerin vaccine, tuberculosis, caused by infection with Mycobacterium tuberculosis , remains a leading cause of morbidity and mortality worldwide. As CD8 + T cells are critical to tuberculosis host defense and a phase 2b vaccine trial of modified vaccinia Ankara expressing Ag85a that failed to demonstrate efficacy, also failed to induce a CD8 + T cell response, an effective tuberculosis vaccine may need to induce CD8 + T cells. However, little is known about CD8, as compared to CD4, antigens in tuberculosis. Herein, we report the results of the first ever HLA allele independent genome-wide CD8 antigen discovery program. Using CD8 + T cells derived from humans with latent tuberculosis infection or tuberculosis and an interferon-γ ELISPOT assay, we screened a synthetic peptide library representing 10% of the Mycobacterium tuberculosis proteome, selected to be enriched for Mycobacterium tuberculosis antigens. We defined a set of immunodominant CD8 antigens including part or all of 74 Mycobacterium tuberculosis proteins, only 16 of which are previously known CD8 antigens. Immunogenicity was associated with the degree of expression of mRNA and protein. Immunodominant antigens were enriched in cell wall proteins with preferential recognition of Esx protein family members, and within proteins comprising the Mycobacterium tuberculosis secretome. A validation study of immunodominant antigens demonstrated that these antigens were strongly recognized in Mycobacterium tuberculosis -infected individuals from a tuberculosis endemic region in Africa. The tuberculosis vaccine field will likely benefit from this greatly increased known repertoire of CD8 immunodominant antigens and definition of properties of Mycobacterium tuberculosis proteins important for CD8 antigenicity.

Large-scale expansion of γδ T cells and peptide-specific cytotoxic T cells using zoledronate for adoptive immunotherapy.

PubMed

Yoshikawa, Toshiaki; Takahara, Masashi; Tomiyama, Mai; Nieda, Mie; Maekawa, Ryuji; Nakatsura, Tetsuya

2014-11-01

Specific cellular immunotherapy for cancer requires efficient generation and expansion of cytotoxic T lymphocytes (CTLs) that recognize tumor-associated antigens. However, it is difficult to isolate and expand functionally active T-cells ex vivo. In this study, we investigated the efficacy of a new method to induce expansion of antigen-specific CTLs for adoptive immunotherapy. We used tumor-associated antigen glypican-3 (GPC3)-derived peptide and cytomegalovirus (CMV)-derived peptide as antigens. Treatment of human peripheral blood mononuclear cells (PBMCs) with zoledronate is a method that enables large-scale γδ T-cell expansion. To induce expansion of γδ T cells and antigen-specific CTLs, the PBMCs of healthy volunteers or patients vaccinated with GPC3 peptide were cultured with both peptide and zoledronate for 14 days. The expansion of γδ T cells and peptide-specific CTLs from a few PBMCs using zoledronate yields cell numbers sufficient for adoptive transfer. The rate of increase of GPC3‑specific CTLs was approximately 24- to 170,000-fold. These CD8(+) cells, including CTLs, showed GPC3-specific cytotoxicity against SK-Hep-1/hGPC3 and T2 pulsed with GPC3 peptide, but not against SK-Hep-1/vec and T2 pulsed with human immunodeficiency virus peptide. On the other hand, CD8(-) cells, including γδ T cells, showed cytotoxicity against SK-Hep-1/hGPC3 and SK-Hep-1/vec, but did not show GPC3 specificity. Furthermore, adoptive cell transfer of CD8(+) cells, CD8(-) cells, and total cells after expansion significantly inhibited tumor growth in an NOD/SCID mouse model. This study indicates that simultaneous expansion of γδ T cells and peptide-specific CTLs using zoledronate is useful for adoptive immunotherapy.

Unusual antigen presentation offers new insight into HIV vaccine design.

PubMed

McMichael, Andrew J; Picker, Louis J

2017-06-01

Recent findings with a rhesus monkey cytomegalovirus based simian immunodeficiency virus vaccine have identified strong CD8+ T cell responses that are restricted by MHC-E. Also mycobacteria specific CD8+ T cells, that are MHC-E restricted, have been identified. MHC-E therefore can present a wide range of epitope peptides to CD8+ T cells, alongside its well defined role in presenting a conserved MHC-class I signal peptide to the NKG2A/C-CD94 receptor on natural killer cells. Here we explore the antigen processing pathways involved in these atypical T cell responses. Copyright © 2017 Elsevier Ltd. All rights reserved.

Rational design of nanoparticles towards targeting antigen-presenting cells and improved T cell priming.

PubMed

Zupančič, Eva; Curato, Caterina; Paisana, Maria; Rodrigues, Catarina; Porat, Ziv; Viana, Ana S; Afonso, Carlos A M; Pinto, João; Gaspar, Rogério; Moreira, João N; Satchi-Fainaro, Ronit; Jung, Steffen; Florindo, Helena F

2017-07-28

Vaccination is a promising strategy to trigger and boost immune responses against cancer or infectious disease. We have designed, synthesized and characterized aliphatic-polyester (poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) to investigate how the nature of protein association (adsorbed versus entrapped) and polymer/surfactant concentrations impact on the generation and modulation of antigen-specific immune responses. The ability of the NP formulations to target dendritic cells (DC), be internalized and activate the T cells was characterized and optimized in vitro and in vivo using markers of DC activation and co-stimulatory molecules. Ovalbumin (OVA) was used as a model antigen in combination with the engraftment of CD4 + and CD8 + T cells, carrying a transgenic OVA-responding T cell receptor (TCR), to trace and characterize the activation of antigen-specific CD4 + and CD8 + lymph node T cells upon NP vaccination. Accordingly, the phenotype and frequency of immune cell stimulation induced by the NP loaded with OVA, isolated or in combination with synthetic unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotide (ODN) motifs, were characterized. DC-NP interactions increased with incubation time, presenting internalization values between 50 and 60% and 30-40%, in vitro and in vivo, respectively. Interestingly, animal immunization with antigen-adsorbed NP up-regulated major histocompatibility complex (MHC) class II (MHCII), while NP entrapping the antigen up-regulated MHCI, suggesting a more efficient cross-presentation. On the other hand, rather surprisingly, the surfactant used in the NP formulation had a major impact on the activation of antigen presenting cells (APC). In fact, DC collected from lymph nodes of animals immunized with NP prepared using poly(vinil alcohol) (PVA), as a surfactant, expressed significantly higher levels of CD86, MHCI and MHCII. In addition, those NP prepared with PVA and co-entrapping OVA and the toll-like receptor (TLR) ligand CpG, induced the most profound antigen-specific T cell response, by both CD4 + and CD8 + T cells, in vivo. Overall, our data reveal the impact of NP composition and surface properties on the type and extension of induced immune responses. Deeper understanding on the NP-immune cell crosstalk can guide the rational development of nano-immunotherapeutic systems with improved and specific therapeutic efficacy and avoiding off-target effects. Copyright © 2017 Elsevier B.V. All rights reserved.

T Cell Receptor-Major Histocompatibility Complex Interaction Strength Defines Trafficking and CD103+ Memory Status of CD8 T Cells in the Brain.

PubMed

Sanecka, Anna; Yoshida, Nagisa; Kolawole, Elizabeth Motunrayo; Patel, Harshil; Evavold, Brian D; Frickel, Eva-Maria

2018-01-01

T cell receptor-major histocompatibility complex (TCR-MHC) affinities span a wide range in a polyclonal T cell response, yet it is undefined how affinity shapes long-term properties of CD8 T cells during chronic infection with persistent antigen. Here, we investigate how the affinity of the TCR-MHC interaction shapes the phenotype of memory CD8 T cells in the chronically Toxoplasma gondii- infected brain. We employed CD8 T cells from three lines of transnuclear (TN) mice that harbor in their endogenous loci different T cell receptors specific for the same Toxoplasma antigenic epitope ROP7. The three TN CD8 T cell clones span a wide range of affinities to MHCI-ROP7. These three CD8 T cell clones have a distinct and fixed hierarchy in terms of effector function in response to the antigen measured as proliferation capacity, trafficking, T cell maintenance, and memory formation. In particular, the T cell clone of lowest affinity does not home to the brain. The two higher affinity T cell clones show differences in establishing resident-like memory populations (CD103 + ) in the brain with the higher affinity clone persisting longer in the host during chronic infection. Transcriptional profiling of naïve and activated ROP7-specific CD8 T cells revealed that Klf2 encoding a transcription factor that is known to be a negative marker for T cell trafficking is upregulated in the activated lowest affinity ROP7 clone. Our data thus suggest that TCR-MHC affinity dictates memory CD8 T cell fate at the site of infection.

Impact of Leukocyte Function-Associated Antigen-1 Blockade on Endogenous Allospecific T Cells to Multiple Minor Histocompatibility Antigen Mismatched Cardiac Allograft.

PubMed

Kwun, Jean; Farris, Alton B; Song, Hyunjin; Mahle, William T; Burlingham, William J; Knechtle, Stuart J

2015-12-01

Blocking leukocyte function-associated antigen (LFA)-1 in organ transplant recipients prolongs allograft survival. However, the precise mechanisms underlying the therapeutic potential of LFA-1 blockade in preventing chronic rejection are not fully elucidated. Cardiac allograft vasculopathy (CAV) is the preeminent cause of late cardiac allograft failure characterized histologically by concentric intimal hyperplasia. Anti-LFA-1 monoclonal antibody was used in a multiple minor antigen-mismatched, BALB.B (H-2B) to C57BL/6 (H-2B), cardiac allograft model. Endogenous donor-specific CD8 T cells were tracked down using major histocompatibility complex multimers against the immunodominant H4, H7, H13, H28, and H60 minor Ags. The LFA-1 blockade prevented acute rejection and preserved palpable beating quality with reduced CD8 T-cell graft infiltration. Interestingly, less CD8 T cell infiltration was secondary to reduction of T-cell expansion rather than less trafficking. The LFA-1 blockade significantly suppressed the clonal expansion of minor histocompatibility antigen-specific CD8 T cells during the expansion and contraction phase. The CAV development was evaluated with morphometric analysis at postoperation day 100. The LFA-1 blockade profoundly attenuated neointimal hyperplasia (61.6 vs 23.8%; P < 0.05), CAV-affected vessel number (55.3 vs 15.9%; P < 0.05), and myocardial fibrosis (grade 3.29 vs 1.8; P < 0.05). Finally, short-term LFA-1 blockade promoted long-term donor-specific regulation, which resulted in attenuated transplant arteriosclerosis. Taken together, LFA-1 blockade inhibits initial endogenous alloreactive T-cell expansion and induces more regulation. Such a mechanism supports a pulse tolerance induction strategy with anti-LFA-1 rather than long-term treatment.

Lambda phage-based vaccine induces antitumor immunity in hepatocellular carcinoma.

PubMed

Iwagami, Yoshifumi; Casulli, Sarah; Nagaoka, Katsuya; Kim, Miran; Carlson, Rolf I; Ogawa, Kosuke; Lebowitz, Michael S; Fuller, Steve; Biswas, Biswajit; Stewart, Solomon; Dong, Xiaoqun; Ghanbari, Hossein; Wands, Jack R

2017-09-01

Hepatocellular carcinoma (HCC) is a difficult to treat tumor with a poor prognosis. Aspartate β-hydroxylase (ASPH) is a highly conserved enzyme overexpressed on the cell surface of both murine and human HCC cells. We evaluated therapeutic effects of nanoparticle lambda (λ) phage vaccine constructs against ASPH expressing murine liver tumors. Mice were immunized before and after subcutaneous implantation of a syngeneic BNL HCC cell line. Antitumor actively was assessed by generation of antigen specific cellular immune responses and the identification of tumor infiltrating lymphocytes. Prophylactic and therapeutic immunization significantly delayed HCC growth and progression. ASPH-antigen specific CD4+ and CD8+ lymphocytes were identified in the spleen of tumor bearing mice and cytotoxicity was directed against ASPH expressing BNL HCC cells. Furthermore, vaccination generated antigen specific Th1 and Th2 cytokine secretion by immune cells. There was widespread necrosis with infiltration of CD3+ and CD8+ T cells in HCC tumors of λ phage vaccinated mice compared to controls. Moreover, further confirmation of anti-tumor effects on ASPH expressing tumor cell growth were obtained in another murine syngeneic vaccine model with pulmonary metastases. These observations suggest that ASPH may serve as a highly antigenic target for immunotherapy.

Inflammatory impact of IFN-γ in CD8+ T cell-mediated lung injury is mediated by both Stat1-dependent and -independent pathways

PubMed Central

Ramana, Chilakamarti V.; DeBerge, Matthew P.; Kumar, Aseem; Alia, Christopher S.; Durbin, Joan E.

2015-01-01

Influenza infection results in considerable pulmonary pathology, a significant component of which is mediated by CD8+ T cell effector functions. To isolate the specific contribution of CD8+ T cells to lung immunopathology, we utilized a nonviral murine model in which alveolar epithelial cells express an influenza antigen and injury is initiated by adoptive transfer of influenza-specific CD8+ T cells. We report that IFN-γ production by adoptively transferred influenza-specific CD8+ T cells is a significant contributor to acute lung injury following influenza antigen recognition, in isolation from its impact on viral clearance. CD8+ T cell production of IFN-γ enhanced lung epithelial cell expression of chemokines and the subsequent recruitment of inflammatory cells into the airways. Surprisingly, Stat1 deficiency in the adoptive-transfer recipients exacerbated the lung injury that was mediated by the transferred influenza-specific CD8+ T cells but was still dependent on IFN-γ production by these cells. Loss of Stat1 resulted in sustained activation of Stat3 signaling, dysregulated chemokine expression, and increased infiltration of the airways by inflammatory cells. Taken together, these data identify important roles for IFN-γ signaling and Stat1-independent IFN-γ signaling in regulating CD8+ T cell-mediated acute lung injury. This is the first study to demonstrate an anti-inflammatory effect of Stat1 on CD8+ T cell-mediated lung immunopathology without the complication of differences in viral load. PMID:25617378

CD4/CD8/Dendritic cell complexes in the spleen: CD8+ T cells can directly bind CD4+ T cells and modulate their response

PubMed Central

Barinov, Aleksandr; Galgano, Alessia; Krenn, Gerald; Tanchot, Corinne; Vasseur, Florence

2017-01-01

CD4+ T cell help to CD8+ T cell responses requires that CD4+ and CD8+ T cells interact with the same antigen presenting dendritic cell (Ag+DC), but it remains controversial whether helper signals are delivered indirectly through a licensed DC and/or involve direct CD4+/CD8+ T cell contacts and/or the formation of ternary complexes. We here describe the first in vivo imaging of the intact spleen, aiming to evaluate the first interactions between antigen-specific CD4+, CD8+ T cells and Ag+DCs. We show that in contrast to CD4+ T cells which form transient contacts with Ag+DC, CD8+ T cells form immediate stable contacts and activate the Ag+DC, acquire fragments of the DC membranes by trogocytosis, leading to their acquisition of some of the DC properties. They express MHC class II, and become able to present the specific Marilyn peptide to naïve Marilyn CD4+ T cells, inducing their extensive division. In vivo, these CD8+ T cells form direct stable contacts with motile naïve CD4+ T cells, recruiting them to Ag+DC binding and to the formation of ternary complexes, where CD4+ and CD8+ T cells interact with the DC and with one another. The presence of CD8+ T cells during in vivo immune responses leads to the early activation and up-regulation of multiple functions by CD4+ T lymphocytes. Thus, while CD4+ T cell help is important to CD8+ T cell responses, CD8+ T cells can interact directly with naïve CD4+ T cells impacting their recruitment and differentiation. PMID:28686740

Tumor-associated antigen human chorionic gonadotropin beta contains numerous antigenic determinants recognized by in vitro-induced CD8+ and CD4+ T lymphocytes.

PubMed

Dangles, Virginie; Halberstam, Ilan; Scardino, Antonio; Choppin, Jeannine; Wertheimer, Mireille; Richon, Sophie; Quelvennec, Erwann; Moirand, Romain; Guillet, Jean-Gérard; Kosmatopoulos, Kostas; Bellet, Dominique; Zeliszewski, Dominique

2002-02-01

The beta subunit of human chorionic gonadotropin (hCG beta) is markedly overexpressed by neoplastic cells of differing histological origin including those present in colon, breast, prostate and bladder tumors. We have previously shown that some patients with hCG beta-producing urothelial tumors have circulating T cells that proliferate in response to hCG beta. To make a comprehensive study of hCG beta as a potential target for cancer immunotherapy, we investigated whether hCG beta peptides could induce CD4+ or CD8+ T-cell responses in vitro. By stimulating peripheral blood mononuclear cells (PBMCs) from three donors with mixtures of overlapping 16-mer synthetic peptides analogous to portions of either the hCG beta 20-71 or the hCG beta 102-129 region, we established six CD4+ T-cell lines that proliferated specifically in response to five distinct determinants located within these two hCG beta regions. Three antigenic determinants (hCG beta 52-67, 106-121 and 114-125) were presented by HLA-DR molecules, while the two other antigenic determinants (hCG beta 48-63 and 56-67) were presented by HLA-DQ molecules. Interestingly, one T-cell line specific for peptide hCG beta 106-121 recognized hCG beta peptides comprising, at position 117, either an alanine or an aspartic acid residue, with the latter residue being present within the protein expressed by some tumor cells. In addition, three other hCG beta-derived peptides that exhibited HLA-A*0201 binding ability were able to stimulate CD8+ cytotoxic T cells from two HLA-A*0201 donors. These three immunogenic peptides corresponded to regions hCG beta 40-48, hCG beta 44-52 and hCG beta 75-84. Our results indicate that the tumor-associated antigen hCG beta possesses numerous antigenic determinants liable to stimulate CD4+ and CD8+ T lymphocytes, and might thus be an effective target antigen for the immunotherapy of hCG beta-producing tumors.

Therapeutic targeting of regulatory T cells enhances tumor-specific CD8+ T cell responses in Epstein–Barr virus associated nasopharyngeal carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fogg, Mark; Murphy, John R.; Lorch, Jochen

Epstein–Barr virus (EBV) is associated with multiple malignancies including nasopharyngeal carcinoma (NPC). In nasopharynx cancer, CD8+ T cells specific for EBV Nuclear Antigen-1 (EBNA-1) and Latent Membrane Protein 2 (LMP2) are important components of anti-tumor immunity since both are consistently expressed in NPC. We have previously shown that EBNA-1-specific CD8+ T cell responses were suppressed in NPC patients compared to healthy controls. We now find that CD8+ T cell responses specific for LMP2 are also abnormal in NPC patients, and both EBNA-1- and LMP2-specific responses are suppressed by regulatory T cells (Treg). EBNA-1 and LMP2-specific CD8+ T cell responses, asmore » well as immune control of EBV-infected cells in vitro, could be restored by the depletion of Tregs and by use of a clinically approved drug targeting Tregs. Thus, in vivo modulation of Tregs may be an effective means of enhancing these anti-tumor immune responses in NPC patients. - Highlights: • Viral proteins are tumor antigens in Epstein–Barr virus associated Nasopharyngeal Carcinoma. • CD8+ T cell responses against EBV proteins EBNA-1 and LMP2 are suppressed in NPC patients. • T regulatory cells are responsible for suppressing EBV immunity in NPC patients. • Depletion of Tregs with Ontak can rescue EBV-specific CD8+ T cell responses in NPC patients. • This clinically approved drug may be effective for enhancing anti-tumor immunity in NPC patients.« less

The biological and practical significance of antigenic variability in protective T cell responses against Theileria parva.

PubMed

Morrison, W I

2007-08-19

The evolution of antigenically distinct pathogen strains that fail to cross-protect is well documented for pathogens controlled primarily by humoral immune responses. Unlike antibodies, which recognise native proteins, protective T cells can potentially recognise epitopes in a variety of proteins that are not necessarily displayed on the pathogen surface. Moreover, individual hosts of different MHC genotypes generally respond to different sets of epitopes. It is therefore less easy to envisage how strain restricted immunity can arise for pathogens controlled by T cell responses, particularly in antigenically complex parasites. Nevertheless, strain restricted immunity is clearly a feature of a number of parasitic infections, where immunity is known to be mediated by T cell responses. One such parasite is Theileria parva which induces potent CD8 T cell responses that play an important role in immunity. CD8 T cells specific for parasitized lymphoblasts exhibit strain specificity, which appears to correlate with the ability of parasite strains to cross-protect. Studies using recently identified T. parva antigens recognised by CD8 T cells have shown that the strain restricted nature of immunity is a consequence of the CD8 T cell response in individual animals being focused on a limited number of dominant polymorphic antigenic determinants. Responses in animals of different MHC genotypes are often directed to different parasite antigens, indicating that, at the host population level, a larger number of parasite proteins can serve as targets for the protective T cell response. Nevertheless, the finding that parasite strains show overlapping antigenic profiles, probably as a consequence of sexual recombination, suggests that induction of responses to an extended but limited set of antigens in individual animals may overcome the strain restricted nature of immunity.

Hyaluronic Acid-Modified Cationic Lipid-PLGA Hybrid Nanoparticles as a Nanovaccine Induce Robust Humoral and Cellular Immune Responses.

PubMed

Liu, Lanxia; Cao, Fengqiang; Liu, Xiaoxuan; Wang, Hai; Zhang, Chao; Sun, Hongfan; Wang, Chun; Leng, Xigang; Song, Cunxian; Kong, Deling; Ma, Guilei

2016-05-18

Here, we investigated the use of hyaluronic acid (HA)-decorated cationic lipid-poly(lactide-co-glycolide) acid (PLGA) hybrid nanoparticles (HA-DOTAP-PLGA NPs) as vaccine delivery vehicles, which were originally developed for the cytosolic delivery of genes. Our results demonstrated that after the NPs uptake by dendritic cells (DCs), some of the antigens that were encapsulated in HA-DOTAP-PLGA NPs escaped to the cytosolic compartment, and whereas some of the antigens remained in the endosomal/lysosomal compartment, where both MHC-I and MHC-II antigen presentation occurred. Moreover, HA-DOTAP-PLGA NPs led to the up-regulation of MHC, costimulatory molecules, and cytokines. In vivo experiments further revealed that more powerful immune responses were induced from mice immunized with HA-DOTAP-PLGA NPs when compared with cationic lipid-PLGA nanoparticles and free ovalbumin (OVA); the responses included antigen-specific CD4(+) and CD8(+) T-cell responses, the production of antigen-specific IgG antibodies and the generation of memory CD4(+) and CD8(+) T cells. Overall, these data demonstrate the high potential of HA-DOTAP-PLGA NPs for use as vaccine delivery vehicles to elevate cellular and humoral immune responses.

Effector CD8+ T cell IFN-γ production and cytotoxicity are enhanced by mild hyperthermia

PubMed Central

Mace, Thomas A.; Zhong, Lingwen; Kokolus, Kathleen M.; Repasky, Elizabeth A.

2012-01-01

Purpose Clinical trials combining hyperthermia with radiation and/or chemotherapy for cancer treatment have resulted in improved overall survival and control of local recurrences. The contribution of thermally enhanced anti-immune function in these effects is of considerable interest, but not understood; studies on the fundamental effects of elevated temperature on immune effector cells are needed. The goal of this study is to investigate the potential of mild hyperthermia to impact tumor antigen-specific (Ag) effector CD8+ T cell functions. Method Pmel-1 Ag-specific CD8+ T cells were exposed to mild hyperthermia and tested for changes in IFN-γ production and cytotoxicity. Additionally, overall plasma membrane organization and the phosphorylation of signaling proteins were also investigated following heat treatment. Results Exposing effector Pmel-1 specific CD8+ T cells to mild hyperthermia (39.5°C) resulted in significantly enhanced Ag-specific IFN-γ production and tumor target cell killing compared to that seen using lower temperatures (33 and 37°C). Further, inhibition of protein synthesis during hyperthermia did not reduce subsequent Ag-induced IFN-γ production by CD8+ T cells. Correlated with these effects, we observed a distinct clustering of GM1+ lipid microdomains at the plasma membrane and enhanced phosphorylation of LAT and PKCθ which may be related to an observed enhancement of Ag-specific effector CD8+ T cell IFN-γ gene transcription following mild hyperthermia. However, mitogen–mediated production of IFN-γ, which bypasses T cell receptor activation with antigen, was not enhanced. Conclusions Antigen-dependent effector T cell activity is enhanced following mild hyperthermia. These effects could potentially occur in patients being treated with thermal therapies. These data also provide support for the use of thermal therapy as an adjuvant for immunotherapies to improve CD8+ effector cell function. PMID:22235780

Identification of a dendritic cell receptor that couples sensing of necrosis to immunity

PubMed Central

Sancho, David; Joffre, Olivier P.; Keller, Anna M.; Rogers, Neil C.; Martinez, Dolores; Hernanz-Falcón, Patricia; Rosewell, Ian; Reis e Sousa, Caetano

2009-01-01

Injury or impaired clearance of apoptotic cells leads to the pathological accumulation of necrotic corpses, which induce an inflammatory response that initiates tissue repair1. In addition, antigens present within necrotic cells can sometimes provoke a specific immune response2-4 and it has been argued that necrosis could explain adaptive immunity in seemingly infection-free situations, such as after allograft transplantation or in spontaneous and therapy-induced tumour rejection5, 6. In the mouse, the CD8α+ subset of dendritic cells (DC) phagocytoses dead cell remnants and crossprimes CD8+ T cells against cell-associated antigens7. Here, we show that CD8α+ DC utilise CLEC9A (DNGR-1), a recently-characterised C-type lectin8-10, to recognise a preformed signal that is exposed on necrotic cells. Loss or blockade of CLEC9A does not impair uptake of necrotic cell material by CD8α+ DC but specifically reduces crosspresentation of dead cell-associated antigens in vitro and decreases the immunogenicity of necrotic cells in vivo. The function of CLEC9A requires a key tyrosine residue within its intracellular tail that allows recruitment and activation of the tyrosine kinase Syk, which is also essential for crosspresentation of dead cell-associated antigens. Thus, CLEC9A functions as a Syk-coupled C-type lectin receptor to mediate sensing of necrosis by the principal DC subset involved in regulating crosspriming to cell-associated antigens. PMID:19219027

Cluster Intradermal DNA Vaccination Rapidly Induces E7-specific CD8+ T Cell Immune Responses Leading to Therapeutic Antitumor Effects

PubMed Central

Peng, Shiwen; Trimble, Cornelia; Alvarez, Ronald D.; Huh, Warner K.; Lin, Zhenhua; Monie, Archana; Hung, Chien-Fu; Wu, T.-C.

2010-01-01

Intradermal administration of DNA vaccines via a gene gun represents a feasible strategy to deliver DNA directly into the professional antigen-presenting cells (APCs) in the skin. This helps to facilitate the enhancement of DNA vaccine potency via strategies that modify the properties of APCs. We have previously demonstrated that DNA vaccines encoding human papillomavirus type 16 (HPV-16) E7 antigen linked to calreticulin (CRT) are capable of enhancing the E7-specific CD8+ T cell immune responses and antitumor effects against E7-expressing tumors. It has also been shown that cluster (short-interval) DNA vaccination regimen generates potent immune responses in a minimal timeframe. Thus, in the current study we hypothesize that the cluster intradermal CRT/E7 DNA vaccination will generate significant antigen-specific CD8+ T cell infiltrates in E7-expressing tumors in tumor-bearing mice, leading to an increase in apoptotic tumor cell death. We found that cluster intradermal CRT/E7 DNA vaccination is capable of rapidly generating a significant number of E7-specific CD8+ T cells, resulting in significant therapeutic antitumor effects in vaccinated mice. We also observed that cluster intradermal CRT/E7 DNA vaccination in the presence of tumor generates significantly higher E7-specific CD8+ T cell immune responses in the systemic circulation as well as in the tumors. In addition, this vaccination regimen also led to significantly lower levels of CD4+Foxp3+ T regulatory cells and myeloid suppressor cells compared to vaccination with CRT DNA in peripheral blood and in tumor infiltrating lymphocytes, resulting in an increase in apoptotic tumor cell death. Thus, our study has significant potential for future clinical translation. PMID:18401437

Brucella β 1,2 Cyclic Glucan Is an Activator of Human and Mouse Dendritic Cells

PubMed Central

Martirosyan, Anna; Pérez-Gutierrez, Camino; Banchereau, Romain; Dutartre, Hélène; Lecine, Patrick; Dullaers, Melissa; Mello, Marielle; Pinto Salcedo, Suzana; Muller, Alexandre; Leserman, Lee; Levy, Yves; Zurawski, Gerard; Zurawski, Sandy; Moreno, Edgardo; Moriyón, Ignacio; Klechevsky, Eynav; Banchereau, Jacques; Oh, SangKon; Gorvel, Jean-Pierre

2012-01-01

Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella β 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella β 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8+ T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4+ and CD8+ T cell responses including cross-presentation by different human DC subsets. Brucella β 1,2 cyclic glucans increased the memory CD4+ T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis. Thus cyclic glucans represent a new class of adjuvants, which might contribute to the development of effective antimicrobial therapies. PMID:23166489

Comprehensive Analysis of Cytomegalovirus pp65 Antigen-Specific CD8+ T Cell Responses According to Human Leukocyte Antigen Class I Allotypes and Intraindividual Dominance.

PubMed

Hyun, Seung-Joo; Sohn, Hyun-Jung; Lee, Hyun-Joo; Lee, Seon-Duk; Kim, Sueon; Sohn, Dae-Hee; Hong, Cheol-Hwa; Choi, Haeyoun; Cho, Hyun-Il; Kim, Tai-Gyu

2017-01-01

To define whether individual human leukocyte antigen (HLA) class I allotypes are used preferentially in human cytomegalovirus (CMV)-specific cytotoxic T lymphocyte responses, CD8 + T cell responses restricted by up to six HLA class I allotypes in an individual were measured in parallel using K562-based artificial antigen-presenting cells expressing both CMV pp65 antigen and one of 32 HLA class I allotypes (7 HLA-A, 14 HLA-B, and 11 HLA-C) present in 50 healthy Korean donors. The CD8 + T cell responses to pp65 in the HLA-C allotypes were lower than responses to those in HLA-A and -B allotypes and there was no difference between the HLA-A and HLA-B loci. HLA-A*02:01, -B*07:02, and -C*08:01 showed the highest magnitude and frequency of immune responses to pp65 at each HLA class I locus. However, HLA-A*02:07, -B*59:01, -B*58:01, -B*15:11, -C*03:02, and -C*02:02 did not show any immune responses. Although each individual has up to six different HLA allotypes, 46% of the donors showed one allotype, 24% showed two allotypes, and 2% showed three allotypes that responded to pp65. Interestingly, the frequencies of HLA-A alleles were significantly correlated with the positivity of specific allotypes. Our results demonstrate that specific HLA class I allotypes are preferentially used in the CD8 + T cell immune response to pp65 and that a hierarchy among HLA class I allotypes is present in an individual.

Restoration of Viral Immunity in Immunodeficient Humans by the Adoptive Transfer of T Cell Clones

NASA Astrophysics Data System (ADS)

Riddell, Stanley R.; Watanabe, Kathe S.; Goodrich, James M.; Li, Cheng R.; Agha, Mounzer E.; Greenberg, Philip D.

1992-07-01

The adoptive transfer of antigen-specific T cells to establish immunity is an effective therapy for viral infections and tumors in animal models. The application of this approach to human disease would require the isolation and in vitro expansion of human antigen-specific T cells and evidence that such T cells persist and function in vivo after transfer. Cytomegalovirus-specific CD8^+ cytotoxic T cell (CTL) clones could be isolated from bone marrow donors, propagated in vitro, and adoptively transferred to immunodeficient bone marrow transplant recipients. No toxicity developed and the clones provided persistent reconstitution of CD8^+ cytomegalovirus-specific CTL responses.

Human CD8 T lymphocytes recognize Mycobacterium tuberculosis antigens presented by HLA-E during active tuberculosis and express type 2 cytokines.

PubMed

Caccamo, Nadia; Pietra, Gabriella; Sullivan, Lucy C; Brooks, Andrew G; Prezzemolo, Teresa; La Manna, Marco P; Di Liberto, Diana; Joosten, Simone A; van Meijgaarden, Krista E; Di Carlo, Paola; Titone, Lucina; Moretta, Lorenzo; Mingari, Maria C; Ottenhoff, Tom H M; Dieli, Francesco

2015-04-01

CD8 T cells contribute to protective immunity against Mycobacterium tuberculosis. In humans, M. tuberculosis reactive CD8 T cells typically recognize peptides associated to classical MHC class Ia molecules, but little information is available on CD8 T cells recognizing M. tuberculosis Ags presented by nonclassical MHC class Ib molecules. We show here that CD8 T cells from tuberculosis (TB) patients recognize HLA-E-binding M. tuberculosis peptides in a CD3/TCR αβ mediated and CD8-dependent manner, and represent an additional type of effector cells playing a role in immune response to M. tuberculosis during active infection. HLA-E-restricted recognition of M. tuberculosis peptides is detectable by a significant enhanced ex vivo frequency of tetramer-specific circulating CD8 T cells during active TB. These CD8 T cells produce type 2 cytokines upon antigenic in vitro stimulation, help B cells for Ab production, and mediate limited TRAIL-dependent cytolytic and microbicidal activity toward M. tuberculosis infected target cells. Our results, together with the finding that HLA-E/M. tuberculosis peptide specific CD8 T cells are detected in TB patients with or without HIV coinfection, suggest that this is a new human T-cell population that participates in immune response in TB. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Antibody-Independent Control of γ-Herpesvirus Latency via B Cell Induction of Anti-Viral T Cell Responses

PubMed Central

McClellan, Kelly B; Gangappa, Shivaprakash; Speck, Samuel H; Virgin, Herbert W.

2006-01-01

B cells can use antibody-dependent mechanisms to control latent viral infections. It is unknown whether this represents the sole function of B cells during chronic viral infection. We report here that hen egg lysozyme (HEL)-specific B cells can contribute to the control of murine γ-herpesvirus 68 (γHV68) latency without producing anti-viral antibody. HEL-specific B cells normalized defects in T cell numbers and proliferation observed in B cell−/− mice during the early phase of γHV68 latency. HEL-specific B cells also reversed defects in CD8 and CD4 T cell cytokine production observed in B cell−/− mice, generating CD8 and CD4 T cells necessary for control of latency. Furthermore, HEL-specific B cells were able to present virally encoded antigen to CD8 T cells. Therefore, B cells have antibody independent functions, including antigen presentation, that are important for control of γ-herpesvirus latency. Exploitation of this property of B cells may allow enhanced vaccine responses to chronic virus infection. PMID:16789842

The Activation and Inactivation of Mature CD4 T cells: A Case for Peripheral Self–Nonself Discrimination

PubMed Central

Bretscher, P A

2014-01-01

The establishment of central tolerance to most self-antigens results in a repertoire of mature peripheral lymphocytes specific for foreign and peripheral self-antigens. The framework that single, mature lymphocytes are inactivated by antigen, whereas their activation requires lymphocyte cooperation, accounts for diverse observations and incorporates a mechanism of peripheral tolerance. This framework accounts for the generalizations that the sustained activation by antigen of most B cells and CD8 T cells requires CD4 T helper cells; in the absence of CD4 T cells, antigen can inactivate these B cells and CD8 T cells. In this sense, CD4 T cells are the guardians of the fate of most B cells and CD8 T cells when they encounter antigen. I argue here that the single-lymphocyte/multiple-lymphocyte framework for the inactivation/activation of lymphocytes also applies to CD4 T cells. I consider within this framework a model for the activation/inactivation of CD4 T cells that is consistent with the large majority of contemporary observations, including significant clinical observations. I outline the grounds why I feel this model is more plausible than the contemporary and predominant pathogen-associated molecular pattern (PAMP) and Danger Models for CD4 T cell activation. These models are based upon what I consider the radical premise that self–nonself discrimination does not exist at the level of mature CD4 T cells. I explain why I feel this feature renders the PAMP and Danger Models somewhat implausible. The model I propose, in contrast, is conservative in that it embodies such a process of self–nonself discrimination. PMID:24684567

Functional characterization of BK virus-specific CD4+ T cells with cytotoxic potential in seropositive adults.

PubMed

Zhou, Wendi; Sharma, Madeva; Martinez, Joy; Srivastava, Tumul; Diamond, Don J; Knowles, Wendy; Lacey, Simon F

2007-09-01

BK polyomavirus (BKV) reactivation is associated with a failure of T cell immunity in kidney transplant patients, and may lead to BKV-associated nephropathy (BKVN) and loss of the allograft. BKV reactivation in hematopoietic stem cell transplant recipients is associated with hemorrhagic cystitis. We have investigated T cell responses to overlapping peptide mixtures corresponding to the whole BKV major T antigen (TAg) and major capsid protein (VP1) in peripheral blood mononuclear cell samples from a cohort of healthy BKV-seropositive subjects. The majority of these individuals possessed populations of both CD8(+) and CD4(+) T cells specific for these BKV antigens. After expansion in culture, the majority of the BKV-specific CD4(+) T cells, in addition to expressing CD40L (CD154), secreted both interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, contained both granzyme A and granzyme B, and degranulated/mobilized CD107 in response to antigen-specific stimulation. These T cells thus represent potentially functional BKV-specific cytotoxic CD4(+) T lymphocytes. Secretion of both TNF-alpha and IFN-gamma by CD154(+)CD4(+) T cells on BKV-specific stimulation was associated with higher levels of granzyme B and a higher proportion of degranulating cells compared with CD154(+)CD4(+) T cells producing only IFN-gamma or neither cytokine. These healthy subjects also harbored populations of functional CD8(+) T cells specific for one or more of three newly defined HLA-A 02-restricted cytotoxic T lymphocyte epitopes within the BKV TAg as well as two HLA-A 02-restricted epitopes within the BKV VP1 we have previously described. The BKV-specific CD4(+) T cells characterized in this study may play a part in maintaining persistent memory T cell responses to the virus and thus contribute to the immune control of BKV in healthy individuals.

T Cells with Low Avidity for a Tissue-Restricted Antigen Routinely Evade Central and Peripheral Tolerance and Cause Autoimmunity

PubMed Central

Zehn, Dietmar; Bevan, Michael J.

2009-01-01

Summary T cells causing autoimmunity must escape tolerance. We observed that CD8+ T cells with high avidity for an antigen expressed in the pancreas, kidney, and thymic medulla were efficiently removed from a polyclonal repertoire by central and peripheral tolerance mechanisms. However, both mechanisms spared low-avidity T cells from elimination. Neither the introduction of activated, self-antigen-specific CD4+ helper T cells nor a global inflammatory stimulus were sufficient to activate the low-avidity CD8+ T cells and did not break tolerance. In contrast, challenge with a recombinant bacterium expressing the self antigen primed the low-avidity T cells, and the animals rapidly developed autoimmune diabetes. We suggest that whereas thymic and peripheral tolerance mechanisms remove cells that can be primed by endogenous amounts of self antigen, they do not guard against tissue destruction by low-avidity effector T cells, which have been primed by higher amounts of self antigen or by crossreactive antigens. PMID:16879996

Plasmacytoid Dendritic Cells Require Direct Infection To Sustain the Pulmonary Influenza A Virus-Specific CD8 T Cell Response.

PubMed

Hemann, Emily A; Sjaastad, Louisa E; Langlois, Ryan A; Legge, Kevin L

2015-12-30

Following influenza A virus (IAV) infection, development of a robust IAV-specific CD8 T cell response is required for clearance of primary infection and enhances memory protection. Following IAV infection, plasmacytoid dendritic cells (pDC) or CD8α(+) DC regulate pulmonary effector CD8 T cell responses within the lung. Without this DC-T cell interaction, insufficient effector CD8 T cells are maintained in the lungs, leading to enhanced morbidity and mortality. Previous studies have demonstrated that pDC are capable of classical presentation or cross-presentation of IAV antigens and could potentially regulate IAV-specific CD8 T cell responses through either mechanism. Our results demonstrate that pDC from the lungs of donor mice infected with an IAV that is not able to replicate in hematopoietic cells (142t-IAV), unlike donor pDC isolated from the lungs of control infected mice, are not able to rescue the host IAV-specific CD8 T cell response from apoptosis. This indicates that pDC must utilize the direct presentation pathway for this rescue. This inability of pDC from 142t-IAV donors to rescue the IAV-specific CD8 T cell response is not due to differences in the overall ability of 142t-IAV to replicate within the lungs or generate defective viral genomes or to differences in levels of costimulatory molecules required for this interaction. We further demonstrate that bypassing the antigen presentation pathway by coating the 142t-IAV pDC with IAV peptide epitopes restores their ability to rescue the IAV-specific CD8 T cell response. IAV continues to be a global health burden, infecting 5 to 20% of the global population annually. Continued investigation into the mechanisms that mediate protective immune responses against IAV is important to improving current vaccination and antiviral strategies antagonistic toward IAV. Our findings presented herein demonstrate a key requirement for pDC promotion of effector CD8 T cell survival: that rather than utilizing cross-presentation, pDC must be infected and utilize the endogenous pathway for presentation of antigens to CD8 T cells during in vivo IAV infections. This suggests that targeting presentation via the endogenous pathway in pDC could be important for the development of unique antiviral cellular therapies. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Conventional CD4+ T cells present bacterial antigens to induce cytotoxic and memory CD8+ T cell responses.

PubMed

Cruz-Adalia, Aránzazu; Ramirez-Santiago, Guillermo; Osuna-Pérez, Jesús; Torres-Torresano, Mónica; Zorita, Virgina; Martínez-Riaño, Ana; Boccasavia, Viola; Borroto, Aldo; Martínez Del Hoyo, Gloria; González-Granado, José María; Alarcón, Balbino; Sánchez-Madrid, Francisco; Veiga, Esteban

2017-11-17

Bacterial phagocytosis and antigen cross-presentation to activate CD8 + T cells are principal functions of professional antigen presenting cells. However, conventional CD4 + T cells also capture and kill bacteria from infected dendritic cells in a process termed transphagocytosis (also known as transinfection). Here, we show that transphagocytic T cells present bacterial antigens to naive CD8 + T cells, which proliferate and become cytotoxic in response. CD4 + T-cell-mediated antigen presentation also occurs in vivo in the course of infection, and induces the generation of central memory CD8 + T cells with low PD-1 expression. Moreover, transphagocytic CD4 + T cells induce protective anti-tumour immune responses by priming CD8 + T cells, highlighting the potential of CD4 + T cells as a tool for cancer immunotherapy.

Vaccination and the TAP-independent antigen processing pathways.

PubMed

López, Daniel; Lorente, Elena; Barriga, Alejandro; Johnstone, Carolina; Mir, Carmen

2013-09-01

The cytotoxic CD8(+) T lymphocyte-mediated cellular response is important for the elimination of virus-infected cells and requires the prior recognition of short viral peptide antigens previously translocated to the endoplasmic reticulum by the transporter associated with antigen processing (TAP). However, individuals with nonfunctional TAP complexes or infected cells with TAP molecules blocked by specific viral proteins, such as the cowpoxvirus, a component of the first source of early empirical vaccination against smallpox, are still able to present several HLA class I ligands generated by the TAP-independent antigen processing pathways to specific cytotoxic CD8(+) T lymphocytes. Currently, bioterrorism and emerging infectious diseases have renewed interest in poxviruses. Recent works that have identified HLA class I ligands and epitopes in virus-infected TAP-deficient cells have implications for the study of both the effectiveness of early empirical vaccination and the analysis of HLA class I antigen processing in TAP-deficient subjects.

Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection

PubMed Central

Nunes-Alves, Cláudio; Booty, Matthew G.; Carpenter, Stephen M.; Rothchild, Alissa C.; Martin, Constance J.; Desjardins, Danielle; Steblenko, Katherine; Kløverpris, Henrik N.; Madansein, Rajhmun; Ramsuran, Duran; Leslie, Alasdair; Correia-Neves, Margarida; Behar, Samuel M.

2015-01-01

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRβ bias. Using a retrogenic model of TB10.44-11-specific CD8+ T cells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity. PMID:25945999

Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection.

PubMed

Nunes-Alves, Cláudio; Booty, Matthew G; Carpenter, Stephen M; Rothchild, Alissa C; Martin, Constance J; Desjardins, Danielle; Steblenko, Katherine; Kløverpris, Henrik N; Madansein, Rajhmun; Ramsuran, Duran; Leslie, Alasdair; Correia-Neves, Margarida; Behar, Samuel M

2015-05-01

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRβ bias. Using a retro genic model of TB10.44-11-specific CD8+ Tcells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.

CD8+ T Cells Orchestrate pDC-XCR1+ Dendritic Cell Spatial and Functional Cooperativity to Optimize Priming.

PubMed

Brewitz, Anna; Eickhoff, Sarah; Dähling, Sabrina; Quast, Thomas; Bedoui, Sammy; Kroczek, Richard A; Kurts, Christian; Garbi, Natalio; Barchet, Winfried; Iannacone, Matteo; Klauschen, Frederick; Kolanus, Waldemar; Kaisho, Tsuneyasu; Colonna, Marco; Germain, Ronald N; Kastenmüller, Wolfgang

2017-02-21

Adaptive cellular immunity is initiated by antigen-specific interactions between T lymphocytes and dendritic cells (DCs). Plasmacytoid DCs (pDCs) support antiviral immunity by linking innate and adaptive immune responses. Here we examined pDC spatiotemporal dynamics during viral infection to uncover when, where, and how they exert their functions. We found that pDCs accumulated at sites of CD8 + T cell antigen-driven activation in a CCR5-dependent fashion. Furthermore, activated CD8 + T cells orchestrated the local recruitment of lymph node-resident XCR1 chemokine receptor-expressing DCs via secretion of the XCL1 chemokine. Functionally, this CD8 + T cell-mediated reorganization of the local DC network allowed for the interaction and cooperation of pDCs and XCR1 + DCs, thereby optimizing XCR1 + DC maturation and cross-presentation. These data support a model in which CD8 + T cells upon activation create their own optimal priming microenvironment by recruiting additional DC subsets to the site of initial antigen recognition. Published by Elsevier Inc.

Semi-allogeneic dendritic cells can induce antigen-specific T-cell activation, which is not enhanced by concurrent alloreactivity.

PubMed

Wells, James W; Cowled, Chris J; Darling, David; Guinn, Barbara-Ann; Farzaneh, Farzin; Noble, Alistair; Galea-Lauri, Joanna

2007-12-01

Alloreactive T-cell responses are known to result in the production of large amounts of proinflammatory cytokines capable of activating and maturing dendritic cells (DC). However, it is unclear whether these allogeneic responses could also act as an adjuvant for concurrent antigen-specific responses. To examine effects of simultaneous alloreactive and antigen-specific T-cell responses induced by semi-allogeneic DC. Semi-allogeneic DC were generated from the F(1) progeny of inbred strains of mice (C57BL/6 and C3H, or C57BL/6 and DBA). We directly primed antigen-specific CD8(+) and CD4(+) T-cells from OT-I and OT-II mice, respectively, in the absence of allogeneic responses, in vitro, and in the presence or absence of alloreactivity in vivo. In vitro, semi-allogeneic DC cross-presented ovalbumin (OVA) to naïve CD8(+) OT-I transgenic T-cells, primed naïve CD4(+) OT-II transgenic T-cells and could stimulate strong alloreactive T-cell proliferation in a primary mixed lymphocyte reaction (MLR). In vivo, semi-allogeneic DC migrated efficiently to regional lymph nodes but did not survive there as long as autologous DC. In addition, they were not able to induce cytotoxic T-lymphocyte (CTL) activity to a target peptide, and only weakly stimulated adoptively transferred OT-II cells. The CD4(+) response was unchanged in allo-tolerized mice, indicating that alloreactive T-cell responses could not provide help for concurrently activated antigen-specific responses. In an EL4 tumour-treatment model, vaccination with semi-allogeneic DC/EL4 fusion hybrids, but not allogeneic DC/EL4 hybrids, significantly increased mouse survival. Expression of self-Major histocompatibility complex (MHC) by semi-allogeneic DC can cause the induction of antigen-specific immunity, however, concurrently activated allogeneic bystander responses do not provide helper or adjuvant effects.

Identification of a dendritic cell receptor that couples sensing of necrosis to immunity.

PubMed

Sancho, David; Joffre, Olivier P; Keller, Anna M; Rogers, Neil C; Martínez, Dolores; Hernanz-Falcón, Patricia; Rosewell, Ian; Reis e Sousa, Caetano

2009-04-16

Injury or impaired clearance of apoptotic cells leads to the pathological accumulation of necrotic corpses, which induce an inflammatory response that initiates tissue repair. In addition, antigens present in necrotic cells can sometimes provoke a specific immune response and it has been argued that necrosis could explain adaptive immunity in seemingly infection-free situations, such as after allograft transplantation or in spontaneous and therapy-induced tumour rejection. In the mouse, the CD8alpha+ subset of dendritic cells phagocytoses dead cell remnants and cross-primes CD8+ T cells against cell-associated antigens. Here we show that CD8alpha+ dendritic cells use CLEC9A (also known as DNGR-1), a recently-characterized C-type lectin, to recognize a preformed signal that is exposed on necrotic cells. Loss or blockade of CLEC9A does not impair the uptake of necrotic cell material by CD8+ dendritic cells, but specifically reduces cross-presentation of dead-cell-associated antigens in vitro and decreases the immunogenicity of necrotic cells in vivo. The function of CLEC9A requires a key tyrosine residue in its intracellular tail that allows the recruitment and activation of the tyrosine kinase SYK, which is also essential for cross-presentation of dead-cell-associated antigens. Thus, CLEC9A functions as a SYK-coupled C-type lectin receptor to mediate sensing of necrosis by the principal dendritic-cell subset involved in regulating cross-priming to cell-associated antigens.

HLA-B*35-Restricted CD8+-T-Cell Epitope in Mycobacterium tuberculosis Rv2903c

PubMed Central

Klein, Michèl R.; Hammond, Abdulrahman S.; Smith, Steve M.; Jaye, Assan; Lukey, Pauline T.; McAdam, Keith P. W. J.

2002-01-01

Few human CD8+ T-cell epitopes in mycobacterial antigens have been described to date. Here we have identified a novel HLA-B*35-restricted CD8+ T-cell epitope in Mycobacterium tuberculosis Rv2903c based on a reverse immunogenetics approach. Peptide-specific CD8 T cells were able to kill M. tuberculosis-infected macrophages and produce gamma interferon and tumor necrosis factor alpha. PMID:11796635

Epitope diversification driven by non-tumor epitope-specific Th1 and Th17 mediates potent antitumor reactivity.

PubMed

Ichikawa, Kosuke; Kagamu, Hiroshi; Koyama, Kenichi; Miyabayashi, Takao; Koshio, Jun; Miura, Satoru; Watanabe, Satoshi; Yoshizawa, Hirohisa; Narita, Ichiei

2012-09-21

MHC class I-restricted peptide-based vaccination therapies have been conducted to treat cancer patients, because CD8⁺ CTL can efficiently induce apoptosis of tumor cells in an MHC class I-restricted epitope-specific manner. Interestingly, clinical responders are known to demonstrate reactivity to epitopes other than those used for vaccination; however, the mechanism underlying how antitumor T cells with diverse specificity are induced is unclear. In this study, we demonstrated that dendritic cells (DCs) that engulfed apoptotic tumor cells in the presence of non-tumor MHC class II-restricted epitope peptides, OVA(323-339), efficiently presented tumor-associated antigens upon effector-dominant CD4⁺ T cell balance against regulatory T cells (Treg) for the OVA(323-339) epitope. Th1 and Th17 induced tumor-associated antigens presentation of DC, while Th2 ameliorated tumor-antigen presentation for CD8⁺ T cells. Blocking experiments with anti-IL-23p19 antibody and anti-IL-23 receptor indicated that an autocrine mechanism of IL-23 likely mediated the diverted tumor-associated antigens presentation of DC. Tumor-associated antigens presentation of DC induced by OVA(323-339) epitope-specific CD4⁺ T cells resulted in facilitated antitumor immunity in both priming and effector phase in vivo. Notably, this immunotherapy did not require pretreatment to reduce Treg induced by tumor. This strategy may have clinical implications for designing effective antitumor immunotherapies. Copyright © 2012 Elsevier Ltd. All rights reserved.

Urushiol (poison ivy)-triggered suppressor T cell clone generated from peripheral blood.

PubMed Central

Kalish, R S; Morimoto, C

1988-01-01

Allergic contact dermatitis to Toxicodendron radicans (poison ivy) is mediated by the hapten urushiol. An urushiol-specific, interleukin 2 (IL-2)-dependent T cell clone (RLB9-7) was generated from the peripheral blood of a patient with a history of allergic contact dermatitis to T. radicans. This clone proliferated specifically to both leaf extract and pure urushiol. Although the clone had the phenotype CD3+CD4+CD8+, proliferation to antigen was blocked by anti-CD8 and anti-HLA-A, B, C, but not by anti-CD4, suggesting that CD4 was not functionally associated with the T cell receptor. Furthermore, studies with antigen-presenting cells from MHC-typed donors indicated that the clone was MHC class 1 restricted. RLB9-7 was WT31 positive, indicating it bears the alpha beta T cell receptor. The clone lacked significant natural killer cell activity and produced only low levels of IL-2 or gamma-interferon upon antigen stimulation. Addition of RLB9-7 to autologous peripheral blood mononuclear cells in the presence of urushiol inhibited the pokeweed mitogen-driven IgG synthesis. This suppression was resistant to irradiation (2,000 rad) and was not seen when RLB9-7 was added to allogeneic cells, even in the presence of irradiated autologous antigen-presenting cells, suggesting that suppression was MHC restricted and not mediated by nonspecific soluble factors. However, RLB9-7 cells in the presence of urushiol inhibited the synthesis of tetanus toxoid-specific IgG by autologous lymphocytes, indicating that the suppression, although triggered specifically by urushiol, was nonspecific. PMID:2458387

Generation of dendritic cell-based vaccine using high hydrostatic pressure for non-small cell lung cancer immunotherapy

PubMed Central

Hradilova, Nada; Sadilkova, Lenka; Palata, Ondrej; Mysikova, Dagmar; Mrazkova, Hana; Lischke, Robert; Spisek, Radek; Adkins, Irena

2017-01-01

High hydrostatic pressure (HHP) induces immunogenic death of tumor cells which confer protective anti-tumor immunity in vivo. Moreover, DC pulsed with HHP-treated tumor cells induced therapeutic effect in mouse cancer model. In this study, we tested the immunogenicity, stability and T cell stimulatory activity of human monocyte-derived dendritic cell (DC)-based HHP lung cancer vaccine generated in GMP compliant serum free medium using HHP 250 MPa. DC pulsed with HHP-killed lung cancer cells and poly(I:C) enhanced DC maturation, chemotactic migration and production of pro-inflammatory cytokines after 24h. Moreover, DC-based HHP lung cancer vaccine showed functional plasticity after transfer into serum-containing media and stimulation with LPS or CD40L after additional 24h. LPS and CD40L stimulation further differentially enhanced the expression of costimulatory molecules and production of IL-12p70. DC-based HHP lung cancer vaccine decreased the number of CD4+CD25+Foxp3+ T regulatory cells and stimulated IFN-γ-producing tumor antigen-specific CD4+ and CD8+ T cells from non-small cell lung cancer (NSCLC) patients. Tumor antigen specific CD8+ and CD4+ T cell responses were detected in NSCLC patient’s against a selected tumor antigens expressed by lung cancer cell lines used for the vaccine generation. We also showed for the first time that protein antigen from HHP-killed lung cancer cells is processed and presented by DC to CD8+ T cells. Our results represent important preclinical data for ongoing NSCLC Phase I/II clinical trial using DC-based active cellular immunotherapy (DCVAC/LuCa) in combination with chemotherapy and immune enhancers. PMID:28187172

Poly-functional and long-lasting anticancer immune response elicited by a safe attenuated Pseudomonas aeruginosa vector for antigens delivery

PubMed Central

Chauchet, Xavier; Hannani, Dalil; Djebali, Sophia; Laurin, David; Polack, Benoit; Marvel, Jacqueline; Buffat, Laurent; Toussaint, Bertrand; Le Gouëllec, Audrey

2016-01-01

Live-attenuated bacterial vectors for antigens delivery have aroused growing interest in the field of cancer immunotherapy. Their potency to stimulate innate immunity and to promote intracellular antigen delivery into antigen-presenting cells could be exploited to elicit a strong and specific cellular immune response against tumor cells. We previously described genetically-modified and attenuated Pseudomonas aeruginosa vectors able to deliver in vivo protein antigens into antigen-presenting cells, through Type 3 secretion system of the bacteria. Using this approach, we managed to protect immunized mice against aggressive B16 melanoma development in both a prophylactic and therapeutic setting. In this study, we further investigated the antigen-specific CD8+ T cell response, in terms of phenotypic and functional aspects, obtained after immunizations with a killed but metabolically active P. aeruginosa attenuated vector. We demonstrated that P. aeruginosa vaccine induces a highly functional pool of antigen-specific CD8+ T cell able to infiltrate the tumor. Furthermore, multiple immunizations allowed the development of a long-lasting immune response, represented by a pool of predominantly effector memory cells which protected mice against late tumor challenge. Overall, killed but metabolically active P. aeruginosa vector is a safe and promising approach for active and specific antitumor immunotherapy. PMID:28035332

Non-equivalent antigen presenting capabilities of dendritic cells and macrophages in generating brain-infiltrating CD8 + T cell responses.

PubMed

Malo, Courtney S; Huggins, Matthew A; Goddery, Emma N; Tolcher, Heather M A; Renner, Danielle N; Jin, Fang; Hansen, Michael J; Pease, Larry R; Pavelko, Kevin D; Johnson, Aaron J

2018-02-12

The contribution of antigen-presenting cell (APC) types in generating CD8 + T cell responses in the central nervous system (CNS) is not fully defined, limiting the development of vaccines and understanding of immune-mediated neuropathology. Here, we generate a transgenic mouse that enables cell-specific deletion of the H-2Kb MHC class I molecule. By deleting H-2K b on dendritic cells and macrophages, we compare the effect of each APC in three distinct models of neuroinflammation: picornavirus infection, experimental cerebral malaria, and a syngeneic glioma. Dendritic cells and macrophages both activate CD8 + T cell responses in response to these CNS immunological challenges. However, the extent to which each of these APCs contributes to CD8 + T cell priming varies. These findings reveal distinct functions for dendritic cells and macrophages in generating CD8 + T cell responses to neurological disease.

The generation of CD8+ T-cell population specific for vaccinia virus epitope involved in the antiviral protection against ectromelia virus challenge.

PubMed

Gierynska, Malgorzata; Szulc-Dabrowska, Lidia; Dzieciatkowski, Tomasz; Golke, Anna; Schollenberger, Ada

2015-12-01

Eradication of smallpox has led to cessation of vaccination programs. This has rendered the human population increasingly susceptible not only to variola virus infection but also to infections with other representatives of Poxviridae family that cause zoonotic variola-like diseases. Thus, new approaches for designing improved vaccine against smallpox are required. Discovering that orthopoxviruses, e.g. variola virus, vaccinia virus, ectromelia virus, share common immunodominant antigen, may result in the development of such a vaccine. In our study, the generation of antigen-specific CD8(+) T cells in mice during the acute and memory phase of the immune response was induced using the vaccinia virus immunodominant TSYKFESV epitope and CpG oligodeoxynucleotides as adjuvants. The role of the generated TSYKFESV-specific CD8(+) T cells was evaluated in mice during ectromelia virus infection using systemic and mucosal model. Moreover, the involvement of dendritic cells subsets in the adaptive immune response stimulation was assessed. Our results indicate that the TSYKFESV epitope/TLR9 agonist approach, delivered systemically or mucosally, generated strong CD8(+) T-cell response when measured 10 days after immunization. Furthermore, the TSYKFESV-specific cell population remained functionally active 2 months post-immunization, and gave cross-protection in virally challenged mice, even though the numbers of detectable antigen-specific T cells decreased. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Targeted suppression of autoreactive CD8+ T-cell activation using blocking anti-CD8 antibodies.

PubMed

Clement, Mathew; Pearson, James A; Gras, Stephanie; van den Berg, Hugo A; Lissina, Anya; Llewellyn-Lacey, Sian; Willis, Mark D; Dockree, Tamsin; McLaren, James E; Ekeruche-Makinde, Julia; Gostick, Emma; Robertson, Neil P; Rossjohn, Jamie; Burrows, Scott R; Price, David A; Wong, F Susan; Peakman, Mark; Skowera, Ania; Wooldridge, Linda

2016-10-17

CD8 + T-cells play a role in the pathogenesis of autoimmune diseases such as multiple sclerosis and type 1 diabetes. However, drugs that target the entire CD8 + T-cell population are not desirable because the associated lack of specificity can lead to unwanted consequences, most notably an enhanced susceptibility to infection. Here, we show that autoreactive CD8 + T-cells are highly dependent on CD8 for ligand-induced activation via the T-cell receptor (TCR). In contrast, pathogen-specific CD8 + T-cells are relatively CD8-independent. These generic differences relate to an intrinsic dichotomy that segregates self-derived and exogenous antigen-specific TCRs according to the monomeric interaction affinity with cognate peptide-major histocompatibility complex class I (pMHCI). As a consequence, "blocking" anti-CD8 antibodies can suppress autoreactive CD8 + T-cell activation in a relatively selective manner. These findings provide a rational basis for the development and in vivo assessment of novel therapeutic strategies that preferentially target disease-relevant autoimmune responses within the CD8 + T-cell compartment.

Antigen presentation under the influence of 'immune evasion' proteins and its modulation by interferon-gamma: implications for immunotherapy of cytomegalovirus infection with antiviral CD8 T cells.

PubMed

Fink, Annette; Lemmermann, Niels A W; Gillert-Marien, Dorothea; Thomas, Doris; Freitag, Kirsten; Böhm, Verena; Wilhelmi, Vanessa; Reifenberg, Kurt; Reddehase, Matthias J; Holtappels, Rafaela

2012-11-01

Cytomegalovirus (CMV) disease with multiple organ manifestations is the most feared viral complication limiting the success of hematopoietic cell transplantation as a therapy of hematopoietic malignancies. A timely endogenous reconstitution of CD8 T cells controls CMV infection, and adoptive transfer of antiviral CD8 T cells is a therapeutic option to prevent CMV disease by bridging the gap between an early CMV reactivation and delayed endogenous reconstitution of protective immunity. Preclinical research in murine models has provided 'proof of concept' for CD8 T-cell therapy of CMV disease. Protection by CD8 T cells appears to be in conflict with the finding that CMVs encode proteins that inhibit antigen presentation to CD8 T cells by interfering with the constitutive trafficking of peptide-loaded MHC class I molecules (pMHC-I complexes) to the cell surface. Here, we have systematically explored antigen presentation in the presence of the three currently noted immune evasion proteins of murine CMV in all possible combinations and its modulation by pre-treatment of cells with interferon-gamma (IFN-γ). The data reveal improvement in antigen processing by pre-treatment with IFN-γ can almost overrule the inhibitory function of immune evasion molecules in terms of pMHC-I expression levels capable of triggering most of the specific CD8 T cells, though the intensity of stimulation did not retrieve their full functional capacity. Notably, an in vivo conditioning of host tissue cells with IFN-γ in adoptive cell transfer recipients constitutively overexpressing IFN-γ (B6-SAP-IFN-γ mice) enhanced the antiviral efficiency of CD8 T cells in this transgenic cytoimmunotherapy model.

CD30 antigen in embryonal carcinoma and embryogenesis and release of the soluble molecule.

PubMed Central

Latza, U.; Foss, H. D.; Dürkop, H.; Eitelbach, F.; Dieckmann, K. P.; Loy, V.; Unger, M.; Pizzolo, G.; Stein, H.

1995-01-01

The expression, serological detection, and possible functional role of the CD30 antigen in Hodgkin's disease and anaplastic large cell lymphoma is well documented. In embryonal carcinoma (EC), the expression of this cytokine receptor has been demonstrated only by immunohistology. Because the CD30 monoclonal antibody Ki-1 was found to cross-react with an unrelated molecule, we examined by in situ hybridization testicular germ cell neoplasms for the presence of CD30-specific transcripts. CD30 mRNA was detectable in the tumor cells of 9 of 9 cases of EC or mixed germ cell tumors with an EC component but in no other nonlymphoid tumors. Thus, the CD30 transcript expression pattern proved to be identical to the immunostaining pattern seen with the CD30-specific monoclonal antibody Ber-H2. By Northern blot analysis, CD30 transcripts could be demonstrated in the EC cell line Tera-2. Employing a highly sensitive second generation sandwich enzyme-linked immunosorbent assay, we could detect the soluble CD30 molecule in 8 of 8 sera from patients with a diagnosis of EC but not in 8 of 10 sera from patients with other testicular germ cell tumors. In fetal tissue, no CD30-expressing germ cells or epithelial cells could be observed. Thus, the cellularly expressed CD30 marker for testicular neoplasms of EC type. Moreover, the serum levels of soluble CD30 antigen seem to be a promising parameter for monitoring patients with EC. Images Figure 1 Figure 2 PMID:7856755

Formulation of the bivalent prostate cancer vaccine with surgifoam elicits antigen-specific effector T cells in PSA-transgenic mice.

PubMed

Karan, Dev

2017-10-13

We previously developed and characterized an adenoviral-based prostate cancer vaccine for simultaneous targeting of prostate-specific antigen (PSA) and prostate stem cell antigen (PSCA). We also demonstrated that immunization of mice with the bivalent vaccine (Ad 5 -PSA+PSCA) inhibited the growth of established prostate tumors. However, there are multiple challenges hindering the success of immunological therapies in the clinic. One of the prime concerns has been to overcome the immunological tolerance and maintenance of long-term effector T cells. In this study, we further characterized the use of the bivalent vaccine (Ad 5 -PSA+PSCA) in a transgenic mouse model expressing human PSA in the mouse prostate. We demonstrated the expression of PSA analyzed at the mRNA level (by RT-PCR) and protein level (by immunohistochemistry) in the prostate lobes harvested from the PSA-transgenic (PSA-Tg) mice. We established that the administration of the bivalent vaccine in surgifoam to the PSA-Tg mice induces strong PSA-specific effector CD8 + T cells as measured by IFN-γ secretion and in vitro cytotoxic T-cell assay. Furthermore, the use of surgifoam with Ad 5 -PSA+PSCA vaccine allows multiple boosting vaccinations with a significant increase in antigen-specific CD8 + T cells. These observations suggest that the formulation of the bivalent prostate cancer vaccine (Ad 5 -PSA+PSCA) with surgifoam bypasses the neutralizing antibody response, thus allowing multiple boosting. This formulation is also helpful for inducing an antigen-specific immune response in the presence of self-antigen, and maintains long-term effector CD8 + T cells. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Microenvironmental stresses induce HLA-E/Qa-1 surface expression and thereby reduce CD8(+) T-cell recognition of stressed cells.

PubMed

Sasaki, Takanori; Kanaseki, Takayuki; Shionoya, Yosuke; Tokita, Serina; Miyamoto, Sho; Saka, Eri; Kochin, Vitaly; Takasawa, Akira; Hirohashi, Yoshihiko; Tamura, Yasuaki; Miyazaki, Akihiro; Torigoe, Toshihiko; Hiratsuka, Hiroyoshi; Sato, Noriyuki

2016-04-01

Hypoxia and glucose deprivation are often observed in the microenvironment surrounding solid tumors in vivo. However, how they interfere with MHC class I antigen processing and CD8(+) T-cell responses remains unclear. In this study, we analyzed the production of antigenic peptides presented by classical MHC class I in mice, and showed that it is quantitatively decreased in the cells exposed to either hypoxia or glucose deprivation. In addition, we unexpectedly found increased surface expression of HLA-E in human and Qa-1 in mouse tumor cells exposed to combined oxygen and glucose deprivation. The induced Qa-1 on the stressed tumor model interacted with an inhibitory NKG2/CD94 receptor on activated CD8(+) T cells and attenuated their specific response to the antigen. Our results thus suggest that microenvironmental stresses modulate not only classical but also nonclassical MHC class I presentation, and confer the stressed cells the capability to escape from the CD8(+) T-cell recognition. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development.

PubMed

Taniuchi, Ichiro; Osato, Motomi; Egawa, Takeshi; Sunshine, Mary Jean; Bae, Suk Chul; Komori, Toshihisa; Ito, Yoshiaki; Littman, Dan R

2002-11-27

T lymphocytes differentiate in discrete stages within the thymus. Immature thymocytes lacking CD4 and CD8 coreceptors differentiate into double-positive cells (CD4(+)CD8(+)), which are selected to become either CD4(+)CD8(-)helper cells or CD4(-)CD8(+) cytotoxic cells. A stage-specific transcriptional silencer regulates expression of CD4 in both immature and CD4(-)CD8(+) thymocytes. We show here that binding sites for Runt domain transcription factors are essential for CD4 silencer function at both stages, and that different Runx family members are required to fulfill unique functions at each stage. Runx1 is required for active repression in CD4(-)CD8(-) thymocytes whereas Runx3 is required for establishing epigenetic silencing in cytotoxic lineage thymocytes. Runx3-deficient cytotoxic T cells, but not helper cells, have defective responses to antigen, suggesting that Runx proteins have critical functions in lineage specification and homeostasis of CD8-lineage T lymphocytes.

HIV-1 alters the cytokine microenvironment and effector function of CD8+T cells upon antigen-specific activation with mycobacteria

USDA-ARS?s Scientific Manuscript database

Tuberculosis is the most common opportunistic infection in individuals living with human immunodeficiency virus (HIV). In addition to CD4+ T cell depletion, HIV infection compromises the function of CD8+ T cell-mediated immunity to Mycobacterium tuberculosis (M.tb). These effects on susceptibility ...

Extending antigen release from particulate vaccines results in enhanced antitumor immune response.

PubMed

Kapadia, Chintan H; Tian, Shaomin; Perry, Jillian L; Sailer, David; Christopher Luft, J; DeSimone, Joseph M

2018-01-10

Tumor-specific CD8 + cytotoxic T lymphocytes (CTLs) play a critical role in an anti-tumor immune response. However, vaccination intended to elicit a potent CD8 + T cell responses employing tumor-associated peptide antigens, are typically ineffective due to poor immunogenicity. Previously, we engineered a polyethylene glycol (PEG) hydrogel-based subunit vaccine for the delivery of an antigenic peptide and CpG (adjuvant) to elicit potent CTLs. In this study, we further examined the effect of antigen release kinetics on their induced immune responses. A CD8 + T cell epitope peptide from OVA (CSIINFEKL) and CpG were co-conjugated to nanoparticles utilizing either a disulfide or a thioether linkage. Subsequent studies comparing peptide release rates as a function of linker, determined that the thioether linkage provided sustained release of peptide over 72h. Ability to control the release of peptide resulted in both higher and prolonged antigen presentation when compared to disulfide-linked peptide. Both NP vaccine formulations resulted in activation and maturation of bone marrow derived dendritic cells (BMDCs) and induced potent CD8 + T cell responses when compared to soluble antigen and soluble CpG. Immunization with either disulfide or thioether linked vaccine constructs effectively inhibited EG7-OVA tumor growth in mice, however only treatment with the thioether linked vaccine construct resulted in enhanced survival. Copyright © 2017. Published by Elsevier B.V.

Neutral Polymer Micelle Carriers with pH-Responsive, Endosome-Releasing Activity Modulate Antigen Trafficking to Enhance CD8 T-Cell Responses

PubMed Central

Keller, Salka; Wilson, John T; Patilea, Gabriela I; Kern, Hanna B; Convertine, Anthony J; Stayton, Patrick S

2014-01-01

Synthetic subunit vaccines need to induce CD8+ cytotoxic T-cell (CTL) responses for effective vaccination against intracellular pathogens. Most subunit vaccines primarily generate humoral immune responses, with a weaker than desired CD8+ cytotoxic T-cell response. Here, a neutral, pH-responsive polymer micelle carrier that alters intracellular antigen trafficking was shown to enhance CD8+ T-cell responses with a correlated increase in cytosolic delivery and a decrease in exocytosis. Polymer diblock carriers consisted of a N-(2-hydroxypropyl) methacrylamide corona block with pendant pyridyl disulfide groups for reversible conjugation of thiolated ovalbumin, and a tercopolymer ampholytic core-forming block composed of propylacrylic acid (PAA), dimethylaminoethyl methacrylate (DMAEMA), and butyl methacrylate (BMA). The diblock copolymers self-assembled into 25–30 nm diameter micellar nanoparticles. Conjugation of ovalbumin to the micelles significantly enhanced antigen cross-presentation in vitro relative to free ovalbumin, an unconjugated physical mixture of ovalbumin and polymer, and a non pH-responsive micelle-ovalbumin control. Mechanistic studies in a murine dendritic cell line (DC2.4) demonstrated micelle-mediated enhancements in intracellular antigen retention and cytosolic antigen accumulation. Approximately 90% of initially internalized ovalbumin-conjugated micelles were retained in cells after 1.5 h, compared to only ~40% for controls. Furthermore, cells dosed with conjugates displayed 67-fold higher cytosolic antigen levels relative to soluble ovalbumin 4 h post uptake. Subcutaneous immunization of mice with ovalbumin-polymer conjugates significantly enhanced antigen-specific CD8+ T cell responses (0.4 % IFN-γ+ of CD8+) compared to immunization with soluble protein, ovalbumin and polymer mixture, and the control micelle without endosome-releasing activity. Additionally, pH-responsive carrier facilitated antigen delivery to antigen presenting cells in the draining lymph nodes. As early as 90 min post injection ova-micelle conjugates were associated with 28% and 55% of dendritic cells and macrophages, respectively. After 24 h, conjugates preferentially associated with dendritic cells, affording 30-, 3-, and 3-fold enhancements in uptake relative to free protein, physical mixture, and the non pH-responsive conjugate controls, respectively. These results demonstrate the potential of pH-responsive polymeric micelles for use in vaccine applications that rely on CD8+ T cell activation. PMID:24698946

Neutral polymer micelle carriers with pH-responsive, endosome-releasing activity modulate antigen trafficking to enhance CD8(+) T cell responses.

PubMed

Keller, Salka; Wilson, John T; Patilea, Gabriela I; Kern, Hanna B; Convertine, Anthony J; Stayton, Patrick S

2014-10-10

Synthetic subunit vaccines need to induce CD8(+) cytotoxic T cell (CTL) responses for effective vaccination against intracellular pathogens. Most subunit vaccines primarily generate humoral immune responses, with a weaker than desired CD8(+) cytotoxic T cell response. Here, a neutral, pH-responsive polymer micelle carrier that alters intracellular antigen trafficking was shown to enhance CD8(+) T cell responses with a correlated increase in cytosolic delivery and a decrease in exocytosis. Polymer diblock carriers consisted of a N-(2-hydroxypropyl) methacrylamide corona block with pendent pyridyl disulfide groups for reversible conjugation of thiolated ovalbumin, and a tercopolymer ampholytic core-forming block composed of propylacrylic acid (PAA), dimethylaminoethyl methacrylate (DMAEMA), and butyl methacrylate (BMA). The diblock copolymers self-assembled into 25-30nm diameter micellar nanoparticles. Conjugation of ovalbumin to the micelles significantly enhanced antigen cross-presentation in vitro relative to free ovalbumin, an unconjugated physical mixture of ovalbumin and polymer, and a non-pH-responsive micelle-ovalbumin control. Mechanistic studies in a murine dendritic cell line (DC 2.4) demonstrated micelle-mediated enhancements in intracellular antigen retention and cytosolic antigen accumulation. Approximately 90% of initially internalized ovalbumin-conjugated micelles were retained in cells after 1.5h, compared to only ~40% for controls. Furthermore, cells dosed with conjugates displayed 67-fold higher cytosolic antigen levels relative to soluble ovalbumin 4h post uptake. Subcutaneous immunization of mice with ovalbumin-polymer conjugates significantly enhanced antigen-specific CD8(+) T cell responses (0.4% IFN-γ(+) of CD8(+)) compared to immunization with soluble protein, ovalbumin and polymer mixture, and the control micelle without endosome-releasing activity. Additionally, pH-responsive carrier facilitated antigen delivery to antigen presenting cells in the draining lymph nodes. As early as 90min post injection, ova-micelle conjugates were associated with 28% and 55% of dendritic cells and macrophages, respectively. After 24h, conjugates preferentially associated with dendritic cells, affording 30-, 3-, and 3-fold enhancements in uptake relative to free protein, physical mixture, and the non-pH-responsive conjugate controls, respectively. These results demonstrate the potential of pH-responsive polymeric micelles for use in vaccine applications that rely on CD8(+) T cell activation. Copyright © 2014 Elsevier B.V. All rights reserved.

Distinct Effects of Saracatinib on Memory CD8+ T-cell Differentiation

PubMed Central

Takai, Shinji; Sabzevari, Helen; Farsaci, Benedetto; Schlom, Jeffrey; Greiner, John W.

2012-01-01

Immunologic memory involving CD8+ T-cells is a hallmark of an adaptive antigen-specific immune response and comprises a critical component of protective immunity. Designing approaches that enhance long-term T-cell memory would, for the most part, fortify vaccines and enhance host protection against infectious diseases and, perhaps, cancer immunotherapy. A better understanding of the cellular programs involved in the antigen-specific T-cell response has led to new approaches that target the magnitude and quality of the memory T-cell response. Here we show that T-cells from T-cell receptor transgenic mice for the nucleoprotein of influenza virus NP68 exhibit the distinct phases priming, expansion, contraction, memory - of an antigen-specific T-cell response when exposed in vitro to the cognate peptide. Saracatinib, a specific inhibitor of Src family kinases, administered at low doses during the expansion or contraction phases, increased CD62Lhigh/CD44high central memory CD8+ T-cells and IFN-γ production, while suppressing immunity when added during the priming phase. These effects by saracatinib were not accompanied by the expected decline of Src family kinases, but were accompanied by Akt-mTOR suppression and/or mediated via another pathway. Increased central memory cells by saracatinib were recapitulated in mice using a poxvirus-based influenza vaccine, thus underscoring the importance of dose and timing of the inhibitor in the context of memory T-cell differentiation. Finally, vaccine plus saracatinib treatment showed better protection against tumor challenge. The immune-potentiating effects on CD8+ T-cells by a low dose of saracatinib might afford better protection from pathogen or cancer when combined with vaccine. PMID:22450814

A Higher Activation Threshold of Memory CD8+ T Cells Has a Fitness Cost That Is Modified by TCR Affinity during Tuberculosis.

PubMed

Carpenter, Stephen M; Nunes-Alves, Cláudio; Booty, Matthew G; Way, Sing Sing; Behar, Samuel M

2016-01-01

T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8+ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8+ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8+ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8+ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRβ deep sequencing to track TB10.4-specific CD8+ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3β sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and -independent factors affect the fitness of memory CD8+ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection.

A Higher Activation Threshold of Memory CD8+ T Cells Has a Fitness Cost That Is Modified by TCR Affinity during Tuberculosis

PubMed Central

Carpenter, Stephen M.; Nunes-Alves, Cláudio; Booty, Matthew G.; Way, Sing Sing; Behar, Samuel M.

2016-01-01

T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8+ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8+ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8+ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8+ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRβ deep sequencing to track TB10.4-specific CD8+ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3β sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and -independent factors affect the fitness of memory CD8+ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection. PMID:26745507

Administration of sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate conjugated GP100{sub 25–33} peptide-coupled spleen cells effectively mounts antigen-specific immune response against mouse melanoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Xiaoli; Xia, Chang-Qing, E-mail: cqx65@yahoo.com; Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL32610

It remains a top research priority to develop immunotherapeutic approaches to induce potent antigen-specific immune responses against tumors. However, in spite of some promising results, most strategies are ineffective because they generate low numbers of tumor-reactive cytotoxic T lymphocytes (CTLs). Here we designed a strategy to enhance antigen-specific immune response via administering sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate (sulfo-SMCC)-conjugated melanoma tumor antigen GP100{sub 25–33} peptide-coupled syngeneic spleen cells in a mouse model of melanoma. We found that infusion of GP100{sub 25–33} peptide-coupled spleen cells significantly attenuated the growth of melanoma in prophylactic and therapeutic immunizations. Consistent with these findings, the adoptive transfer of spleenmore » cells from immunized mice to naïve syngeneic mice was able to transfer anti-tumor effect, suggesting that GP100{sub 25–33} peptide-specific immune response was induced. Further studies showed that, CD8+ T cell proliferation and the frequency of interferon (IFN)-γ-producing CD8+ T cells upon ex vivo stimulation by GP100{sub 25–33} were significantly increased compared to control groups. Tumor antigen, GP100{sub 25–23} specific immune response was also confirmed by ELISpot and GP100-tetramer assays. This approach is simple, easy-handled, and efficiently delivering antigens to lymphoid tissues. Our study offers an opportunity for clinically translating this approach into tumor immunotherapy. - Highlights: • Infusion of GP100{sub 25–33}-coupled spleen cells leads to potent anti-melanoma immunity. • GP100{sub 25–33}-coupled spleen cell treatment induces antigen-specific IFN-γ-producing CD8 T cells. • This approach takes advantage of homing nature of immune cells.« less

IFNγ producing CD8+ T cells modified to resist major immune checkpoints induce regression of MHC class I-deficient melanomas

PubMed Central

Buferne, Michel; Chasson, Lionel; Grange, Magali; Mas, Amandine; Arnoux, Fanny; Bertuzzi, Mélanie; Naquet, Philippe; Leserman, Lee; Schmitt-Verhulst, Anne-Marie; Auphan-Anezin, Nathalie

2015-01-01

Tumors with reduced expression of MHC class I (MHC-I) molecules may be unrecognized by tumor antigen-specific CD8+ T cells and thus constitute a challenge for cancer immunotherapy. Here we monitored development of autochthonous melanomas in TiRP mice that develop tumors expressing a known tumor antigen as well as a red fluorescent protein (RFP) reporter knock in gene. The latter permits non-invasive monitoring of tumor growth by biofluorescence. One developing melanoma was deficient in cell surface expression of MHC-I, but MHC-I expression could be rescued by exposure of these cells to IFNγ. We show that CD8+ T cells specific for tumor antigen/MHC-I were efficient at inducing regression of the MHC-I-deficient melanoma, provided that the T cells were endowed with properties permitting their migration into the tumor and their efficient production of IFNγ. This was the case for CD8+ T cells transfected to express an active form of STAT5 (STAT5CA). The amount of IFNγ produced ex vivo from T cells present in tumors after adoptive transfer of the CD8+ T cells was correlated with an increase in surface expression of MHC-I molecules by the tumor cells. We also show that these CD8+ T cells expressed PD-1 and upregulated its ligand PDL-1 on melanoma cells within the tumor. Despite upregulation of this immunosuppressive pathway, efficient IFNγ production in the melanoma microenvironment was found associated with resistance of STAT5CA-expressing CD8+ T cells to inhibition both by PD-1/PDL-1 engagement and by TGFβ1, two main immune regulatory mechanisms hampering the efficiency of immunotherapy in patients. PMID:25949872

Activation of Antigen-Specific CD8(+) T Cells by Poly-DL-Lactide/Glycolide (PLGA) Nanoparticle-Primed Gr-1(high) Cells.

PubMed

Luo, Wen-Hui; Yang, Ya-Wun

2016-04-01

The aim of this study was to investigate the induction of antigen-specific T cell activation and cell cycle modulation by a poly-DL-lactide/glycolide (PLGA) nanoparticle (NP)-primed CD11b(+)Gr-1(high) subset isolated from mouse bone marrow. PLGA NPs containing the ovalbumin (OVA) antigen were prepared using the double emulsion and solvent evaporation method, and protein release rate and cell viability were determined. The Lin2(¯)CD11b(+)Gr-1(high)Ly6c(low) (Gr-1(high)) subset was sorted from the bone marrow of C57BL/6 J mice by fluorescence-activated cell sorting (FACS) and co-cultured with OT-I CD8(+) splenic T cells. Proliferation of OT-I CD8(+) T cells was monitored, and cell cycles were determined by 5-bromo-2'-deoxyuridine (BrdU) labeling. Treatment of Gr-1(high) cells with PLGA/OVA NPs upregulated expression of the SIINFEKL-H2K(b) complex in the context of MHC I. Co-cultures of OT-I CD8(+) T cells with the PLGA/OVA NP-primed Gr-1(high) cells induced the proliferation of T cells in vitro and modulated cell division and morphology. Treatment of Gr-1(high) cells with PLGA/OVA NPs also induced cell apoptosis and necrosis. This study demonstrated the function of PLGA/OVA NPs in the activation of OT-I CD8(+) T cells and the capability of cross-presentation via the Gr-1(high) polymorphonuclear subset from mouse bone marrow.

Upregulation of interleukin 7 receptor alpha and programmed death 1 marks an epitope-specific CD8+ T-cell response that disappears following primary Epstein-Barr virus infection.

PubMed

Sauce, Delphine; Larsen, Martin; Abbott, Rachel J M; Hislop, Andrew D; Leese, Alison M; Khan, Naeem; Papagno, Laura; Freeman, Gordon J; Rickinson, Alan B

2009-09-01

In immunocompetent individuals, the stability of the herpesvirus-host balance limits opportunities to study the disappearance of a virus-specific CD8(+) T-cell response. However, we noticed that in HLA-A 0201-positive infectious mononucleosis (IM) patients undergoing primary Epstein-Barr virus (EBV) infection, the initial CD8 response targets three EBV lytic antigen-derived epitopes, YVLDHLIVV (YVL), GLCTLVAML (GLC), and TLDYKPLSV (TLD), but only the YVL and GLC reactivities persist long-term; the TLD response disappears within 10 to 27 months. While present, TLD-specific cells remained largely indistinguishable from YVL and GLC reactivities in many phenotypic and functional respects but showed unique temporal changes in two markers of T-cell fate, interleukin 7 receptor alpha (IL-7Ralpha; CD127) and programmed death 1 (PD-1). Thus, following the antigen-driven downregulation of IL-7Ralpha seen on all populations in acute IM, in every case, the TLD-specific population recovered expression unusually quickly post-IM. As well, in four of six patients studied, TLD-specific cells showed very strong PD-1 upregulation in the last blood sample obtained before the cells' disappearance. Our data suggest that the disappearance of this individual epitope reactivity from an otherwise stable EBV-specific response (i) reflects a selective loss of cognate antigen restimulation (rather than of IL-7-dependent signals) and (ii) is immediately preceded, and perhaps mediated, by PD-1 upregulation to unprecedented levels.

CD8 Memory Cells Develop Unique DNA Repair Mechanisms Favoring Productive Division.

PubMed

Galgano, Alessia; Barinov, Aleksandr; Vasseur, Florence; de Villartay, Jean-Pierre; Rocha, Benedita

2015-01-01

Immune responses are efficient because the rare antigen-specific naïve cells are able to proliferate extensively and accumulate upon antigen stimulation. Moreover, differentiation into memory cells actually increases T cell accumulation, indicating improved productive division in secondary immune responses. These properties raise an important paradox: how T cells may survive the DNA lesions necessarily induced during their extensive division without undergoing transformation. We here present the first data addressing the DNA damage responses (DDRs) of CD8 T cells in vivo during exponential expansion in primary and secondary responses in mice. We show that during exponential division CD8 T cells engage unique DDRs, which are not present in other exponentially dividing cells, in T lymphocytes after UV or X irradiation or in non-metastatic tumor cells. While in other cell types a single DDR pathway is affected, all DDR pathways and cell cycle checkpoints are affected in dividing CD8 T cells. All DDR pathways collapse in secondary responses in the absence of CD4 help. CD8 T cells are driven to compulsive suicidal divisions preventing the propagation of DNA lesions. In contrast, in the presence of CD4 help all the DDR pathways are up regulated, resembling those present in metastatic tumors. However, this up regulation is present only during the expansion phase; i.e., their dependence on antigen stimulation prevents CD8 transformation. These results explain how CD8 T cells maintain genome integrity in spite of their extensive division, and highlight the fundamental role of DDRs in the efficiency of CD8 immune responses.

Engaging the CD40-CD40L pathway augments T-helper cell responses and improves control of Mycobacterium tuberculosis infection

PubMed Central

Bizzell, Erica; Madan-Lala, Ranjna

2017-01-01

Mycobacterium tuberculosis (Mtb) impairs dendritic cell (DC) functions and induces suboptimal antigen-specific CD4 T cell immune responses that are poorly protective. Mucosal T-helper cells producing IFN-γ (Th1) and IL-17 (Th17) are important for protecting against tuberculosis (TB), but the mechanisms by which DCs generate antigen-specific T-helper responses during Mtb infection are not well defined. We previously reported that Mtb impairs CD40 expression on DCs and restricts Th1 and Th17 responses. We now demonstrate that CD40-dependent costimulation is required to generate IL-17 responses to Mtb. CD40-deficient DCs were unable to induce antigen-specific IL-17 responses after Mtb infection despite the production of Th17-polarizing innate cytokines. Disrupting the interaction between CD40 on DCs and its ligand CD40L on antigen-specific CD4 T cells, genetically or via antibody blockade, significantly reduced antigen-specific IL-17 responses. Importantly, engaging CD40 on DCs with a multimeric CD40 agonist (CD40LT) enhanced antigen-specific IL-17 generation in ex vivo DC-T cell co-culture assays. Further, intratracheal instillation of Mtb-infected DCs treated with CD40LT significantly augmented antigen-specific Th17 responses in vivo in the lungs and lung-draining lymph nodes of mice. Finally, we show that boosting CD40-CD40L interactions promoted balanced Th1/Th17 responses in a setting of mucosal DC transfer, and conferred enhanced control of lung bacterial burdens following aerosol challenge with Mtb. Our results demonstrate that CD40 costimulation by DCs plays an important role in generating antigen-specific Th17 cells and targeting the CD40-CD40L pathway represents a novel strategy to improve adaptive immunity to TB. PMID:28767735

Dietary gluten triggers concomitant activation of CD4+ and CD8+ αβ T cells and γδ T cells in celiac disease

PubMed Central

Han, Arnold; Newell, Evan W.; Glanville, Jacob; Fernandez-Becker, Nielsen; Khosla, Chaitan; Chien, Yueh-hsiu; Davis, Mark M.

2013-01-01

Celiac disease is an intestinal autoimmune disease driven by dietary gluten and gluten-specific CD4+ T-cell responses. In celiac patients on a gluten-free diet, exposure to gluten induces the appearance of gluten-specific CD4+ T cells with gut-homing potential in the peripheral blood. Here we show that gluten exposure also induces the appearance of activated, gut-homing CD8+ αβ and γδ T cells in the peripheral blood. Single-cell T-cell receptor sequence analysis indicates that both of these cell populations have highly focused T-cell receptor repertoires, indicating that their induction is antigen-driven. These results reveal a previously unappreciated role of antigen in the induction of CD8+ αβ and γδ T cells in celiac disease and demonstrate a coordinated response by all three of the major types of T cells. More broadly, these responses may parallel adaptive immune responses to viral pathogens and other systemic autoimmune diseases. PMID:23878218

CD8+ gamma-delta TCR+ and CD4+ T cells produce IFN-γ at 5-7 days after yellow fever vaccination in Indian rhesus macaques, before the induction of classical antigen-specific T cell responses.

PubMed

Neves, Patrícia C C; Rudersdorf, Richard A; Galler, Ricardo; Bonaldo, Myrna C; de Santana, Marlon Gilsepp Veloso; Mudd, Philip A; Martins, Maurício A; Rakasz, Eva G; Wilson, Nancy A; Watkins, David I

2010-11-29

The yellow fever 17D (YF-17D) vaccine is one of the most efficacious vaccines developed to date. Interestingly, vaccination with YF-17D induces IFN-γ production early after vaccination (days 5-7) before the development of classical antigen-specific CD8(+) and CD4(+) T cell responses. Here we investigated the cellular source of this early IFN-γ production. At days 5 and 7 post-vaccination activated CD8(+) gamma-delta TCR T cells produced IFN-γ and TNF-α. Activated CD4(+) T cells produced IFN-γ and TNF-α at day 7 post-vaccination. This early IFN-γ production was also induced after vaccination with recombinant YF-17D (rYF-17D), but was not observed after recombinant Adenovirus type 5 (rAd5) vaccination. Early IFN-γ production, therefore, might be an important aspect of yellow fever vaccination. Copyright © 2010 Elsevier Ltd. All rights reserved.

Cationic liposomes promote antigen cross-presentation in dendritic cells by alkalizing the lysosomal pH and limiting the degradation of antigens

PubMed Central

Gao, Jie; Ochyl, Lukasz J; Yang, Ellen; Moon, James J

2017-01-01

Cationic liposomes (CLs) have been widely examined as vaccine delivery nanoparticles since they can form complexes with biomacromolecules, promote delivery of antigens and adjuvant molecules to antigen-presenting cells (APCs), and mediate cellular uptake of vaccine components. CLs are also known to trigger antigen cross-presentation – the process by which APCs internalize extracellular protein antigens, degrade them into minimal CD8+ T-cell epitopes, and present them in the context of major histocompatibility complex-I (MHC-I). However, the precise mechanisms behind CL-mediated induction of cross-presentation and cross-priming of CD8+ T-cells remain to be elucidated. In this study, we have developed two distinct CL systems and examined their impact on the lysosomal pH in dendritic cells (DCs), antigen degradation, and presentation of peptide:MHC-I complexes to antigen-specific CD8+ T-cells. To achieve this, we have used 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as the prototypical components of CLs with tertiary amine groups and compared the effect of CLs and anionic liposomes on lysosomal pH, antigen degradation, and cross-presentation by DCs. Our results showed that CLs, but not anionic liposomes, elevated the lysosomal pH in DCs and reduced antigen degradation, thereby promoting cross-presentation and cross-priming of CD8+ T-cell responses. These studies shed new light on CL-mediated cross-presentation and suggest that intracellular fate of vaccine components and subsequent immunological responses can be controlled by rational design of nanomaterials. PMID:28243087

Role of PD-1 during effector CD8 T cell differentiation.

PubMed

Ahn, Eunseon; Araki, Koichi; Hashimoto, Masao; Li, Weiyan; Riley, James L; Cheung, Jeanne; Sharpe, Arlene H; Freeman, Gordon J; Irving, Bryan A; Ahmed, Rafi

2018-05-01

PD-1 (programmed cell death-1) is the central inhibitory receptor regulating CD8 T cell exhaustion during chronic viral infection and cancer. Interestingly, PD-1 is also expressed transiently by activated CD8 T cells during acute viral infection, but the role of PD-1 in modulating T cell effector differentiation and function is not well defined. To address this question, we examined the expression kinetics and role of PD-1 during acute lymphocytic choriomeningitis virus (LCMV) infection of mice. PD-1 was rapidly up-regulated in vivo upon activation of naive virus-specific CD8 T cells within 24 h after LCMV infection and in less than 4 h after peptide injection, well before any cell division had occurred. This rapid PD-1 expression by CD8 T cells was driven predominantly by antigen receptor signaling since infection with a LCMV strain with a mutation in the CD8 T cell epitope did not result in the increase of PD-1 on antigen-specific CD8 T cells. Blockade of the PD-1 pathway using anti-PD-L1 or anti-PD-1 antibodies during the early phase of acute LCMV infection increased mTOR signaling and granzyme B expression in virus-specific CD8 T cells and resulted in faster clearance of the infection. These results show that PD-1 plays an inhibitory role during the naive-to-effector CD8 T cell transition and that the PD-1 pathway can also be modulated at this stage of T cell differentiation. These findings have implications for developing therapeutic vaccination strategies in combination with PD-1 blockade.

A fully human IgG1 anti-PD-L1 MAb in an in vitro assay enhances antigen-specific T-cell responses

PubMed Central

Grenga, Italia; Donahue, Renee N; Lepone, Lauren M; Richards, Jacob; Schlom, Jeffrey

2016-01-01

Monoclonal antibodies (MAbs) that interfere with checkpoint molecules are being investigated for the treatment of infectious diseases and cancer, with the aim of enhancing the function of an impaired immune system. Avelumab (MSB0010718C) is a fully human IgG1 MAb targeting programmed death-ligand 1 (PD-L1), which differs from other checkpoint-blocking antibodies in its ability to mediate antibody-dependent cell-mediated cytotoxicity. These studies were conducted to define whether avelumab could enhance the detection of antigen-specific immune response in in vitro assays. Peripheral blood mononuclear cells from 17 healthy donors were stimulated in vitro, with and without avelumab, with peptide pools encoding for cytomegalovirus, Epstein–Barr virus, influenza and tetanus toxin or the negative peptide control encoding for human leukocyte antigen. These studies show for the first time that the addition of avelumab to an antigen-specific IVS assay (a) increased the frequency of activated antigen-specific CD8+ T lymphocytes, and did so to a greater extent than that seen with commercially available PD-L1-blocking antibodies, (b) reduced CD4+ T-cell proliferation and (c) induced a switch in the production of Th2 to Th1 cytokines. Moreover, there was an inverse correlation between the enhancement of CD8+ T-cell activation and reduction in CD4+ T-cell proliferation induced by avelumab. These findings provide the rationale for the use of avelumab anti-PD-L1 in in vitro assays to monitor patient immune responses to immunotherapies. PMID:27350882

A fully human IgG1 anti-PD-L1 MAb in an in vitro assay enhances antigen-specific T-cell responses.

PubMed

Grenga, Italia; Donahue, Renee N; Lepone, Lauren M; Richards, Jacob; Schlom, Jeffrey

2016-05-01

Monoclonal antibodies (MAbs) that interfere with checkpoint molecules are being investigated for the treatment of infectious diseases and cancer, with the aim of enhancing the function of an impaired immune system. Avelumab (MSB0010718C) is a fully human IgG1 MAb targeting programmed death-ligand 1 (PD-L1), which differs from other checkpoint-blocking antibodies in its ability to mediate antibody-dependent cell-mediated cytotoxicity. These studies were conducted to define whether avelumab could enhance the detection of antigen-specific immune response in in vitro assays. Peripheral blood mononuclear cells from 17 healthy donors were stimulated in vitro, with and without avelumab, with peptide pools encoding for cytomegalovirus, Epstein-Barr virus, influenza and tetanus toxin or the negative peptide control encoding for human leukocyte antigen. These studies show for the first time that the addition of avelumab to an antigen-specific IVS assay (a) increased the frequency of activated antigen-specific CD8(+) T lymphocytes, and did so to a greater extent than that seen with commercially available PD-L1-blocking antibodies, (b) reduced CD4(+) T-cell proliferation and (c) induced a switch in the production of Th2 to Th1 cytokines. Moreover, there was an inverse correlation between the enhancement of CD8(+) T-cell activation and reduction in CD4(+) T-cell proliferation induced by avelumab. These findings provide the rationale for the use of avelumab anti-PD-L1 in in vitro assays to monitor patient immune responses to immunotherapies.

Co-delivery of PSA and PSMA DNA vaccines with electroporation induces potent immune responses.

PubMed

Ferraro, Bernadette; Cisper, Neil J; Talbott, Kendra T; Philipson-Weiner, Lindsey; Lucke, Colleen E; Khan, Amir S; Sardesai, Niranjan Y; Weiner, David B

2011-01-01

Prostate cancer (PCa) remains a significant public health problem. Current treatment modalities for PCa can be useful, but may be accompanied by deleterious side effects and often do not confer long-term control. Accordingly, additional modalities, such as immunotherapy, may represent an important approach for PCa treatment. The identification of tissue-specific antigens engenders PCa an attractive target for immunotherapeutic approaches. Delivery of DNA vaccines with electroporation has shown promising results for prophylactic and therapeutic targets in a variety of species including humans. Application of this technology for PCa immunotherapy strategies has been limited to single antigen and epitope targets. We sought to test the hypothesis that a broader collection of antigens would improve the breadth and effectiveness of a PCa immune therapy approach. We therefore developed highly optimized DNA vaccines encoding prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) as a dual antigen approach to immune therapy of PCa. PSA-and PSMA-specific cellular immunogenicity was evaluated in a mouse model for co-delivery and single antigen vaccination. Mice received 2 immunizations spaced 2 weeks apart and immunogenicity was evaluated 1 week after the second vaccination. Both the PSA and PSMA vaccines induced robust antigen-specific IFNγ responses by ELISpot. Further characterization of cellular immunogenicity by flow cytometry indicated strong antigen-specific TNFα production by CD4+ T cells and IFNγ and IL-2 secretion by both CD4+ and CD8+ T cells. There was also a strong humoral response as determined by PSA-specific seroconversion. These data support further study of this novel approach to immune therapy of PCa.

Most Do, but Some Do Not: CD4+CD25− T Cells, but Not CD4+CD25+ Treg Cells, Are Cytolytic When Redirected by a Chimeric Antigen Receptor (CAR)

PubMed Central

Hombach, Andreas A.; Abken, Hinrich

2017-01-01

Evidences are accumulating that CD4+ T cells can physiologically mediate antigen specific target cell lysis. By circumventing major histocompatibility complex (MHC)-restrictions through an engineered chimeric antigen receptor (CAR), CD4+ T cells lyse defined target cells as efficiently as do CD8+ T cells. However, the cytolytic capacity of redirected CD4+CD25− T cells, in comparison with CD4+CD25+ regulatory T (Treg) cells was so far not thoroughly defined. Treg cells require a strong CD28 signal together with CD3ζ for activation. We consequently used a CAR with combined CD28­CD3ζ signalling for redirecting CD4+CD25− T cells and CD4+CD25+ Treg cells from the same donor. CAR redirected activation of these T cell subsets and induced a distinct cytokine pattern with high IL-10 and a lack of IL-2 release by Treg cells. Despite strong antigen-specific activation, CAR Treg cells produced only weak target cell lysis, whereas CD4+CD25− CAR T cells were potent killers. Cytolysis did not correlate with the target cell sensitivity to Fas/FasL mediated killing; CD4+CD25− T cells upregulated perforin and granzyme B upon CAR activation, whereas Treg cells did less. The different cytolytic capacities of CAR redirected conventional CD4+ cells and Treg cells imply their use for different purposes in cell therapy. PMID:28850063

Increased expression of activation antigens on CD8+ T lymphocytes in Myalgic Encephalomyelitis/chronic fatigue syndrome: inverse associations with lowered CD19+ expression and CD4+/CD8+ ratio, but no associations with (auto)immune, leaky gut, oxidative and nitrosative stress biomarkers.

PubMed

Maes, Michael; Bosmans, Eugene; Kubera, Marta

2015-01-01

There is now evidence that specific subgroups of patients with Myalgic Encephalomyelitis / chronic fatigue syndrome (ME/CFS) suffer from a neuro-psychiatric-immune disorder. This study was carried out to delineate the expression of the activation markers CD38 and human leukocyte antigen (HLA) DR on CD4+ and CD8+ peripheral blood lymphocytes in ME/CFS. Proportions and absolute numbers of peripheral lymphocytes expressing CD3+, CD19+, CD4+, CD8+, CD38+ and HLA-DR+ were measured in ME/CFS (n=139), chronic fatigue (CF, n=65) and normal controls (n=40). The proportions of CD3+, CD8+, CD8+CD38+ and CD8+HLA-DR+ were significantly higher in ME/CFS patients than controls, while CD38+, CD8+CD38+, CD8+HLA-DR+ and CD38+HLA-DR+ were significantly higher in ME/CFS than CF. The percentage of CD19+ cells and the CD4+/CD8+ ratio were significantly lower in ME/CFS and CF than in controls. There were highly significant inverse correlations between the increased expression of CD38+, especially that of CD8+CD38+, and the lowered CD4+/CD8+ ratio and CD19+ expression. There were no significant associations between the flow cytometric results and severity or duration of illness and peripheral blood biomarkers of oxidative and nitrosative stress (O&NS, i.e. IgM responses to O&N modified epitopes), leaky gut (IgM or IgA responses to LPS of gut commensal bacteria), cytokines (interleukin-1, tumor necrosis factor-α), neopterin, lysozyme and autoimmune responses to serotonin. The results support that a) increased CD38 and HLA-DR expression on CD8+ T cells are biomarkers of ME/CFS; b) increased CD38 antigen expression may contribute to suppression of the CD4+/CD8+ ratio and CD19+ expression; c) there are different immune subgroups of ME/CFS patients, e.g. increased CD8+ activation marker expression versus inflammation or O&NS processes; and d) viral infections or reactivation may play a role in a some ME/CFS patients.

The CD8+ memory T-cell state of readiness is actively maintained and reversible

PubMed Central

Allam, Atef; Conze, Dietrich B.; Giardino Torchia, Maria Letizia; Munitic, Ivana; Yagita, Hideo; Sowell, Ryan T.; Marzo, Amanda L.

2009-01-01

The ability of the adaptive immune system to respond rapidly and robustly upon repeated antigen exposure is known as immunologic memory, and it is thought that acquisition of memory T-cell function is an irreversible differentiation event. In this study, we report that many phenotypic and functional characteristics of antigen-specific CD8 memory T cells are lost when they are deprived of contact with dendritic cells. Under these circumstances, memory T cells reverted from G1 to the G0 cell-cycle state and responded to stimulation like naive T cells, as assessed by proliferation, dependence upon costimulation, and interferon-γ production, without losing cell surface markers associated with memory. The memory state was maintained by signaling via members of the tumor necrosis factor receptor superfamily, CD27 and 4-1BB. Foxo1, a transcription factor involved in T-cell quiescence, was reduced in memory cells, and stimulation of naive CD8 cells via CD27 caused Foxo1 to be phosphorylated and emigrate from the nucleus in a phosphatidylinositol-3 kinase–dependent manner. Consistent with these results, maintenance of G1 in vivo was compromised in antigen-specific memory T cells in vesicular stomatitis virus-infected CD27-deficient mice. Therefore, sustaining the functional phenotype of T memory cells requires active signaling and maintenance. PMID:19617575

Vaginal type-II mucosa is an inductive site for primary CD8+ T-cell mucosal immunity

PubMed Central

Wang, Yichuan; Sui, Yongjun; Kato, Shingo; Hogg, Alison E.; Steel, Jason C.; Morris, John C.; Berzofsky, Jay A.

2014-01-01

The structured lymphoid tissues are considered the only inductive sites where primary T cell immune responses occur. The naïve T cells in structured lymphoid tissues, once being primed by antigen -bearing dendritic cells, differentiate into memory T cells and traffic back to the mucosal sites through the bloodstream. Contrary to this belief, here we show that the vaginal type-II mucosa itself, despite lack of structured lymphoid tissues, can act as an inductive site during primary CD8+ T cell immune responses. We provide evidence that the vaginal mucosa supports both the local immune priming of naïve CD8+ T cells and the local expansion of antigen-specific CD8+ T cells, thereby demonstrating a different paradigm for primary mucosal T cell immune induction. PMID:25600442

Homologous Prime-Boost Vaccination with OVA Entrapped in Self-Adjuvanting Archaeosomes Induces High Numbers of OVA-Specific CD8+ T Cells that Protect Against Subcutaneous B16-OVA Melanoma

PubMed Central

Stark, Felicity C.; McCluskie, Michael J.; Krishnan, Lakshmi

2016-01-01

Homologous prime-boost vaccinations with live vectors typically fail to induce repeated strong CD8+ T cell responses due to the induction of anti-vector immunity, highlighting the need for alternative delivery vehicles. The unique ether lipids of archaea may be constituted into liposomes, archaeosomes, which do not induce anti-carrier responses, making them an ideal candidate for use in repeat vaccination systems. Herein, we evaluated in mice the maximum threshold of antigen-specific CD8+ T cell responses that may be induced by multiple homologous immunizations with ovalbumin (OVA) entrapped in archaeosomes derived from the ether glycerolipids of the archaeon Methanobrevibacter smithii (MS-OVA). Up to three immunizations with MS-OVA administered in optimized intervals (to allow for sufficient resting of the primed cells prior to boosting), induced a potent anti-OVA CD8+ T cell response of up to 45% of all circulating CD8+ T cells. Additional MS-OVA injections did not add any further benefit in increasing the memory of CD8+ T cell frequency. In contrast, OVA expressed by Listeria monocytogenes (LM-OVA), an intracellular bacterial vector failed to evoke a boosting effect after the second injection, resulting in significantly reduced antigen-specific CD8+ T cell frequencies. Furthermore, repeated vaccination with MS-OVA skewed the response increasingly towards an effector memory (CD62low) phenotype. Vaccinated animals were challenged with B16-OVA at late time points after vaccination (+7 months) and were afforded protection compared to control. Therefore, archaeosomes constituted a robust particulate delivery system to unravel the kinetics of CD8+ T cell response induction and memory maintenance and constitute an efficient vaccination regimen optimized for tumor protection. PMID:27869670

Dose-dependent modulation of CD8 and functional avidity as a result of peptide encounter

PubMed Central

Kroger, Charles J; Alexander-Miller, Martha A

2007-01-01

The generation of an optimal CD8+ cytotoxic T lymphocyte (CTL) response is critical for the clearance of many intracellular pathogens. Previous studies suggest that one contributor to an optimal immune response is the presence of CD8+ cells exhibiting high functional avidity. In this regard, CD8 expression has been shown to contribute to peptide sensitivity. Here, we investigated the ability of naive splenocytes to modulate CD8 expression according to the concentration of stimulatory peptide antigen. Our results showed that the level of CD8 expressed was inversely correlated with the amount of peptide used for the primary stimulation, with higher concentrations of antigen resulting in lower expression of both CD8α and CD8β. Importantly the ensuing CD8low and CD8high CTL populations were not the result of the selective outgrowth of naive CD8+ T-cell subpopulations expressing distinct levels of CD8. Subsequent encounter with peptide antigen resulted in continued modulation of both the absolute level and the isoform of CD8 expressed and in the functional avidity of the responding cells. We propose that CD8 cell surface expression is not a static property, but can be modulated to ‘fine tune’ the sensitivity of responding CTL to a defined concentration of antigen. PMID:17484768

Influenza A Virus Infection of Human Primary Dendritic Cells Impairs Their Ability to Cross-Present Antigen to CD8 T Cells

PubMed Central

Smed-Sörensen, Anna; Chalouni, Cécile; Chatterjee, Bithi; Cohn, Lillian; Blattmann, Peter; Nakamura, Norihiro; Delamarre, Lélia; Mellman, Ira

2012-01-01

Influenza A virus (IAV) infection is normally controlled by adaptive immune responses initiated by dendritic cells (DCs). We investigated the consequences of IAV infection of human primary DCs on their ability to function as antigen-presenting cells. IAV was internalized by both myeloid DCs (mDCs) and plasmacytoid DCs but only mDCs supported viral replication. Although infected mDCs efficiently presented endogenous IAV antigens on MHC class II, this was not the case for presentation on MHC class I. Indeed, cross-presentation by uninfected cells of minute amounts of endocytosed, exogenous IAV was ∼300-fold more efficient than presentation of IAV antigens synthesized by infected cells and resulted in a statistically significant increase in expansion of IAV-specific CD8 T cells. Furthermore, IAV infection also impaired cross-presentation of other exogenous antigens, indicating that IAV infection broadly attenuates presentation on MHC class I molecules. Our results suggest that cross-presentation by uninfected mDCs is a preferred mechanism of antigen-presentation for the activation and expansion of CD8 T cells during IAV infection. PMID:22412374

A live attenuated BCG vaccine overexpressing multistage antigens Ag85B and HspX provides superior protection against Mycobacterium tuberculosis infection.

PubMed

Yuan, Xuefeng; Teng, Xindong; Jing, Yukai; Ma, Jilei; Tian, Maopeng; Yu, Qi; Zhou, Lei; Wang, Ruibo; Wang, Weihua; Li, Li; Fan, Xionglin

2015-12-01

Tuberculosis (TB) remains one of the most menacing infectious diseases, although attenuated Mycobacterium bovis Bacillus Calmette-Guerin (BCG) vaccine has been widely used to protect children against primary TB. There are increasing evidences that rapid growing and dormant Mycobacterium tuberculosis (M. tuberculosis) coexist in vivo after infection. However, BCG vaccine only elicits cell-mediated immune responses to secretory antigens expressed by rapid growing pathogen. BCG vaccine is thus unable to thwart the reactivation of latent tuberculosis infection (LTBI), and its protection wanes over age after neonatal immunization. In order to extend its ability for a durable protection, a novel recombinant BCG (rBCG) strain, named rBCG::XB, was constructed by overexpressing immunodominant multistage antigens of Ag85B and HspX, which are expressed by both rapid replicating and dormant M. tuberculosis. Long-term protective effect and immunogenicity of rBCG::XB were compared with the parental BCG in vaccinated C57BL/6 mice. Our results demonstrated that rBCG::XB provided the stronger and long-lasting protection against M. tuberculosis H37Rv intranasal infection than BCG. The rBCG::XB not only elicited the more durable multistage antigen-specific CD4(+)Th1-biased immune responses and specific polyfunctional CD4(+)T cells but also augmented the CD8(+) CTL effects against Ag85B in vivo. In particular, higher levels of CD4(+) TEM and CD8(+) TCM cells, dominated by IL2(+) CD4(+) and CD8(+) TCM cells, were obtained in the spleen of rBCG::XB vaccinated mice. Therefore, our findings indicate that rBCG::XB is a promising candidate to improve the efficacy of BCG.

B-Cell and T-Cell Immune Responses to Experimental Helicobacter pylori Infection in Humans

PubMed Central

Nurgalieva, Zhannat Z.; Conner, Margaret E.; Opekun, Antone R.; Zheng, Carl Q.; Elliott, Susan N.; Ernst, Peter B.; Osato, Michael; Estes, Mary K.; Graham, David Y.

2005-01-01

The acute antibody and T-cell immune response to Helicobacter pylori infection in humans has not been studied systematically. Serum from H. pylori-naive volunteers challenged with H. pylori and cured after 4 or 12 weeks was tested by enzyme-linked immunosorbent assays for anti-H. pylori-specific immunoglobulin M (IgM) and IgA established using bacterial lysates from homologous (the infecting strain) and heterologous H. pylori. Proteins recognized by IgM antibody were identified by mass spectrometry of immunoreactive bands separated by two-dimensional gel electrophoresis. Mucosal T-cell subsets (CD4, CD8, CD3, and CD30 cells) were assessed by immunohistochemistry. All 18 infected volunteers developed H. pylori-specific IgM responses to both homologous or heterologous H. pylori antigens. H. pylori antigens reacted with IgM antibody at 4 weeks postinfection. IgM Western blotting showed immunoreactivity of postinfection serum samples to multiple H. pylori proteins with molecular weights ranging between 9,000 (9K) to 150K with homologous strains but only a 70K band using heterologous antigens. Two-dimensional electrophoresis demonstrated that production of H. pylori-specific IgM antibodies was elicited by H. pylori flagellins A and B, urease B, ABC transporter binding protein, heat shock protein 70 (DnaK), and alkyl hydroperoxide reductase. Mucosal CD3, CD4, and CD8 T-cell numbers increased following infection. IgM antibody responses were detected to a range of homologous H. pylori antigens 2 to 4 weeks postchallenge. The majority of H. pylori proteins were those involved in motility and colonization and may represent targets for vaccine development. PMID:15845507

Evaluation of novel synthetic TLR7/8 agonists as vaccine adjuvants

PubMed Central

Smith, Alyson J.; Li, Yufeng; Bazin, Hélène G.; St-Jean, Julien R.; Larocque, Daniel; Evans, Jay T.; Baldridge, Jory R.

2016-01-01

Small-molecule adjuvants that boost and direct adaptive immunity provide a powerful means to increase the effectiveness of vaccines. Through rational design several novel imidazoquinoline and oxoadenine TLR7/8 agonists, each with unique molecular modifications, were synthesized and assessed for their ability to augment adaptive immunity. All agonists bound human TLR7 and TLR8 and induced maturation of both human mDCs and pDCs. All agonists prompted production of type I interferon and/or proinflammatory cytokines, albeit with varying potencies. In most in vitro assays, the oxoadenine class of agonists proved more potent than the imidazoquinolines. Therefore, an optimized oxoadenine TLR7/8 agonist that demonstrated maximal activity in the in vitro assays was further assessed in a vaccine study with the CRM197 antigen in a porcine model. Antigen-specific antibody production was greatly enhanced in a dose dependent manner, with antibody titers increased 800-fold compared to titers from pigs vaccinated with the non-adjuvanted vaccine. Moreover, pigs vaccinated with antigen containing the highest dose of adjuvant promoted a 13-fold increase in the percentage of antigen-specific CD3+/CD8+ T cells over pigs vaccinated with antigen alone. Together this work demonstrates the promise of these novel TLR7/8 agonists as effective human vaccine adjuvants. PMID:27402566

Frequency and reactivity of antigen-specific T cells were concurrently measured through the combination of artificial antigen-presenting cell, MACS and ELISPOT.

PubMed

Shen, Chuanlai; Xu, Tao; Wu, You; Li, Xiaoe; Xia, Lingzhi; Wang, Wei; Shahzad, Khawar Ali; Zhang, Lei; Wan, Xin; Qiu, Jie

2017-11-27

Conventional peptide-major histocompatibility complex (pMHC) multimer staining, intracellular cytokine staining, and enzyme-linked immunospot (ELISPOT) assay cannot concurrently determine the frequency and reactivity of antigen-specific T cells (AST) in a single assay. In this report, pMHC multimer, magnetic-activated cell sorting (MACS), and ELISPOT techniques have been integrated into a micro well by coupling pMHC multimers onto cell-sized magnetic beads to characterize AST cell populations in a 96-well microplate which pre-coated with cytokine-capture antibodies. This method, termed AAPC-microplate, allows the enumeration and local cytokine production of AST cells in a single assay without using flow cytometry or fluorescence intensity scanning, thus will be widely applicable. Here, ovalbumin 257-264 -specific CD8 + T cells from OT-1 T cell receptor (TCR) transgenic mice were measured. The methodological accuracy, specificity, reproducibility, and sensitivity in enumerating AST cells compared well with conventional pMHC multimer staining. Furthermore, the AAPC-microplate was applied to detect the frequency and reactivity of Hepatitis B virus (HBV) core antigen 18-27 - and surface antigen 183-191 -specific CD8 + T cells for the patients, and was compared with conventional method. This method without the need of high-end instruments may facilitate the routine analysis of patient-specific cellular immune response pattern to a given antigen in translational studies.

A monoclonal cytolytic T-lymphocyte response observed in a melanoma patient vaccinated with a tumor-specific antigenic peptide encoded by gene MAGE-3

PubMed Central

Coulie, Pierre G.; Karanikas, Vaios; Colau, Didier; Lurquin, Christophe; Landry, Claire; Marchand, Marie; Dorval, Thierry; Brichard, Vincent; Boon, Thierry

2001-01-01

Vaccination of melanoma patients with tumor-specific antigens recognized by cytolytic T lymphocytes (CTL) produces significant tumor regressions in a minority of patients. These regressions appear to occur in the absence of massive CTL responses. To detect low-level responses, we resorted to antigenic stimulation of blood lymphocyte cultures in limiting dilution conditions, followed by tetramer analysis, cloning of the tetramer-positive cells, and T-cell receptor (TCR) sequence analysis of the CTL clones that showed strict specificity for the tumor antigen. A monoclonal CTL response against a MAGE-3 antigen was observed in a melanoma patient, who showed partial rejection of a large metastasis after treatment with a vaccine containing only the tumor-specific antigenic peptide. Tetramer analysis after in vitro restimulation indicated that about 1/40,000 postimmunization CD8+ blood lymphocytes were directed against the antigen. The same TCR was present in all of the positive microcultures. TCR evaluation carried out directly on blood lymphocytes by PCR amplification led to a similar frequency estimate after immunization, whereas the TCR was not found among 2.5 × 106 CD8+ lymphocytes collected before immunization. Our results prove unambiguously that vaccines containing only a tumor-specific antigenic peptide can elicit a CTL response. Even though they provide no information about the effector mechanisms responsible for the observed reduction in tumor mass in this patient, they would suggest that low-level CTL responses can initiate tumor rejection. PMID:11517302

Role of heterogeneous cell population on modulation of dendritic cell phenotype and activation of CD8 T cells for use in cell-based immunotherapies.

PubMed

Frizzell, Hannah; Park, Jaehyung; Comandante Lou, Natacha; Woodrow, Kim A

2017-01-01

Dendritic cell (DC)-based immunotherapies have much utility in their ability to prime antigen-specific adaptive immune responses. However, there does not yet exist a consensus standard to how DCs should be primed. In this study, we aimed to determine the role of heterogeneous co-cultures, composed of both CD11c+ (DCs) and CD11c- cells, in combination with monophosphoryl lipid A (MPLA) stimulation on DC phenotype and function. Upon DC priming in different co-culture ratios, we observed reduced expression of MHCII and CD86 and increased antigen uptake among CD11c+ cells in a CD11c- dependent manner. DCs from all culture conditions were induced to mature by MPLA treatment, as determined by secretion of pro-inflammatory cytokines IL-12 and TNF-α. Antigen-specific stimulation of CD4+ T cells was not modulated by co-culture composition, in terms of proliferation nor levels of IFN-γ. However, the presence of CD11c- cells enhanced cross-presentation to CD8+ T cells compared to purified CD11c+ cells, resulting in increased cell proliferation along with higher IFN-γ production. These findings demonstrate the impact of cell populations present during DC priming, and point to the use of heterogeneous cultures of DCs and innate immune cells to enhance cell-mediated immunity. Copyright © 2016 Elsevier Inc. All rights reserved.

Viral Sequestration of Antigen Subverts Cross Presentation to CD8+ T Cells

PubMed Central

Tewalt, Eric F.; Grant, Jean M.; Granger, Erica L.; Palmer, Douglas C.; Heuss, Neal D.; Gregerson, Dale S.; Restifo, Nicholas P.; Norbury, Christopher C.

2009-01-01

Virus-specific CD8+ T cells (TCD8+) are initially triggered by peptide-MHC Class I complexes on the surface of professional antigen presenting cells (pAPC). Peptide-MHC complexes are produced by two spatially distinct pathways during virus infection. Endogenous antigens synthesized within virus-infected pAPC are presented via the direct-presentation pathway. Many viruses have developed strategies to subvert direct presentation. When direct presentation is blocked, the cross-presentation pathway, in which antigen is transferred from virus-infected cells to uninfected pAPC, is thought to compensate and allow the generation of effector TCD8+. Direct presentation of vaccinia virus (VACV) antigens driven by late promoters does not occur, as an abortive infection of pAPC prevents production of these late antigens. This lack of direct presentation results in a greatly diminished or ablated TCD8+ response to late antigens. We demonstrate that late poxvirus antigens do not enter the cross-presentation pathway, even when identical antigens driven by early promoters access this pathway efficiently. The mechanism mediating this novel means of viral modulation of antigen presentation involves the sequestration of late antigens within virus factories. Early antigens and cellular antigens are cross-presented from virus-infected cells, as are late antigens that are targeted to compartments outside of the virus factories. This virus-mediated blockade specifically targets the cross-presentation pathway, since late antigen that is not cross-presented efficiently enters the MHC Class II presentation pathway. These data are the first to describe an evasion mechanism employed by pathogens to prevent entry into the cross-presentation pathway. In the absence of direct presentation, this evasion mechanism leads to a complete ablation of the TCD8+ response and a potential replicative advantage for the virus. Such mechanisms of viral modulation of antigen presentation must also be taken into account during the rational design of antiviral vaccines. PMID:19478869

Lovastatin inhibits T cell proliferation while preserving the cytolytic function of EBV-, CMV- and MART-1-specific CTLs

PubMed Central

Li, Dan; Li, Yufeng; Hernandez, Jessica A.; Patenia, Rebecca; Kim, Tae Kon; Khalili, Jahan; Dougherty, Mark C.; Hanley, Patrick J.; Bollard, Catherine M.; Komanduri, Krishna V.; Hwu, Patrick; Champlin, Richard E.; Radvanyi, Laszlo G.; Molldrem, Jeffrey J.; Ma, Qing

2016-01-01

Statin treatment has been shown to reduce graft-versus-host disease (GVHD) while preserving graft-versus-tumor (GVT) effect in allogeneic stem cell transplantation (allo-HCT). Herein, we investigated whether lovastatin treatment affects the function of human cytolytic T lymphocytes (CTLs). Upon TCR stimulation, lovastatin significantly inhibited the proliferation of both CD4+ and CD8+ T cells from healthy donors while their intracellular cytokine production including IFN-γ and TNF-α remained the same with a slight decrease of IL-2. Moreover, the specific lysis of target cells by CTL lines derived from patients and normal donors specific for EBV-encoded antigen LMP2 or CMV-encoded antigen pp65 was uncompromised in the presence of lovastatin. In addition, we evaluated the effect of lovastatin on the proliferation and effector function of the CD8+ tumor–infiltrating lymphocytes (TILs) derived from melanoma patients specific for MART-1 antigen. Lovastatin significantly reduced the expansion of antigen-specific TILs upon MART-1 stimulation. However, the effector function of TILs, including the specific lysis of target cells and secretion of cytokine IFN-γ, remained intact with lovastatin treatment. Taken together, these data demonstrated that lovastatin inhibits the proliferation of EBV-, CMV- and MART-1-specific CTLs without affecting cytolytic capacity. The differential effect of lovastatin on the proliferation versus cytoxicity of CTLs might shed some light on elucidating the possible mechanisms of GVHD and GVT effect elicited by alloimmune responses. PMID:20948439

Enhancement of Gag-specific but reduction of Env- and Pol-specific CD8+ T cell responses by simian immunodeficiency virus nonstructural proteins in mice.

PubMed

Zhang, Yinfeng; Sun, Caijun; Feng, Liqiang; Xiao, Lijun; Chen, Ling

2012-04-01

Accessory and regulatory proteins (nonstructural proteins) have received increasing attention as components in novel HIV/SIV vaccine design. However, the complicated interactions between nonstructural proteins and structural proteins remain poorly understood, especially their effects on immunogenicity. In this study, the immunogenicity of structural proteins in the presence and absence of nonstructural proteins was compared. First, a series of recombinant plasmids and adenoviral vectors carrying various SIVmac239 nonstructural and structural genes was constructed. Then mice were primed with DNA plasmids and boosted with corresponding Ad5 vectors of different combinations, and the resulting immune responses were measured. Our results demonstrated that when the individual Gag, Pol, or Env gene products were coimmunized with the whole repertoire of nonstructural proteins, the Gag-specific CD8(+) T response was greatly enhanced, while the Env- and Pol-specific CD8(+) T responses were significantly reduced. The same pattern was not observed in CD4(+) T cell responses. Antibody responses against both the Gag and Env proteins were elicited more effectively when these structural antigens were immunized together with nonstructural antigens. These findings may provide helpful insights into the development of novel HIV/SIV vaccines.

Administration of HPV DNA vaccine via electroporation elicits the strongest CD8+ T cell immune responses compared to intramuscular injection and intradermal gene gun delivery

PubMed Central

Best, Simon R.; Peng, Shiwen; Juang, Chi-Mou; Hung, Chien-Fu; Hannaman, Drew; Saunders, John R.; Wu, T.-C.; Pai, Sara I.

2009-01-01

DNA vaccines are an attractive approach to eliciting antigen-specific immunity. Intracellular targeting of tumor antigens through its linkage to immunostimulatory molecules such as calreticulin (CRT) can improve antigen processing and presentation through the MHC Class I pathway and increase cytotoxic CD8+ T cell production. However, even with these enhancements, the efficacy of such immunotherapeutic strategies is dependent on the identification of an effective route and method of DNA administration. Electroporation and gene gun-mediated particle delivery are leading methods of DNA vaccine delivery that can generate protective and therapeutic levels of immune responses in experimental models. In this study, we perform a head-to-head comparison of three methods of vaccination – conventional intramuscular injection, electroporation mediated intramuscular delivery, and epidermal gene gun-mediated particle delivery - in the ability to generate antigen specific cytotoxic CD8+ T cell responses as well as anti-tumor immune responses against an HPV-16 E7 expressing tumor cell line using the pNGVL4a-CRT/E7(detox) DNA vaccine. Vaccination via electroporation generated the highest number of E7-specific cytotoxic CD8+ T cells, which correlated to improved outcomes in the treatment of growing tumors. In addition, we demonstrate that electroporation results in significantly higher levels of circulating protein compared to gene gun or intramuscular vaccination, which likely enhances calreticulin’s role as a local tumor anti-angiogenesis agent. We conclude that electroporation is a promising method for delivery of HPV DNA vaccines and should be considered for DNA vaccine delivery in human clinical trials. PMID:19622402

Upregulation of Interleukin 7 Receptor Alpha and Programmed Death 1 Marks an Epitope-Specific CD8+ T-Cell Response That Disappears following Primary Epstein-Barr Virus Infection▿ †

PubMed Central

Sauce, Delphine; Larsen, Martin; Abbott, Rachel J. M.; Hislop, Andrew D.; Leese, Alison M.; Khan, Naeem; Papagno, Laura; Freeman, Gordon J.; Rickinson, Alan B.

2009-01-01

In immunocompetent individuals, the stability of the herpesvirus-host balance limits opportunities to study the disappearance of a virus-specific CD8+ T-cell response. However, we noticed that in HLA-A*0201-positive infectious mononucleosis (IM) patients undergoing primary Epstein-Barr virus (EBV) infection, the initial CD8 response targets three EBV lytic antigen-derived epitopes, YVLDHLIVV (YVL), GLCTLVAML (GLC), and TLDYKPLSV (TLD), but only the YVL and GLC reactivities persist long-term; the TLD response disappears within 10 to 27 months. While present, TLD-specific cells remained largely indistinguishable from YVL and GLC reactivities in many phenotypic and functional respects but showed unique temporal changes in two markers of T-cell fate, interleukin 7 receptor alpha (IL-7Rα; CD127) and programmed death 1 (PD-1). Thus, following the antigen-driven downregulation of IL-7Rα seen on all populations in acute IM, in every case, the TLD-specific population recovered expression unusually quickly post-IM. As well, in four of six patients studied, TLD-specific cells showed very strong PD-1 upregulation in the last blood sample obtained before the cells’ disappearance. Our data suggest that the disappearance of this individual epitope reactivity from an otherwise stable EBV-specific response (i) reflects a selective loss of cognate antigen restimulation (rather than of IL-7-dependent signals) and (ii) is immediately preceded, and perhaps mediated, by PD-1 upregulation to unprecedented levels. PMID:19605492

Aberrant phenotypes in peripheral T cell lymphomas.

PubMed Central

Hastrup, N; Ralfkiaer, E; Pallesen, G

1989-01-01

Seventy six peripheral T cell lymphomas were examined immunohistologically to test their reactivity with a panel of monoclonal antibodies against 11 T cell associated antigens (CD1-8, CD27, UCHL1, and the T cell antigen receptor). Sixty two (82%) lymphomas showed aberrant phenotypes, and four main categories were distinguished as follows: (i) lack of one or several pan-T cell antigens (49, 64% of the cases); (ii) loss of both the CD4 and CD8 antigens (11, 15% of the cases); (iii) coexpression of the CD4 and CD8 antigens (13, 17% of the cases); and (iv) expression of the CD1 antigen (eight, 11% of the cases). No correlation was seen between the occurrence of aberrant phenotypes and the histological subtype. It is concluded that the demonstration of an aberrant phenotype is a valuable supplement to histological assessment in the diagnosis of peripheral T cell lymphomas. It is recommended that the panel of monoclonal antibodies against T cell differentiation antigens should be fairly large, as apparently any antigen may be lost in the process of malignant transformation. Images Figure PMID:2469701

pH-Responsive Nanoparticle Vaccines for Dual-Delivery of Antigens and Immunostimulatory Oligonucleotides

PubMed Central

Wilson, John T.; Keller, Salka; Manganiello, Matthew J.; Cheng, Connie; Lee, Chen-Chang; Opara, Chinonso; Convertine, Anthony; Stayton, Patrick S.

2013-01-01

Protein subunit vaccines offer important potential advantages over live vaccine vectors, but generally elicit weaker and shorter-lived cellular immune responses. Here we investigate the use of pH-responsive, endosomolytic polymer nanoparticles that were originally developed for RNA delivery as vaccine delivery vehicles for enhancing cellular and humoral immune responses. Micellar nanoparticles were assembled from amphiphilic diblock copolymers composed of an ampholytic core-forming block and a re-designed polycationic corona block doped with thiol-reactive pyridyl disulfide groups to enable dual-delivery of antigens and immunostimulatory CpG oligodeoxynucleotide (CpG ODN) adjuvants. Polymers assembled into 23 nm particles with simultaneous packaging of CpG ODN and a thiolated protein antigen, ovalbumin (ova). Conjugation of ova to nanoparticles significantly enhanced antigen cross-presentation in vitro relative to free ova or an unconjugated, physical mixture of the parent compounds. Subcutaneous vaccination of mice with ova-nanoparticle conjugates elicited a significantly higher CD8+ T cell response (0.5% IFN-ɣ+ of CD8+) compared to mice vaccinated with free ova or a physical mixture of the two components. Significantly, immunization with ova-nanoparticle conjugates electrostatically complexed with CpG ODN (dual-delivery) enhanced CD8+ T cell responses (3.4% IFN-ɣ+ of CD8+) 7-, 18-, and 8-fold relative to immunization with conjugates, ova administered with free CpG, or a formulation containing free ova and CpG complexed to micelles, respectively. Similarly, dual-delivery carriers significantly increased CD4+IFN-ɣ+ (Th1) responses, and elicited a balanced IgG1/IgG2c antibody response. Intradermal administration further augmented cellular immune responses, with dual-delivery carriers inducing ~7% antigen-specific CD8+ T cells. This work demonstrates the ability of pH-responsive, endosomolytic nanoparticles to actively promote antigen cross-presentation and augment cellular and humoral immune responses via dual-delivery of protein antigens and CpG ODN. Hence, pH-responsive polymeric nanoparticles offer promise as a delivery platform for protein subunit vaccines. PMID:23590591

A high molecular weight-melanoma associated antigen-specific chimeric antigen receptor redirects lymphocytes to target human melanomas

PubMed Central

Burns, William R.; Zhao, Yangbing; Frankel, Timothy L.; Hinrichs, Christian S.; Zheng, Zhili; Xu, Hui; Feldman, Steven A.; Ferrone, Soldano; Rosenberg, Steven A.; Morgan, Richard A.

2011-01-01

Immunotherapy, particularly the adoptive cell transfer (ACT) of tumor infiltrating lymphocytes (TIL), is a very promising therapy for metastatic melanoma. Some patients unable to receive TIL have been successfully treated with autologous peripheral blood lymphocytes (PBL), genetically modified to express HLA class I antigen restricted, melanoma antigen-reactive T-cell receptors; however, substantial numbers of patients remain ineligible due to the lack of expression of the restricting HLA class I allele. We sought to overcome this limitation by designing a non-MHC-restricted, chimeric antigen receptor (CAR) targeting the high molecular weight-melanoma associated antigen (HMW-MAA), which is highly expressed on over 90% of human melanomas but has a restricted distribution in normal tissues. HMW-MAA-specific CARs containing an antigen recognition domain based on variations of the HMW-MAA-specific monoclonal antibody (mAb) 225.28S and a T-cell activation domain based on combinations of CD28, 4-1BB, and CD3ζ activation motifs were constructed within a retroviral vector to allow stable gene transfer into cells and their progeny. Following optimization of the HMW-MAA-specific CAR for expression and function in human PBL, these gene-modified T cells secreted cytokines, were cytolytic, and proliferated in response to HMW-MAA expressing cell lines. Furthermore, the receptor functioned in both CD4+ and CD8+ cells, was non-MHC-restricted, and reacted against explanted human melanomas. To evaluate this HMW-MAA-specific CAR in patients with metastatic melanoma, we developed a clinical-grade retroviral packaging line. This may represent a novel means to treat the majority of patients with advanced melanoma, most notably those unable to receive current ACT therapies. PMID:20395199

Public clonotype usage identifies protective Gag-specific CD8+ T cell responses in SIV infection

PubMed Central

Asher, Tedi E.; Wilson, Nancy A.; Nason, Martha C.; Brenchley, Jason M.; Metzler, Ian S.; Venturi, Vanessa; Gostick, Emma; Chattopadhyay, Pratip K.; Roederer, Mario; Davenport, Miles P.; Watkins, David I.; Douek, Daniel C.

2009-01-01

Despite the pressing need for an AIDS vaccine, the determinants of protective immunity to HIV remain concealed within the complexity of adaptive immune responses. We dissected immunodominant virus-specific CD8+ T cell populations in Mamu-A*01+ rhesus macaques with primary SIV infection to elucidate the hallmarks of effective immunity at the level of individual constituent clonotypes, which were identified according to the expression of distinct T cell receptors (TCRs). The number of public clonotypes, defined as those that expressed identical TCR β-chain amino acid sequences and recurred in multiple individuals, contained within the acute phase CD8+ T cell population specific for the biologically constrained Gag CM9 (CTPYDINQM; residues 181–189) epitope correlated negatively with the virus load set point. This independent molecular signature of protection was confirmed in a prospective vaccine trial, in which clonotype engagement was governed by the nature of the antigen rather than the context of exposure and public clonotype usage was associated with enhanced recognition of epitope variants. Thus, the pattern of antigen-specific clonotype recruitment within a protective CD8+ T cell population is a prognostic indicator of vaccine efficacy and biological outcome in an AIDS virus infection. PMID:19349463

Ocular surface epithelium induces expression of human mucosal lymphocyte antigen (HML-1) on peripheral blood lymphocytes

PubMed Central

Gomes, J A P; Dua, H S; Rizzo, L V; Nishi, M; Joseph, A; Donoso, L A

2004-01-01

Background/aims: Peripheral blood CD8+ lymphocytes that home to mucosal surfaces express the human mucosal lymphocyte antigen (HML-1). At mucosal surfaces, including the ocular surface, only intraepithelial CD8+ lymphocytes express HML-1. These lymphocytes are retained in the intraepithelial compartment by virtue of the interaction between HML-1 and its natural ligand, E-cadherin, which is expressed on epithelial cells. The purpose of this study was to determine whether ocular surface epithelial cells (ocular mucosa) could induce the expression of human mucosal lymphocyte antigen on peripheral blood lymphocytes. Methods: Human corneal and conjunctival epithelial cells were co-cultured with peripheral blood lymphocytes. Both non-activated and activated lymphocytes were used in the experiments. After 7 days of incubation, lymphocytes were recovered and analysed for the antigens CD8/HML-1, CD4/HML-1, CD3/CD8, CD3/CD4, CD3/CD25, CD8/CD25, and CD4/CD25 by flowcytometry. Results: Significant statistical differences were observed in the CD8/HML-1 expression when conjunctival epithelial cells were co-cultured with non-activated and activated lymphocytes (p = 0.04 for each) and when corneal epithelial cells were co-cultured with non-activated lymphocytes (p = 0.03). Significant statistical difference in CD4/HML-1 expression was observed only when conjunctival epithelial cells were co-cultured with activated lymphocytes (p = 0.02). Conclusion: Ocular surface epithelial cells can induce the expression of human mucosal lymphocyte antigen on CD8+ (and to some extent on CD4+) lymphocytes. This may allow the retention of CD8+ and CD4+ lymphocytes within the epithelial compartment of the conjunctiva and play a part in mucosal homing of lymphocytes. PMID:14736792

Antigen Cross-Presentation of Immune Complexes

PubMed Central

Platzer, Barbara; Stout, Madeleine; Fiebiger, Edda

2014-01-01

The ability of dendritic cells (DCs) to cross-present tumor antigens has long been a focus of interest to physicians, as well as basic scientists, that aim to establish efficient cell-based cancer immune therapy. A prerequisite for exploiting this pathway for therapeutic purposes is a better understanding of the mechanisms that underlie the induction of tumor-specific cytotoxic T-lymphocyte (CTL) responses when initiated by DCs via cross-presentation. The ability of humans DC to perform cross-presentation is of utmost interest, as this cell type is a main target for cell-based immunotherapy in humans. The outcome of a cross-presentation event is guided by the nature of the antigen, the form of antigen uptake, and the subpopulation of DCs that performs presentation. Generally, CD8α+ DCs are considered to be the most potent cross-presenting DCs. This paradigm, however, only applies to soluble antigens. During adaptive immune responses, immune complexes form when antibodies interact with their specific epitopes on soluble antigens. Immunoglobulin G (IgG) immune complexes target Fc-gamma receptors on DCs to shuttle exogenous antigens efficiently into the cross-presentation pathway. This receptor-mediated cross-presentation pathway is a well-described route for the induction of strong CD8+ T cell responses. IgG-mediated cross-presentation is intriguing because it permits the CD8− DCs, which are commonly considered to be weak cross-presenters, to efficiently cross-present. Engaging multiple DC subtypes for cross-presentation might be a superior strategy to boost CTL responses in vivo. We here summarize our current understanding of how DCs use IgG-complexed antigens for the efficient induction of CTL responses. Because of its importance for human cell therapy, we also review the recent advances in the characterization of cross-presentation properties of human DC subsets. PMID:24744762

The CD3-Zeta Chimeric Antigen Receptor Overcomes TCR Hypo-Responsiveness of Human Terminal Late-Stage T Cells

PubMed Central

Awerkiew, Sabine; Schmidt, Annette; Hombach, Andreas A.; Pfister, Herbert; Abken, Hinrich

2012-01-01

Adoptive therapy of malignant diseases with tumor-specific cytotoxic T cells showed remarkable efficacy in recent trials. Repetitive T cell receptor (TCR) engagement of target antigen, however, inevitably ends up in hypo-responsive cells with terminally differentiated KLRG-1+ CD57+ CD7− phenotype limiting their therapeutic efficacy. We here revealed that hypo-responsiveness of CMV-specific late-stage CD8+ T cells is due to reduced TCR synapse formation compared to younger cells. Membrane anchoring of TCR components contributes to T cell hypo-responsiveness since dislocation of galectin-3 from the synapse by swainsonine restored both TCR synapse formation and T cell response. Transgenic expression of a CD3-zeta signaling chimeric antigen receptor (CAR) recovered hypo-responsive T cells to full effector functions indicating that the defect is restricted to TCR membrane components while synapse formation of the transgenic CAR was not blocked. CAR engineered late-stage T cells released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells. Our data provide a rationale for TCR independent, CAR mediated activation in the adoptive cell therapy to avoid hypo-responsiveness of late-stage T cells upon repetitive antigen encounter. PMID:22292024

Uptake routes of tumor-antigen MAGE-A3 by dendritic cells determine priming of naïve T-cell subtypes.

PubMed

Moeller, Ines; Spagnoli, Giulio C; Finke, Jürgen; Veelken, Hendrik; Houet, Leonora

2012-11-01

Induction of tumor-antigen-specific T cells in active cancer immunotherapy is generally difficult due to the very low anti-tumoral precursor cytotoxic T cells. By improving tumor-antigen uptake and presentation by dendritic cells (DCs), this problem can be overcome. Focusing on MAGE-A3 protein, frequently expressed in many types of tumors, we analyzed different DC-uptake routes after additional coating the recombinant MAGE-A3 protein with either a specific monoclonal antibody or an immune complex formulation. Opsonization of the protein with antibody resulted in increased DC-uptake compared to the uncoated rhMAGE-A3 protein. This was partly due to Fcγ receptor-dependent internalization. However, unspecific antigen internalization via macropinocytosis also played a role. When analyzing DC-uptake of MAGE-A3 antigen expressed in multiple myeloma cell line U266, pretreatment with proteasome inhibitor bortezomib resulted in increased apoptosis compared to γ-irradiation. Bortezomib-mediated immunogenic apoptosis, characterized by elevated surface expression of hsp90, triggered higher phagocytosis of U266 cells by DCs involving specific DC-derived receptors. We further investigated the impact of antigen delivery on T-cell priming. Induction of CD8(+) T-cell response was favored by stimulating naïve T cells with either antibody-opsonized MAGE-A3 protein or with the bortezomib-pretreated U266 cells, indicating that receptor-mediated uptake favors cross-presentation of antigens. In contrast, CD4(+) T cells were preferentially induced after stimulation with the uncoated protein or protein in the immune complex, both antigen formulations were preferentially internalized by DCs via macropinocytosis. In summary, receptor-mediated DC-uptake mechanisms favored the induction of CD8(+) T cells, relevant for clinical anti-tumor response.

The potential association of CMV-specific CD8+ T lymphocyte reconstitution with the risk of CMV reactivation and persistency in post allogeneic stem cell transplant patients.

PubMed

Shams El-Din, Ahmed Ali; El-Desoukey, Nermeen Ahmed; Amin Tawadrous, Dalia Gamil; Fouad, Neveen Mohammed Baha El-Din; Abdel-Mooti, Mohammed; Hotar, Said Fathy

2018-01-09

development of cytomegalovirus (CMV)-specific CD8+ T cell response is crucial in preventing symptomatic CMV infection specially, in stem cell transplant (SCT) patients. The aim of this study was to evaluate CMV-specific CD8+ T cell reconstitution in allogeneic SCT recipients and to study the possible association between CMV-specific CD8+ T cell recovery with protection from CMV reactivation and persistency. Human leuKocyte antigen (HLA)-tetramers were used for CMV-specific CD8+ cell quantitation by Flow cytometry in twenty post-allogeneic SCT patients. Nine patients (45%) developed rapid recovery of CMV-specific CD8+ cells, among them; 7 patients (78%) had no CMV reactivation in the first 95 days post-transplant. Five patients had developed persistent CMV viremia; all of them had not developed CMV-specific CD8+ recovery till day 95 post-transplant. Patients with persistent CMV viremia had a statistically significant lower means of CMV-specific CD8+ percent and absolute count compared to those without persistent viremia (p = .001, .015), respectively. The incidence of CMV reactivation and persistency was higher among patients with delayed CMV-specific CD8+ reconstitution in the first 95 days post-transplant. CMV-specific CD8+ cells can help in categorizing patients into risk groups: (early recovery/low risk) and (delayed recovery/increased risk), this tool may guide clinicians in the selection of patients who may profit from prophylactic antiviral therapy and frequent viral monitoring.

PLAC1-specific TCR-engineered T cells mediate antigen-specific antitumor effects in breast cancer

PubMed Central

Li, Qiongshu; Liu, Muyun; Wu, Man; Zhou, Xin; Wang, Shaobin; Hu, Yuan; Wang, Youfu; He, Yixin; Zeng, Xiaoping; Chen, Junhui; Liu, Qubo; Xiao, Dong; Hu, Xiang; Liu, Weibin

2018-01-01

Placenta-specific 1 (PLAC1), a novel cancer-testis antigen (CTA), is expressed in a number of different human malignancies. It is frequently produced in breast cancer, serving a function in tumorigenesis. Adoptive immunotherapy using T cell receptor (TCR)-engineered T cells against CTA mediates objective tumor regression; however, to the best of our knowledge, targeting PLAC1 using engineered T cells has not yet been attempted. In the present study, the cDNAs encoding TCRα- and β-chains specific for human leukocyte antigen (HLA)-A*0201-restricted PLAC1 were cloned from a cytotoxic T-lymphocyte, generated by in vitro by the stimulation of CD8+ T cells using autologous HLA-A2+ dendritic cells loaded with a PLAC1-specific peptide (p28-36, VLCSIDWFM). The TCRα/β-chains were linked by a 2A peptide linker (TCRα-Thosea asigna virus-TCRβ), and the constructs were cloned into the lentiviral vector, followed by transduction into human cytotoxic (CD8+) T cells. The efficiency of transduction was up to 25.16%, as detected by PLAC1 multimers. TCR-transduced CD8+ T cells, co-cultured with human non-metastatic breast cancer MCF-7 cells (PLAC1+, HLA-A2+) and triple-negative breast cancer MDAMB-231 cells (PLAC1+, HLA-A2+), produced interferon γ and tumor necrosis factor α, suggesting TCR activation. Furthermore, the PLAC1 TCR-transduced CD8+ T cells efficiently and specifically identified and annihilated the HLA-A2+/PLAC1+ breast cancer cell lines in a lactate dehydrogenase activity assay. Western blot analysis demonstrated that TCR transduction stimulated the production of mitogen-activated protein kinase signaling molecules, extracellular signal-regulated kinases 1/2 and nuclear factor-κB, through phosphoinositide 3-kinase γ-mediated phosphorylation of protein kinase B in CD8+ T cells. Xenograft mouse assays revealed that PLAC1 TCR-transduced CD8+T cells significantly delayed the tumor progression in mice-bearing breast cancer compared with normal saline or negative control-transduced groups. In conclusion, a novel HLA-A2-restricted and PLAC1-specific TCR was identified. The present study demonstrated PLAC1 to be a potential target for breast cancer treatment; and the usage of PLAC1-specific TCR-engineered T cells may be a novel strategy for PLAC1-positive breast cancer treatment. PMID:29556312

Just-in-time vaccines: Biomineralized calcium phosphate core-immunogen shell nanoparticles induce long-lasting CD8+ T cell responses in mice

PubMed Central

Zhou, Weibin; Moguche, Albanus; Chiu, David; Murali-Krishna, Kaja; Baneyx, François

2014-01-01

Distributed and on-demand vaccine production could be game-changing for infectious disease treatment in the developing world by providing new therapeutic opportunities and breaking the refrigeration “cold chain”. Here, we show that a fusion protein between a calcium phosphate binding domain and the model antigen ovalbumin can mineralize a biocompatible adjuvant in a single step. The resulting 50 nm calcium phosphate core-immunogen shell particles are comparable to soluble protein in inducing ovalbumin-specific antibody response and class switch recombination in mice. However, single dose vaccination with nanoparticles leads to higher expansion of ovalbumin-specific CD8+ T cells upon challenge with an influenza virus bearing the ovalbumin-derived SIINFEKL peptide, and these cells produce high levels of IFN-γ. Furthermore, mice exhibit a robust antigen-specific CD8+ T cell recall response when challenged with virus 8 months post-immunization. These results underscore the promise of immunogen-controlled adjuvant mineralization for just-in-time manufacturing of effective T cell vaccines. PMID:24275478

Recognition and killing of brain tumor stem-like initiating cells by CD8+ cytolytic T cells.

PubMed

Brown, Christine E; Starr, Renate; Martinez, Catalina; Aguilar, Brenda; D'Apuzzo, Massimo; Todorov, Ivan; Shih, Chu-Chih; Badie, Behnam; Hudecek, Michael; Riddell, Stanley R; Jensen, Michael C

2009-12-01

Solid tumors contain a subset of stem-like cells that are resistant to the cytotoxic effects of chemotherapy/radiotherapy, but their susceptibility to cytolytic T lymphocyte (CTL) effector mechanisms has not been well characterized. Using a panel of early-passage human brain tumor stem/initiating cell (BTSC) lines derived from high-grade gliomas, we show that BTSCs are subject to immunologic recognition and elimination by CD8(+) CTLs. Compared with serum-differentiated CD133(low) tumor cells and established glioma cell lines, BTSCs are equivalent with respect to expression levels of HLA class I and ICAM-1, similar in their ability to trigger degranulation and cytokine synthesis by antigen-specific CTLs, and equally susceptible to perforin-dependent CTL-mediated cytolysis. BTSCs are also competent in the processing and presentation of antigens as evidenced by the killing of these cells by CTL when antigen is endogenously expressed. Moreover, we show that CTLs can eliminate all BTSCs with tumor-initiating activity in an antigen-specific manner in vivo. Current models predict that curative therapies for many cancers will require the elimination of the stem/initiating population, and these studies lay the foundation for developing immunotherapeutic approaches to eradicate this tumor population.

Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

PubMed Central

Anthony, Donald D.; Milkovich, Kimberly A.; Zhang, Wenji; Rodriguez, Benigno; Yonkers, Nicole L.; Tary-Lehmann, Magdalena; Lehmann, Paul V.

2012-01-01

Chronic allograft rejection is in part mediated by host T cells that recognize allogeneic antigens on transplanted tissue. One factor that determines the outcome of a T cell response is clonal size, while another is the effector quality. Studies of alloimmune predictors of transplant graft survival have most commonly focused on only one measure of the alloimmune response. Because differing qualities and frequencies of the allospecific T cell response may provide distinctly different information we analyzed the relationship between frequency of soluble antigen and allo-antigen specific memory IFN-γ secreting CD4 and CD8 T cells, their ability to secrete IL-2, and their proliferative capacity, while accounting for cognate and bystander proliferation. The results show proliferative responses primarily reflect on IL-2 production by antigen-specific T cells, and that proliferating cells in such assays entail a considerable fraction of bystander cells. On the other hand, proliferation (and IL-2 production) did not reflect on the frequency of IFN-γ producing memory cells, a finding particularly accentuated in the CD8 T cell compartment. These data provide rationale for considering both frequency and effector function of pre-transplant T cell reactivity when analyzing immune predictors of graft rejection. PMID:24710419

Enhancement of dendritic cell-based vaccine potency by anti-apoptotic siRNAs targeting key pro-apoptotic proteins in cytotoxic CD8(+) T cell-mediated cell death.

PubMed

Kim, Jin Hee; Kang, Tae Heung; Noh, Kyung Hee; Bae, Hyun Cheol; Kim, Seok-Ho; Yoo, Young Do; Seong, Seung-Yong; Kim, Tae Woo

2009-01-29

Dendritic cells (DCs) have become an important measure for the treatment of malignancies. Current DC preparations, however, generate short-lived DCs because they are subject to cell death from various apoptotic pressures. Antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) is one of the main obstacles to limit the DC-mediated immune priming since CTLs can recognize the target antigen expressing DCs as target cells and kill the DCs. CTLs secret perforin and serine protease granzymes during CTL killing. Perforin and serine protease granzymes induce the release of a number of mitochondrial pro-apoptotic factors, which are controlled by members of the BCL-2 family, such as BAK, BAX and BIM. FasL linking to Fas on DCs triggers the activation of caspase-8, which eventually leads to mitochondria-mediated apoptosis via truncation of BID. In this study, we tried to enhance the DC priming capacity by prolonging DC survival using anti-apoptotic siRNA targeting these key pro-apoptotic molecules in CTL killing. Human papillomavirus (HPV)-16 E7 antigen presenting DCs that were transfected with these anti-apoptotic siRNAs showed increased resistance to T cell-mediated death, leading to enhanced E7-specific CD8(+) T cell activation in vitro and in vivo. Among them, siRNA targeting BIM (siBIM) generated strongest E7-specific E7-specific CD8(+) T cell immunity. More importantly, vaccination with E7 presenting DCs transfected with siBIM was capable of generating a marked therapeutic effect in vaccinated mice. Our data indicate that ex vivo manipulation of DCs with siBIM may represent a plausible strategy for enhancing dendritic cell-based vaccine potency.

Loss of DNAM-1 contributes to CD8+ T cell exhaustion in chronic HIV-1 infection

PubMed Central

Cella, Marina; Presti, Rachel; Vermi, William; Lavender, Kerry; Turnbull, Emma; Ochsenbauer-Jambor, Christina; Kappes, John C.; Ferrari, Guido; Kessels, Lisa; Williams, Ian; McMichael, Andrew J.; Haynes, Barton F.; Borrow, Persephone; Colonna, Marco

2011-01-01

Summary The hallmark of chronic viral infections is a progressive exhaustion of antigen specific CD8+ T cells that leads to persisting viral replication. It is generally believed that exhaustion is a consequence of the accumulation of multiple inhibitory receptors on CD8+ T cells that makes them dysfunctional. Here we show that during human chronic HIV-1 infection a CD8+ T cell positive costimulatory pathway mediated by DNAM-1 is also disrupted. Thus, DNAM-1 downregulation on CD8+ T cells aggravates the impairment of CTL effector function in chronic HIV-1 infection. PMID:20201043

Oligoclonal CD8+ T-cell expansion in patients with chronic hepatitis C is associated with liver pathology and poor response to interferon-alpha therapy.

PubMed

Manfras, Burkhard J; Weidenbach, Hans; Beckh, Karl-Heinz; Kern, Peter; Möller, Peter; Adler, Guido; Mertens, Thomas; Boehm, Bernhard O

2004-05-01

The role of CD8(+) T lymphocytes in chronic hepatitis C virus (HCV) infection and in liver injury with subsequent development of fibrosis and cirrhosis is poorly understood. To address this question, we performed a follow-up study including 27 chronically HCV-infected individuals. We determined clonality and phenotypes of circulating CD8(+) T cells employing TCRBV spectratyping. Antigen specificity was tested by rMHC-peptide tetramer staining and stimulation with recombinant HCV antigens. In addition, T-cell clonality and phenotypes were followed during the variable clinical response of interferon- (IFN) alpha treatment. We could demonstrate that CD8(+) T-cell expansions were significantly associated with liver fibrosis and cirrhosis. Likewise, increased oligoclonality of circulating CD8(+) T cells in chronic HCV infection was identified as an indicator for poor clinical response to IFN-alpha therapy. Moreover, we also found that IFN-alpha therapy enhanced the differentiation of CD8(+) T cells towards a late differentiation phenotype (CD28(-) CD57(+)). In cases of virus elimination the disappearance of expanded terminally differentiated CD8(+) cells was observed. Thus, this study identifies an association of clonal expansions of circulating CD8(+) T cells with liver pathology and provides a possible explanation for the fact that response to IFN-alpha therapy diminishes with the duration of infection.

Antigen-capturing nanoparticles improve the abscopal effect and cancer immunotherapy

NASA Astrophysics Data System (ADS)

Min, Yuanzeng; Roche, Kyle C.; Tian, Shaomin; Eblan, Michael J.; McKinnon, Karen P.; Caster, Joseph M.; Chai, Shengjie; Herring, Laura E.; Zhang, Longzhen; Zhang, Tian; Desimone, Joseph M.; Tepper, Joel E.; Vincent, Benjamin G.; Serody, Jonathan S.; Wang, Andrew Z.

2017-09-01

Immunotherapy holds tremendous promise for improving cancer treatment. To administer radiotherapy with immunotherapy has been shown to improve immune responses and can elicit the 'abscopal effect'. Unfortunately, response rates for this strategy remain low. Herein we report an improved cancer immunotherapy approach that utilizes antigen-capturing nanoparticles (AC-NPs). We engineered several AC-NP formulations and demonstrated that the set of protein antigens captured by each AC-NP formulation is dependent on the NP surface properties. We showed that AC-NPs deliver tumour-specific proteins to antigen-presenting cells (APCs) and significantly improve the efficacy of αPD-1 (anti-programmed cell death 1) treatment using the B16F10 melanoma model, generating up to a 20% cure rate compared with 0% without AC-NPs. Mechanistic studies revealed that AC-NPs induced an expansion of CD8+ cytotoxic T cells and increased both CD4+T/Treg and CD8+T/Treg ratios (Treg, regulatory T cells). Our work presents a novel strategy to improve cancer immunotherapy with nanotechnology.

CD8 down-regulation and functional impairment of SIV-specific cytotoxic T lymphocytes in lymphoid and mucosal tissues during SIV infection.

PubMed

Xu, Huanbin; Wang, Xiaolei; Lackner, Andrew A; Veazey, Ronald S

2013-06-01

Functional impairment of virus-specific T cells is a hallmark of HIV/SIV infection, but the underlying mechanisms of this dysfunction are not well understood. To address this, we simultaneously analyzed the expression and intensity of CD8 and inhibitory PD-1 on CTL in blood and lymphoid tissues in SIV-infected rhesus macaques. The intensity (mean channel fluorescence) of CD8 expression was transiently down-regulated in early SIV infection (10-14 dpi), despite an increase in CD8(+) T cell proliferation. In chronic infection, CD8 expression was maintained at low levels on CD8(+) T cells in all tissues. Interestingly, Gag-specific CTLs were clearly divided into CD8high- and CD8low-expressing populations in SIV-infected macaques, and CD8low Gag-specific cells increased with disease progression, especially in lymphoid tissues when compared with peripheral blood or in Gag-vaccinated controls. Moreover, the CD8low CTL population secreted lower levels of cytokines upon SIV antigen stimulation and exhibited lower proliferative capacity during infection compared with the CD8high CTL population. Meanwhile, intensity of PD-1 expression on Gag-specific CTL in chronic infection was significantly higher than in acute SIV infection, although the frequencies of PD-1+ Gag-specific cells were similar in acute and chronic stages. In summary, down-regulation of CD8 expression and higher expression of PD-1 on SIV-specific CTLs could coordinately attenuate SIV-specific CTL responses and their ability to recognize virus-infected target cells, especially in lymphoid tissues, resulting in failure to contain viremia, and continued persistence and replication of HIV in lymphoid tissue reservoirs.

Costimulatory Effects of an Immunodominant Parasite Antigen Paradoxically Prevent Induction of Optimal CD8 T Cell Protective Immunity.

PubMed

Eickhoff, Christopher S; Zhang, Xiuli; Vasconcelos, Jose R; Motz, R Geoffrey; Sullivan, Nicole L; O'Shea, Kelly; Pozzi, Nicola; Gohara, David W; Blase, Jennifer R; Di Cera, Enrico; Hoft, Daniel F

2016-09-01

Trypanosoma cruzi infection is controlled but not eliminated by host immunity. The T. cruzi trans-sialidase (TS) gene superfamily encodes immunodominant protective antigens, but expression of altered peptide ligands by different TS genes has been hypothesized to promote immunoevasion. We molecularly defined TS epitopes to determine their importance for protection versus parasite persistence. Peptide-pulsed dendritic cell vaccination experiments demonstrated that one pair of immunodominant CD4+ and CD8+ TS peptides alone can induce protective immunity (100% survival post-lethal parasite challenge). TS DNA vaccines have been shown by us (and others) to protect BALB/c mice against T. cruzi challenge. We generated a new TS vaccine in which the immunodominant TS CD8+ epitope MHC anchoring positions were mutated, rendering the mutant TS vaccine incapable of inducing immunity to the immunodominant CD8 epitope. Immunization of mice with wild type (WT) and mutant TS vaccines demonstrated that vaccines encoding enzymatically active protein and the immunodominant CD8+ T cell epitope enhance subdominant pathogen-specific CD8+ T cell responses. More specifically, CD8+ T cells from WT TS DNA vaccinated mice were responsive to 14 predicted CD8+ TS epitopes, while T cells from mutant TS DNA vaccinated mice were responsive to just one of these 14 predicted TS epitopes. Molecular and structural biology studies revealed that this novel costimulatory mechanism involves CD45 signaling triggered by enzymatically active TS. This enhancing effect on subdominant T cells negatively regulates protective immunity. Using peptide-pulsed DC vaccination experiments, we have shown that vaccines inducing both immunodominant and subdominant epitope responses were significantly less protective than vaccines inducing only immunodominant-specific responses. These results have important implications for T. cruzi vaccine development. Of broader significance, we demonstrate that increasing breadth of T cell epitope responses induced by vaccination is not always advantageous for host immunity.

Evaluation of novel synthetic TLR7/8 agonists as vaccine adjuvants.

PubMed

Smith, Alyson J; Li, Yufeng; Bazin, Hélène G; St-Jean, Julien R; Larocque, Daniel; Evans, Jay T; Baldridge, Jory R

2016-08-05

Small-molecule adjuvants that boost and direct adaptive immunity provide a powerful means to increase the effectiveness of vaccines. Through rational design several novel imidazoquinoline and oxoadenine TLR7/8 agonists, each with unique molecular modifications, were synthesized and assessed for their ability to augment adaptive immunity. All agonists bound human TLR7 and TLR8 and induced maturation of both human mDCs and pDCs. All agonists prompted production of type I interferon and/or proinflammatory cytokines, albeit with varying potencies. In most in vitro assays, the oxoadenine class of agonists proved more potent than the imidazoquinolines. Therefore, an optimized oxoadenine TLR7/8 agonist that demonstrated maximal activity in the in vitro assays was further assessed in a vaccine study with the CRM197 antigen in a porcine model. Antigen-specific antibody production was greatly enhanced in a dose dependent manner, with antibody titers increased 800-fold compared to titers from pigs vaccinated with the non-adjuvanted vaccine. Moreover, pigs vaccinated with antigen containing the highest dose of adjuvant promoted a 13-fold increase in the percentage of antigen-specific CD3(+)/CD8(+) T cells over pigs vaccinated with antigen alone. Together this work demonstrates the promise of these novel TLR7/8 agonists as effective human vaccine adjuvants. Copyright © 2016 Elsevier Ltd. All rights reserved.

CD8+ T-cell immunosurveillance constrains lymphoid pre-metastatic myeloid cell accumulation

PubMed Central

Li, Wenzhao; Deng, Jiehui; Herrmann, Andreas; Priceman, Saul J.; Liang, Wei; Shen, Shudan; Pal, Sumanta K.; Hoon, Dave S.B.; Yu, Hua

2014-01-01

Increasing evidence suggests that pre-metastatic niches, consisting mainly of myeloid cells, provide microenvironment critical for cancer cell recruitment and survival to facilitate metastasis. While CD8+ T cells exert immunosurveillance in primary human tumors, whether they can exert similar effects on myeloid cells in the pre-metastatic environment is unknown. Here, we show that CD8+ T cells are capable of constraining pre-metastatic myeloid cell accumulation by inducing myeloid cell apoptosis in C57BL/6 mice. Antigen-specific CD8+ T-cell cytotoxicity against myeloid cells in pre-metastatic lymph nodes is compromised by Stat3. We demonstrate here that Stat3 ablation in myeloid cells leads to CD8+ T-cell activation and increased levels of IFN-γ and granzyme B in the pre-metastatic environment. Furthermore, Stat3 negatively regulates soluble antigen cross-presentation by myeloid cells to CD8+ T cells in the pre-metastatic niche. Importantly, in tumor-free lymph nodes of melanoma patients, infiltration of activated CD8+ T cells inversely correlates with STAT3 activity, which is associated with a decrease in number of myeloid cells. Our study suggested a novel role for CD8+ T cells in constraining myeloid cell activity through direct killing in the pre-metastatic environment, and the therapeutic potential by targeting Stat3 in myeloid cells to improve CD8+ T-cell immunosurveillance against metastasis. PMID:25310972

Superior control of HIV-1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover

PubMed Central

Almeida, Jorge R.; Price, David A.; Papagno, Laura; Arkoub, Zaïna Aït; Sauce, Delphine; Bornstein, Ethan; Asher, Tedi E.; Samri, Assia; Schnuriger, Aurélie; Theodorou, Ioannis; Costagliola, Dominique; Rouzioux, Christine; Agut, Henri; Marcelin, Anne-Geneviève; Douek, Daniel; Autran, Brigitte; Appay, Victor

2007-01-01

The key attributes of CD8+ T cell protective immunity in human immunodeficiency virus (HIV) infection remain unclear. We report that CD8+ T cell responses specific for Gag and, in particular, the immunodominant p24 epitope KK10 correlate with control of HIV-1 replication in human histocompatibility leukocyte antigen (HLA)–B27 patients. To understand further the nature of CD8+ T cell–mediated antiviral efficacy, we performed a comprehensive study of CD8+ T cells specific for the HLA-B27–restricted epitope KK10 in chronic HIV-1 infection based on the use of multiparametric flow cytometry together with molecular clonotypic analysis and viral sequencing. We show that B27-KK10–specific CD8+ T cells are characterized by polyfunctional capabilities, increased clonal turnover, and superior functional avidity. Such attributes are interlinked and constitute the basis for effective control of HIV-1 replication. These data on the features of effective CD8+ T cells in HIV infection may aid in the development of successful T cell vaccines. PMID:17893201

Photochemical Internalization of Peptide Antigens Provides a Novel Strategy to Realize Therapeutic Cancer Vaccination

PubMed Central

Haug, Markus; Brede, Gaute; Håkerud, Monika; Nedberg, Anne Grete; Gederaas, Odrun A.; Flo, Trude H.; Edwards, Victoria T.; Selbo, Pål K.; Høgset, Anders; Halaas, Øyvind

2018-01-01

Effective priming and activation of tumor-specific CD8+ cytotoxic T lymphocytes (CTLs) is crucial for realizing the potential of therapeutic cancer vaccination. This requires cytosolic antigens that feed into the MHC class I presentation pathway, which is not efficiently achieved with most current vaccination technologies. Photochemical internalization (PCI) provides an emerging technology to route endocytosed material to the cytosol of cells, based on light-induced disruption of endosomal membranes using a photosensitizing compound. Here, we investigated the potential of PCI as a novel, minimally invasive, and well-tolerated vaccination technology to induce priming of cancer-specific CTL responses to peptide antigens. We show that PCI effectively promotes delivery of peptide antigens to the cytosol of antigen-presenting cells (APCs) in vitro. This resulted in a 30-fold increase in MHC class I/peptide complex formation and surface presentation, and a subsequent 30- to 100-fold more efficient activation of antigen-specific CTLs compared to using the peptide alone. The effect was found to be highly dependent on the dose of the PCI treatment, where optimal doses promoted maturation of immature dendritic cells, thus also providing an adjuvant effect. The effect of PCI was confirmed in vivo by the successful induction of antigen-specific CTL responses to cancer antigens in C57BL/6 mice following intradermal peptide vaccination using PCI technology. We thus show new and strong evidence that PCI technology holds great potential as a novel strategy for improving the outcome of peptide vaccines aimed at triggering cancer-specific CD8+ CTL responses. PMID:29670624

Immaturity associated antigens are lost during induction for T cell lymphoblastic leukemia: implications for minimal residual disease detection

PubMed Central

Roshal, Mikhail; Fromm, Jonathan R; Winter, Stuart; Dunsmore, Kimberly; Wood, Brent

2011-01-01

Background Induction chemotherapy for acute leukemia often leads to antigenic shifts in residual abnormal blast populations. Studies in precursor B cell ALL (B-ALL) and AML have demonstrated that chemotherapy commonly results in the loss of antigens associated with immaturity, limiting their utility for minimal residual disease (MRD) detection. Little information is available about the stability of these antigens in precursor T cell ALL (T-ALL) though it is presumed that CD99 and TdT are highly informative based on limited studies. Methods In a longitudinal investigation, we explored patterns of lineage specific and immaturity associated antigens in T-ALL in a large cohort of patients treated under the multicenter Children's Oncology Group (COG) protocol. All samples were analyzed using multicolor flow cytometry in a standardized fashion at a single institution. Results We report that markers of immaturity particularly, TdT and CD99 dramatically decline on leukemic blasts during therapy. CD34 and CD10 expression is confined to a minority of pre-treatment samples and is also not stable. In contrast, lineage associated markers including CD2, CD3, CD4, CD5, CD7 and CD8 failed to show significant trends. Conclusions Our study strongly argues for expansion of immunophenotyping panels for T-ALL MRD to decrease reliance on immature antigens. This study represents the first demonstration of consistent immunophenotypic shifts in T-ALL. PMID:20155852

T-Cell Surface Antigens and sCD30 as Biomarkers of the Risk of Rejection in Solid Organ Transplantation.

PubMed

Wieland, Eberhard; Shipkova, Maria

2016-04-01

T-cell activation is a characteristic of organ rejection. T cells, located in the draining lymph nodes of the transplant recipient, are faced with non-self-molecules presented by antigen presenting cells and become activated. Activated T cells are characterized by up-regulated surface antigens, such as costimulatory molecules, adhesion molecules, chemokine receptors, and major histocompatibility complex class II molecules. Surface antigen expression can be followed by flow cytometry using monoclonal antibodies in either cell function assays using donor-specific or nonspecific stimulation of isolated cells or whole blood and without stimulation on circulating lymphocytes. Molecules such as CD30 can be proteolytically cleaved off the surface of activated cells in vivo, and the determination of the soluble protein (sCD30) in serum or plasma is performed by immunoassays. As promising biomarkers for rejection and long-term transplant outcome, CD28 (costimulatory receptor for CD80 and CD86), CD154 (CD40 ligand), and sCD30 (tumor necrosis factor receptor superfamily, member 8) have been identified. Whereas cell function assays are time-consuming laboratory-developed tests which are difficult to standardize, commercial assays are frequently available for soluble proteins. Therefore, more data from clinical trials have been published for sCD30 compared with the surface antigens on activated T cells. This short review summarizes the association between selected surface antigens and immunosuppression, and rejection in solid organ transplantation.

Oral dendritic cells mediate antigen-specific tolerance by stimulating TH1 and regulatory CD4+ T cells.

PubMed

Mascarell, Laurent; Lombardi, Vincent; Louise, Anne; Saint-Lu, Nathalie; Chabre, Henri; Moussu, Hélène; Betbeder, Didier; Balazuc, Anne-Marie; Van Overtvelt, Laurence; Moingeon, Philippe

2008-09-01

A detailed characterization of oral antigen-presenting cells is critical to improve second-generation sublingual allergy vaccines. To characterize oral dendritic cells (DCs) within lingual and buccal tissues from BALB/c mice with respect to their surface phenotype, distribution, and capacity to polarize CD4(+) T-cell responses. In situ analysis of oral DCs was performed by immunohistology. Purified DCs were tested in vitro for their capacity to capture, process, and present the ovalbumin antigen to naive CD4(+) T cells. In vivo priming of ovalbumin-specific T cells adoptively transferred to BALB/c mice was analyzed by cytofluorometry in cervical lymph nodes after sublingual administration of mucoadhesive ovalbumin. Three subsets of oral DCs with a distinct tissue distribution were identified: (1) a minor subset of CD207(+) Langerhans cells located in the mucosa itself, (2) a major subpopulation of CD11b(+)CD11c(-) and CD11b(+)CD11c(+) myeloid DCs at the mucosal/submucosal interface, and (3) B220(+)120G8(+) plasmacytoid DCs found in submucosal tissues. Purified myeloid and plasmacytoid oral DCs capture and process the antigen efficiently and are programmed to elicit IFN-gamma and/or IL-10 production together with a suppressive function in naive CD4(+) T cells. Targeting the ovalbumin antigen to oral DCs in vivo by using mucoadhesive particles establishes tolerance in the absence of cell depletion through the stimulation of IFN-gamma and IL-10-producing CD4(+) regulatory T cells in cervical lymph nodes. The oral immune system is composed of various subsets of tolerogenic DCs organized in a compartmentalized manner and programmed to induce T(H)1/regulatory T-cell responses.

Dihydrolipoamide dehydrogenase-Lpd (Rv0462)-specific T cell recall responses are higher in healthy household contacts of TB: a novel immunodominant antigen from M. tuberculosis.

PubMed

Devasundaram, Santhi; Raja, Alamelu

2017-07-01

The partial effectiveness against pulmonary tuberculosis (PTB), displayed by the existing tuberculosis (TB) vaccine, bacillus Calmette-Guérin (BCG), highlights the need for novel vaccines to replace or improve BCG. In TB immunology, antigen-specific cellular immune response is frequently considered indispensable. Latency-associated antigens are intriguing as targets for TB vaccine development. The mycobacterial protein, dihydrolipoamide dehydrogenase (Lpd; Rv0462), the third enzyme of the pyruvate dehydrogenase (PDH) complex, facilitates Mycobacterium tuberculosis to resist host reactive nitrogen intermediates. Multicolor flow cytometry analysis of whole-blood cultures showed higher Lpd-specific Th1 recall response (IFN-γ, TNF-α, and IL-2; P = 0.0006) and memory CD4 + and CD8 + T cells (CCR7 + CD45RA - and CCR7 - CD45RA - ) in healthy household contacts (HHC) of TB ( P < 0.0001), which is comparable with or higher than the standard antigens, ESAT-6 and CFP-10. The frequency of Lpd-specific multifunctional T cells was higher in HHC compared with PTB patients. However, there is no significant statistical correlation. Regulatory T cell (T reg ) analysis of HHCs and active TB patients demonstrated very low Lpd-specific CD4 + T regs relative to ESAT-6 and CFP-10. Our study demonstrates that the Lpd antigen induces a strong cellular immune response in healthy mycobacteria-infected individuals. In consideration of this population having demonstrated immunologic protection against active TB disease development, our data are encouraging about the possible use of Lpd as a target for further TB subunit vaccine development. © Society for Leukocyte Biology.

Expression of the rat CD26 antigen (dipeptidyl peptidase IV) on subpopulations of rat lymphocytes.

PubMed

Gorrell, M D; Wickson, J; McCaughan, G W

1991-04-15

The T cell activation antigen CD26 has been recently identified as the cell surface ectopeptidase dipeptidyl peptidase IV (DPP-IV). DPP-IV is found on many cell types, including lymphocytes, epithelial cells, and certain endothelial cells. The MRC OX61 monoclonal antibody (MAb) which specifically recognises rat DPP-IV was used to examine the expression of CD26/DPP-IV on rat lymphocytes. The molecular nature of the antigen was examined by immunoprecipitation from thymocytes, splenocytes, and hepatocytes. Analysis by one- and two-dimensional gel electrophoresis indicated that the native form of CD26 includes a 220-kDa homodimer. On tissue sections MRC OX61 MAb stained nearly all thymocytes and in the spleen and lymph nodes predominantly stained the T cell areas. However, in immunofluorescence experiments OX61 stained 80 to 87% of lymph node cells and 78 to 85% of spleen cells. Furthermore, two-colour immunofluorescence analysis of the CD4+, CD8+, and Ig+ lymphocyte subsets indicated that only 2 to 5% of each of these subsets lacked OX61 staining. Spleen cells and thymocytes of both CD4+ and CD8+ subsets stained much more intensely with OX61 after these cells were stimulated with phytohemagglutinin. These findings indicate that rat CD26 antigen expression is not confined to the T cell population as has been suggested, but also occurs on B cells, and is increased on T cells following their activation.

Distinct activation phenotype of a highly conserved novel HLA-B57-restricted epitope during dengue virus infection

PubMed Central

Townsley, Elizabeth; Woda, Marcia; Thomas, Stephen J; Kalayanarooj, Siripen; Gibbons, Robert V; Nisalak, Ananda; Srikiatkhachorn, Anon; Green, Sharone; Stephens, Henry AF; Rothman, Alan L; Mathew, Anuja

2014-01-01

Variation in the sequence of T-cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T-cell responses during second heterologous infections. We identified a highly conserved, novel, HLA-B57-restricted epitope on the DENV NS1 protein. We predicted higher frequencies of B57-NS126–34-specific CD8+ T cells in peripheral blood mononuclear cells from individuals undergoing secondary rather than primary DENV infection. However, high tetramer-positive T-cell frequencies during acute infection were seen in only one of nine subjects with secondary infection. B57-NS126–34-specific and other DENV epitope-specific CD8+ T cells, as well as total CD8+ T cells, expressed an activated phenotype (CD69+ and/or CD38+) during acute infection. In contrast, expression of CD71 was largely limited to DENV epitope-specific CD8+ T cells. In vitro stimulation of cell lines indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides. CD71 may represent a useful marker of antigen-specific T-cell activation. PMID:23941420

Distinct activation phenotype of a highly conserved novel HLA-B57-restricted epitope during dengue virus infection.

PubMed

Townsley, Elizabeth; Woda, Marcia; Thomas, Stephen J; Kalayanarooj, Siripen; Gibbons, Robert V; Nisalak, Ananda; Srikiatkhachorn, Anon; Green, Sharone; Stephens, Henry A F; Rothman, Alan L; Mathew, Anuja

2014-01-01

Variation in the sequence of T-cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T-cell responses during second heterologous infections. We identified a highly conserved, novel, HLA-B57-restricted epitope on the DENV NS1 protein. We predicted higher frequencies of B57-NS1(26-34) -specific CD8(+) T cells in peripheral blood mononuclear cells from individuals undergoing secondary rather than primary DENV infection. However, high tetramer-positive T-cell frequencies during acute infection were seen in only one of nine subjects with secondary infection. B57-NS1(26-34) -specific and other DENV epitope-specific CD8(+) T cells, as well as total CD8(+) T cells, expressed an activated phenotype (CD69(+) and/or CD38(+)) during acute infection. In contrast, expression of CD71 was largely limited to DENV epitope-specific CD8(+) T cells. In vitro stimulation of cell lines indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides. CD71 may represent a useful marker of antigen-specific T-cell activation. © 2013 John Wiley & Sons Ltd.

Aberrant lymphoid antigen expression in acute myeloid leukemia in Saudi Arabia.

PubMed

El-Sissy, Azza H; El-Mashari, May A; Bassuni, Wafaa Y; El-Swaayed, Aziza F

2006-09-01

Immunophenotyping improves both accuracy and reproducibility of acute leukemia classification and is considered particularly useful for identifying aberrant lineage association of acute leukemia, biphenotypic and bilineal acute leukemia, as well as monitoring minimal residual disease. Some immunophenotypes correlate with cytogenetic abnormalities and prognosis. Is to determine aberrant lymphoid antigen expression in Saudi acute myeloid leukemia (AML), correlate them with FAB subtypes, evaluate early surface markers CD7 and CD56, and to investigate the role of cytoplasmic CD79a (a B cell marker that is assigned a high score of 2.0 in the WHO classification). Thirty four newly diagnosed AML cases were included in this study, 47% showed aberrant lymphoid antigen expression. CD9 was the most frequently expressed lymphoid antigen (29.4%) followed by CD7 & CD19 (11.8%), CD4 (8.8%) and CD22 (2.9%). CD9 was expressed in 3/6 (50%) of M3 cases, CD7 was expressed in 11.8% and was mostly confined to FAB M1 and M2 and associated with immature antigens CD34, HLA-DR and TdT. CD56 was expressed in 7/34 (20.6%) cases, three of these cases (42.9%) belonged to the monocytic group. CD56 was also detected in 2 cases with 11q23 rearrangement. CD56 was expressed in 2/7 (28.6%) M2 cases, and was associated with t (8;21) (q22;q22) together with CD19. Co-expression of CD56 and CD7 was detected in 2.9% of the cases. CD79a was expressed in one case together with CD19, diagnosed as acute biphenotypic leukemia, and was associated with t(8;21) (q22;q22). Minimal residual disease in AML is very difficult to trace, detection of aberrant expression of lymphoid antigens will make it easier. The high score given to CD79a by EGIL is questionable based on cytogenetic classification.

Leukocyte function-associated antigen-1-dependent lysis of Fas+ (CD95+/Apo-1+) innocent bystanders by antigen-specific CD8+ CTL.

PubMed

Kojima, H; Eshima, K; Takayama, H; Sitkovsky, M V

1997-09-15

Exquisite specificity toward Ag-bearing cells (cognate targets) is one of the most important properties of CD8+ CTL-mediated cytotoxicity. Using highly Ag-specific CD8+ CTL lines and clones, which spare noncognate, Ag-free targets, we found that in the presence of Ag-bearing targets the CTL acquire the ability to lyse noncognate target cells (bystanders). It is shown that the unexpectedly rapid and efficient lysis of bystanders by Ag-activated CTL is mediated by a Fas ligand (FasL)/Fas-based mechanism and does not depend on perforin. The CTL lysed Fas-expressing bystanders, but spared the Fas-negative or anti-Fas mAb-resistant bystander cells. Accordingly, the FasL-deficient gld/gld CTL did not kill bystanders, while perforin-deficient CTL did. Unlike anti-Fas mAb-induced cell death, the lysis of bystanders was not only FasL/Fas dependent but also required adhesion molecule LFA-1 on the surface of the activated CTL. Lysis of bystanders is viewed as acceptable "collateral" damage, but the persistent presence of activated CTL could result in immunopathologies involving functional Fas-expressing tissues.

Critical role for Sec22b-dependent antigen cross-presentation in antitumor immunity

PubMed Central

Rookhuizen, Derek C.; Joannas, Leonel; Carpier, Jean-Marie; Yatim, Nader; Albert, Matthew L.

2017-01-01

CD8+ T cells mediate antigen-specific immune responses that can induce rejection of solid tumors. In this process, dendritic cells (DCs) are thought to take up tumor antigens, which are processed into peptides and loaded onto MHC-I molecules, a process called “cross-presentation.” Neither the actual contribution of cross-presentation to antitumor immune responses nor the intracellular pathways involved in vivo are clearly established because of the lack of experimental tools to manipulate this process. To develop such tools, we generated mice bearing a conditional DC-specific mutation in the sec22b gene, a critical regulator of endoplasmic reticulum–phagosome traffic required for cross-presentation. DCs from these mice show impaired cross-presentation ex vivo and defective cross-priming of CD8+ T cell responses in vivo. These mice are also defective for antitumor immune responses and are resistant to treatment with anti–PD-1. We conclude that Sec22b-dependent cross-presentation in DCs is required to initiate CD8+ T cell responses to dead cells and to induce effective antitumor immune responses during anti–PD-1 treatment in mice. PMID:28663435

Human Macrophages and Dendritic Cells Can Equally Present MART-1 Antigen to CD8+ T Cells after Phagocytosis of Gamma-Irradiated Melanoma Cells

PubMed Central

Barrio, María Marcela; Abes, Riad; Colombo, Marina; Pizzurro, Gabriela; Boix, Charlotte; Roberti, María Paula; Gélizé, Emmanuelle; Rodriguez-Zubieta, Mariana

2012-01-01

Dendritic cells (DC) can achieve cross-presentation of naturally-occurring tumor-associated antigens after phagocytosis and processing of dying tumor cells. They have been used in different clinical settings to vaccinate cancer patients. We have previously used gamma-irradiated MART-1 expressing melanoma cells as a source of antigens to vaccinate melanoma patients by injecting irradiated cells with BCG and GM-CSF or to load immature DC and use them as a vaccine. Other clinical trials have used IFN-gamma activated macrophage killer cells (MAK) to treat cancer patients. However, the clinical use of MAK has been based on their direct tumoricidal activity rather than on their ability to act as antigen-presenting cells to stimulate an adaptive antitumor response. Thus, in the present work, we compared the fate of MART-1 after phagocytosis of gamma-irradiated cells by clinical grade DC or MAK as well as the ability of these cells to cross present MART-1 to CD8+ T cells. Using a high affinity antibody against MART-1, 2A9, which specifically stains melanoma tumors, melanoma cell lines and normal melanocytes, the expression level of MART-1 in melanoma cell lines could be related to their ability to stimulate IFN-gamma production by a MART-1 specific HLA-A*0201-restricted CD8+ T cell clone. Confocal microscopy with Alexa Fluor®647-labelled 2A9 also showed that MART-1 could be detected in tumor cells attached and/or fused to phagocytes and even inside these cells as early as 1 h and up to 24 h or 48 h after initiation of co-cultures between gamma-irradiated melanoma cells and MAK or DC, respectively. Interestingly, MART-1 was cross-presented to MART-1 specific T cells by both MAK and DC co-cultured with melanoma gamma-irradiated cells for different time-points. Thus, naturally occurring MART-1 melanoma antigen can be taken-up from dying melanoma cells into DC or MAK and both cell types can induce specific CD8+ T cell cross-presentation thereafter. PMID:22768350

Treatment with proteasome inhibitor bortezomib enhances antigen-specific CD8+ T cell-mediated antitumor immunity induced by DNA vaccination

PubMed Central

Tseng, Chih Wen; Monie, Archana; Wu, Chao-Yi; Huang, Bruce; Wang, Mei-Cheng; Hung, Chien-Fu; Wu, T.-C.

2008-01-01

There is an urgent need to develop new innovative therapies for the control of cancer. Antigen-specific immunotherapy and the employment of proteasome inhibitors have emerged as two potentially plausible approaches for the control of cancer. In the current study, we explored the combination of the DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 (HPV-16) E7 antigen (CRT/E7) with the proteasome inhibitor; bortezomib for their ability to generate E7-specific immune responses and antitumor effects in vaccinated mice. We found that the combination of treatment with bortezomib and CRT/E7(detox) DNA generated more potent E7-specific CD8+ T cell immune responses and better therapeutic effects against TC-1 tumors in tumor bearing mice compared to monotherapy. Furthermore, we found that treatment with bortezomib led to increased apoptosis of TC-1 tumor cells and could render the TC-1 tumor cells more susceptible to lysis by E7-specific CTLs. Our data has significant implications for future clinical translation. PMID:18542898

Human intraepithelial lymphocytes.

PubMed

Mayassi, Toufic; Jabri, Bana

2018-04-20

The location of intraepithelial lymphocytes (IEL) between epithelial cells, their effector memory, cytolytic and inflammatory phenotype positions them to kill infected epithelial cells and protect the intestine against pathogens. Human TCRαβ + CD8αβ + IEL have the dual capacity to recognize modified self via natural killer (NK) receptors (autoreactivity) as well as foreign antigen via the T cell receptor (TCR), which is accomplished in mouse by two cell subsets, the naturally occurring TCRαβ + CD8αα + and adaptively induced TCRαβ + CD8αβ + IEL subsets, respectively. The private/oligoclonal nature of the TCR repertoire of both human and mouse IEL suggests local environmental factors dictate the specificity of IEL responses. The line between sensing of foreign antigens and autoreactivity is blurred for IEL in celiac disease, where recognition of stress ligands by induced activating NK receptors in conjunction with inflammatory signals such as IL-15 can result in low-affinity TCR/non-cognate antigen and NK receptor/stress ligand interactions triggering destruction of intestinal epithelial cells.

Liposomes-coated gold nanocages with antigens and adjuvants targeted delivery to dendritic cells for enhancing antitumor immune response.

PubMed

Liang, Ruijing; Xie, Jun; Li, Jun; Wang, Ke; Liu, Liping; Gao, Yujie; Hussain, Mubashir; Shen, Guanxin; Zhu, Jintao; Tao, Juan

2017-12-01

For nanovaccine-based cancer immunotherapy, dendritic cells (DCs) are one of the most powerful antigen presenting cells (APCs) that initiate and promote the maturation of antigen-specific cytotoxic T lymphocytes (e.g., CD8 + T cells) to induce the local and systemic antitumor immunity and further suppress the tumor metastasis and produce long-term protection against tumor. Thus, the activation and maturation of DCs is the prerequisite for efficient CD8 + T cell-based antitumor immune responses, which is considered as a primary and promising task for nanovaccine engineering. Herein, we introduce a versatile nanovaccine of liposomes-coated gold nanocages (Lipos-AuNCs) modified with DCs specific antibody aCD11c for targeted delivery of adjuvant MPLA and melanoma antigen peptide TRP2 to promote the activation and maturation of DCs, and enhance tumor specific T lymphocytes responses. Moreover, AuNCs accumulation and AuNCs-engulfed DCs migration to regional lymph nodes (RLNs) became real-time visualization through in vivo fluorescence and photoacoustic (PA) imaging to monitor the immunity process. In vivo experimental results demonstrated that the targeted antigen/adjuvants-loaded AuNCs exhibited enhanced antitumor immune response to inhibit tumor growth and metastasis in both B16-F10 prophylactic and lung metastasis models, which may act as a promising nanoplatform for antitumor immunotherapy and in vivo tracking. Copyright © 2017 Elsevier Ltd. All rights reserved.

PD-1 identifies the patient-specific CD8+ tumor-reactive repertoire infiltrating human tumors

PubMed Central

Gros, Alena; Robbins, Paul F.; Yao, Xin; Li, Yong F.; Turcotte, Simon; Tran, Eric; Wunderlich, John R.; Mixon, Arnold; Farid, Shawn; Dudley, Mark E.; Hanada, Ken-ichi; Almeida, Jorge R.; Darko, Sam; Douek, Daniel C.; Yang, James C.; Rosenberg, Steven A.

2014-01-01

Adoptive transfer of tumor-infiltrating lymphocytes (TILs) can mediate regression of metastatic melanoma; however, TILs are a heterogeneous population, and there are no effective markers to specifically identify and select the repertoire of tumor-reactive and mutation-specific CD8+ lymphocytes. The lack of biomarkers limits the ability to study these cells and develop strategies to enhance clinical efficacy and extend this therapy to other malignancies. Here, we evaluated unique phenotypic traits of CD8+ TILs and TCR β chain (TCRβ) clonotypic frequency in melanoma tumors to identify patient-specific repertoires of tumor-reactive CD8+ lymphocytes. In all 6 tumors studied, expression of the inhibitory receptors programmed cell death 1 (PD-1; also known as CD279), lymphocyte-activation gene 3 (LAG-3; also known as CD223), and T cell immunoglobulin and mucin domain 3 (TIM-3) on CD8+ TILs identified the autologous tumor-reactive repertoire, including mutated neoantigen-specific CD8+ lymphocytes, whereas only a fraction of the tumor-reactive population expressed the costimulatory receptor 4-1BB (also known as CD137). TCRβ deep sequencing revealed oligoclonal expansion of specific TCRβ clonotypes in CD8+PD-1+ compared with CD8+PD-1– TIL populations. Furthermore, the most highly expanded TCRβ clonotypes in the CD8+ and the CD8+PD-1+ populations recognized the autologous tumor and included clonotypes targeting mutated antigens. Thus, in addition to the well-documented negative regulatory role of PD-1 in T cells, our findings demonstrate that PD-1 expression on CD8+ TILs also accurately identifies the repertoire of clonally expanded tumor-reactive cells and reveal a dual importance of PD-1 expression in the tumor microenvironment. PMID:24667641

An Inducible Transgenic Mouse Model for Immune Mediated Hepatitis Showing Clearance of Antigen Expressing Hepatocytes by CD8+ T Cells

PubMed Central

Cebula, Marcin; Ochel, Aaron; Hillebrand, Upneet; Pils, Marina C.; Schirmbeck, Reinhold; Hauser, Hansjörg; Wirth, Dagmar

2013-01-01

The liver has the ability to prime immune responses against neo antigens provided upon infections. However, T cell immunity in liver is uniquely modulated by the complex tolerogenic property of this organ that has to also cope with foreign agents such as endotoxins or food antigens. In this respect, the nature of intrahepatic T cell responses remains to be fully characterized. To gain deeper insight into the mechanisms that regulate the CD8+ T cell responses in the liver, we established a novel OVA_X_CreERT2 mouse model. Upon tamoxifen administration OVA antigen expression is observed in a fraction of hepatocytes, resulting in a mosaic expression pattern. To elucidate the cross-talk of CD8+ T cells with antigen-expressing hepatocytes, we adoptively transferred Kb/OVA257-264-specific OT-I T cells to OVA_X_CreERT2 mice or generated triple transgenic OVA_X CreERT2_X_OT-I mice. OT-I T cells become activated in OVA_X_CreERT2 mice and induce an acute and transient hepatitis accompanied by liver damage. In OVA_X_CreERT2_X_OT-I mice, OVA induction triggers an OT-I T cell mediated, fulminant hepatitis resulting in 50% mortality. Surviving mice manifest a long lasting hepatitis, and recover after 9 weeks. In these experimental settings, recovery from hepatitis correlates with a complete loss of OVA expression indicating efficient clearance of the antigen-expressing hepatocytes. Moreover, a relapse of hepatitis can be induced upon re-induction of cured OVA_X_CreERT2_X_OT-I mice indicating absence of tolerogenic mechanisms. This pathogen-free, conditional mouse model has the advantage of tamoxifen inducible tissue specific antigen expression that reflects the heterogeneity of viral antigen expression and enables the study of intrahepatic immune responses to both de novo and persistent antigen. It allows following the course of intrahepatic immune responses: initiation, the acute phase and antigen clearance. PMID:23869228

Dynamics of the CD8 T-cell response following yellow fever virus 17D immunization

PubMed Central

Co, Mary Dawn T; Kilpatrick, Elizabeth D; Rothman, Alan L

2009-01-01

Management of yellow fever is focused on the prevention of illness by the use of the yellow fever virus (YFV) 17D vaccine. The role of neutralizing antibodies in protection is generally accepted with YFV-specific T cells likely contributing to the control of viral replication. We studied CD8+ T-cell responses to four defined human leucocyte antigen-B35-restricted epitopes in YFV vaccine recipients as a model of the kinetics of cytotoxic T-lymphocyte responses to an acute human viral infection. Multiple features of these epitope-specific responses were analysed after vaccination including magnitude, cytokine production, phenotype and T-cell receptor repertoire. Peak peptide-specific interferon-γ (IFN-γ) responses of almost 1% of CD8+ T cells were seen as early as 2 weeks post-vaccination; however, dominant responses varied between donors. Peptide-specific responses were still detectable at 54 months post-vaccination. Tetramer-positive cells, at high frequencies, were detected as early as 7–9 days, before detectable IFN-γ-producing cells, suggesting a defect in the functional capacity of some antigen-specific cells early post-vaccination. The predominant memory phenotype of the tetramer-positive population was a differentiated effector (CD45RA+ CCR7− CD62L−) phenotype. The T-cell receptor Vβ analysis revealed a diverse oligoclonal repertoire in tetramer-positive T-cell populations in two individuals. These characteristics of the YFV-specific T-cell response could contribute to vaccine effectiveness. PMID:19740333

Dynamics of the CD8 T-cell response following yellow fever virus 17D immunization.

PubMed

Co, Mary Dawn T; Kilpatrick, Elizabeth D; Rothman, Alan L

2009-09-01

Management of yellow fever is focused on the prevention of illness by the use of the yellow fever virus (YFV) 17D vaccine. The role of neutralizing antibodies in protection is generally accepted with YFV-specific T cells likely contributing to the control of viral replication. We studied CD8(+) T-cell responses to four defined human leucocyte antigen-B35-restricted epitopes in YFV vaccine recipients as a model of the kinetics of cytotoxic T-lymphocyte responses to an acute human viral infection. Multiple features of these epitope-specific responses were analysed after vaccination including magnitude, cytokine production, phenotype and T-cell receptor repertoire. Peak peptide-specific interferon-gamma (IFN-gamma) responses of almost 1% of CD8(+) T cells were seen as early as 2 weeks post-vaccination; however, dominant responses varied between donors. Peptide-specific responses were still detectable at 54 months post-vaccination. Tetramer-positive cells, at high frequencies, were detected as early as 7-9 days, before detectable IFN-gamma-producing cells, suggesting a defect in the functional capacity of some antigen-specific cells early post-vaccination. The predominant memory phenotype of the tetramer-positive population was a differentiated effector (CD45RA(+) CCR7(-) CD62L(-)) phenotype. The T-cell receptor Vbeta analysis revealed a diverse oligoclonal repertoire in tetramer-positive T-cell populations in two individuals. These characteristics of the YFV-specific T-cell response could contribute to vaccine effectiveness.

Immune Responses in Acute and Convalescent Patients with Mild, Moderate and Severe Disease during the 2009 Influenza Pandemic in Norway

PubMed Central

Mohn, Kristin G.-I.; Cox, Rebecca Jane; Tunheim, Gro; Berdal, Jan Erik; Hauge, Anna Germundsson; Jul-Larsen, Åsne; Peters, Bjoern; Oftung, Fredrik

2015-01-01

Increased understanding of immune responses influencing clinical severity during pandemic influenza infection is important for improved treatment and vaccine development. In this study we recruited 46 adult patients during the 2009 influenza pandemic and characterized humoral and cellular immune responses. Those included were either acute hospitalized or convalescent patients with different disease severities (mild, moderate or severe). In general, protective antibody responses increased with enhanced disease severity. In the acute patients, we found higher levels of TNF-α single-producing CD4+T-cells in the severely ill as compared to patients with moderate disease. Stimulation of peripheral blood mononuclear cells (PBMC) from a subset of acute patients with peptide T-cell epitopes showed significantly lower frequencies of influenza specific CD8+ compared with CD4+ IFN-γ T-cells in acute patients. Both T-cell subsets were predominantly directed against the envelope antigens (HA and NA). However, in the convalescent patients we found high levels of both CD4+ and CD8+ T-cells directed against conserved core antigens (NP, PA, PB, and M). The results indicate that the antigen targets recognized by the T-cell subsets may vary according to the phase of infection. The apparent low levels of cross-reactive CD8+ T-cells recognizing internal antigens in acute hospitalized patients suggest an important role for this T-cell subset in protective immunity against influenza. PMID:26606759

Homologous Prime-Boost Vaccination with OVA Entrapped in Self-Adjuvanting Archaeosomes Induces High Numbers of OVA-Specific CD8⁺ T Cells that Protect Against Subcutaneous B16-OVA Melanoma.

PubMed

Stark, Felicity C; McCluskie, Michael J; Krishnan, Lakshmi

2016-11-17

Homologous prime-boost vaccinations with live vectors typically fail to induce repeated strong CD8⁺ T cell responses due to the induction of anti-vector immunity, highlighting the need for alternative delivery vehicles. The unique ether lipids of archaea may be constituted into liposomes, archaeosomes, which do not induce anti-carrier responses, making them an ideal candidate for use in repeat vaccination systems. Herein, we evaluated in mice the maximum threshold of antigen-specific CD8⁺ T cell responses that may be induced by multiple homologous immunizations with ovalbumin (OVA) entrapped in archaeosomes derived from the ether glycerolipids of the archaeon Methanobrevibacter smithii (MS-OVA). Up to three immunizations with MS-OVA administered in optimized intervals (to allow for sufficient resting of the primed cells prior to boosting), induced a potent anti-OVA CD8⁺ T cell response of up to 45% of all circulating CD8⁺ T cells. Additional MS-OVA injections did not add any further benefit in increasing the memory of CD8⁺ T cell frequency. In contrast, OVA expressed by Listeria monocytogenes (LM-OVA), an intracellular bacterial vector failed to evoke a boosting effect after the second injection, resulting in significantly reduced antigen-specific CD8⁺ T cell frequencies. Furthermore, repeated vaccination with MS-OVA skewed the response increasingly towards an effector memory (CD62 low ) phenotype. Vaccinated animals were challenged with B16-OVA at late time points after vaccination (+7 months) and were afforded protection compared to control. Therefore, archaeosomes constituted a robust particulate delivery system to unravel the kinetics of CD8⁺ T cell response induction and memory maintenance and constitute an efficient vaccination regimen optimized for tumor protection.

Therapeutic targeting of regulatory T cells enhances tumor-specific CD8+ T cell responses in Epstein–Barr virus associated nasopharyngeal carcinoma

PubMed Central

Fogg, Mark; Murphy, John R.; Lorch, Jochen; Posner, Marshall; Wang, Fred

2013-01-01

Epstein–Barr virus (EBV) is associated with multiple malignancies including nasopharyngeal carcinoma (NPC). In nasopharynx cancer, CD8+ T cells specific for EBV Nuclear Antigen-1 (EBNA-1) and Latent Membrane Protein 2 (LMP2) are important components of anti-tumor immunity since both are consistently expressed in NPC. We have previously shown that EBNA-1-specific CD8+ T cell responses were suppressed in NPC patients compared to healthy controls. We now find that CD8+ T cell responses specific for LMP2 are also abnormal in NPC patients, and both EBNA-1- and LMP2-specific responses are suppressed by regulatory T cells (Treg). EBNA-1 and LMP2-specific CD8+ T cell responses, as well as immune control of EBV-infected cells in vitro, could be restored by the depletion of Tregs and by use of a clinically approved drug targeting Tregs. Thus, in vivo modulation of Tregs may be an effective means of enhancing these anti-tumor immune responses in NPC patients. PMID:23601786

Local HPV Recombinant Vaccinia Boost Following Priming with an HPV DNA Vaccine Enhances Local HPV-Specific CD8+ T Cell Mediated Tumor Control in the Genital Tract

PubMed Central

Sun, Yun-Yan; Peng, Shiwen; Han, Liping; Qiu, Jin; Song, Liwen; Tsai, Yachea; Yang, Benjamin; Roden, Richard B.S.; Trimble, Cornelia L.; Hung, Chien-Fu; Wu, T-C

2015-01-01

Purpose Two viral oncoproteins, E6 and E7, are expressed in all human papillomavirus (HPV)-infected cells, from initial infection in the genital tract to metastatic cervical cancer. Intramuscular vaccination of women with high grade cervical intraepithelial neoplasia (CIN2/3) twice with a naked DNA vaccine, pNGVL4a-sig/E7(detox)/HSP70, and a single boost with HPVE6/E7 recombinant vaccinia vaccine (TA-HPV) elicited systemic HPV-specific CD8 T cell responses that could traffic to the lesion and was associated with regression in some patients (NCT00788164). Experimental Design Here we examine whether alteration of this vaccination regimen by administration of TA-HPV vaccination in the cervicovaginal tract, rather than IM delivery, can more effectively recruit antigen-specific T cells in an orthotopic syngeneic mouse model of HPV16+ cervical cancer (TC-1 luc). Results We found that pNGVL4a-sig/E7(detox)/HSP70 vaccination followed by cervicovaginal vaccination with TA-HPV increased accumulation of total and E7-specific CD8+ T cells in the cervicovaginal tract and better controlled E7-expressing cervicovaginal TC-1 luc tumor than IM administration of TA-HPV. Furthermore, the E7-specific CD8+ T cells in the cervicovaginal tract generated through the cervicovaginal route of vaccination expressed the α4β7 integrin and CCR9, which are necessary for the homing of the E7-specific CD8+ T cells to the cervicovaginal tract. Finally, we show that cervicovaginal vaccination with TA-HPV can induce potent local HPV-16 E7 antigen-specific CD8+ T cell immune responses regardless of whether an HPV DNA vaccine priming vaccination was administered IM or within the cervicovaginal tract. Conclusions Our results support future clinical translation using cervicovaginal TA-HPV vaccination. PMID:26420854

Local HPV Recombinant Vaccinia Boost Following Priming with an HPV DNA Vaccine Enhances Local HPV-Specific CD8+ T-cell-Mediated Tumor Control in the Genital Tract.

PubMed

Sun, Yun-Yan; Peng, Shiwen; Han, Liping; Qiu, Jin; Song, Liwen; Tsai, Yachea; Yang, Benjamin; Roden, Richard B S; Trimble, Cornelia L; Hung, Chien-Fu; Wu, T-C

2016-02-01

Two viral oncoproteins, E6 and E7, are expressed in all human papillomavirus (HPV)-infected cells, from initial infection in the genital tract to metastatic cervical cancer. Intramuscular vaccination of women with high-grade cervical intraepithelial neoplasia (CIN2/3) twice with a naked DNA vaccine, pNGVL4a-sig/E7(detox)/HSP70, and a single boost with HPVE6/E7 recombinant vaccinia vaccine (TA-HPV) elicited systemic HPV-specific CD8 T-cell responses that could traffic to the lesion and was associated with regression in some patients (NCT00788164). Here, we examine whether alteration of this vaccination regimen by administration of TA-HPV vaccination in the cervicovaginal tract, rather than intramuscular (IM) delivery, can more effectively recruit antigen-specific T cells in an orthotopic syngeneic mouse model of HPV16(+) cervical cancer (TC-1 luc). We found that pNGVL4a-sig/E7(detox)/HSP70 vaccination followed by cervicovaginal vaccination with TA-HPV increased accumulation of total and E7-specific CD8(+) T cells in the cervicovaginal tract and better controlled E7-expressing cervicovaginal TC-1 luc tumor than IM administration of TA-HPV. Furthermore, the E7-specific CD8(+) T cells in the cervicovaginal tract generated through the cervicovaginal route of vaccination expressed the α4β7 integrin and CCR9, which are necessary for the homing of the E7-specific CD8(+) T cells to the cervicovaginal tract. Finally, we show that cervicovaginal vaccination with TA-HPV can induce potent local HPV-16 E7 antigen-specific CD8(+) T-cell immune responses regardless of whether an HPV DNA vaccine priming vaccination was administered IM or within the cervicovaginal tract. Our results support future clinical translation using cervicovaginal TA-HPV vaccination. ©2015 American Association for Cancer Research.

A novel dendritic cell-based direct ex vivo assay for detection and enumeration of circulating antigen-specific human T cells.

PubMed

Carrio, Roberto; Zhang, Ge; Drake, Donald R; Schanen, Brian C

2018-05-07

Although a variety of assays have been used to examine T cell responses in vitro, standardized ex vivo detection of antigen-specific CD4 + T cells from human circulatory PBMCs remains constrained by low-dimensional characterization outputs and the need for polyclonal, mitogen-induced expansion methods to generate detectable response signals. To overcome these limitations, we developed a novel methodology utilizing antigen-pulsed autologous human dendritic target cells in a rapid and sensitive assay to accurately enumerate antigen-specific CD4 + T cell precursor frequency by multiparametric flow cytometry. With this approach, we demonstrate the ability to reproducibly quantitate poly-functional T cell responses following both primary and recall antigenic stimulation. Furthermore, this approach enables more comprehensive phenotypic profiling of circulating antigen-specific CD4 + T cells, providing valuable insights into the pre-existing polarization of antigen-specific T cells in humans. Combined, this approach permits sensitive and detailed ex vivo detection of antigen-specific CD4 + T cells delivering an important tool for advancing vaccine, immune-oncology and other therapeutic studies.

Dynamics of Dengue Virus (DENV)–Specific B Cells in the Response to DENV Serotype 1 Infections, Using Flow Cytometry With Labeled Virions

PubMed Central

Woda, Marcia; Friberg, Heather; Currier, Jeffrey R.; Srikiatkhachorn, Anon; Macareo, Louis R.; Green, Sharone; Jarman, Richard G.; Rothman, Alan L.; Mathew, Anuja

2016-01-01

Background. The development of reagents to identify and characterize antigen-specific B cells has been challenging. Methods. We recently developed Alexa Fluor–labeled dengue viruses (AF DENVs) to characterize antigen-specific B cells in the peripheral blood of DENV-immune individuals. Results. In this study, we used AF DENV serotype 1 (AF DENV-1) together with AF DENV-2 on peripheral blood mononuclear cells (PBMCs) from children in Thailand with acute primary or secondary DENV-1 infections to analyze the phenotypes of antigen-specific B cells that reflected their exposure or clinical diagnosis. DENV serotype-specific and cross-reactive B cells were identified in PBMCs from all subjects. Frequencies of AF DENV+ class-switched memory B cells (IgD−CD27+ CD19+ cells) reached up to 8% during acute infection and early convalescence. AF DENV–labeled B cells expressed high levels of CD27 and CD38 during acute infection, characteristic of plasmablasts, and transitioned into memory B cells (CD38−CD27+) at the early convalescent time point. There was higher activation of memory B cells early during acute secondary infection, suggesting reactivation from a previous DENV infection. Conclusions. AF DENVs reveal changes in the phenotype of DENV serotype–specific and cross-reactive B cells during and after natural DENV infection and could be useful in analysis of the response to DENV vaccination. PMID:27443614

Modulation of CD4(+) T cell-dependent specific cytotoxic CD8(+) T cells differentiation and proliferation by the timing of increase in the pathogen load.

PubMed

Tzelepis, Fanny; Persechini, Pedro M; Rodrigues, Mauricio M

2007-04-25

Following infection with viruses, bacteria or protozoan parasites, naïve antigen-specific CD8(+) T cells undergo a process of differentiation and proliferation to generate effector cells. Recent evidences suggest that the timing of generation of specific effector CD8(+) T cells varies widely according to different pathogens. We hypothesized that the timing of increase in the pathogen load could be a critical parameter governing this process. Using increasing doses of the protozoan parasite Trypanosoma cruzi to infect C57BL/6 mice, we observed a significant acceleration in the timing of parasitemia without an increase in mouse susceptibility. In contrast, in CD8 deficient mice, we observed an inverse relationship between the parasite inoculum and the timing of death. These results suggest that in normal mice CD8(+) T cells became protective earlier, following the accelerated development of parasitemia. The evaluation of specific cytotoxic responses in vivo to three distinct epitopes revealed that increasing the parasite inoculum hastened the expansion of specific CD8(+) cytotoxic T cells following infection. The differentiation and expansion of T. cruzi-specific CD8(+) cytotoxic T cells is in fact dependent on parasite multiplication, as radiation-attenuated parasites were unable to activate these cells. We also observed that, in contrast to most pathogens, the activation process of T. cruzi-specific CD8(+) cytotoxic T cells was dependent on MHC class II restricted CD4(+) T cells. Our results are compatible with our initial hypothesis that the timing of increase in the pathogen load can be a critical parameter governing the kinetics of CD4(+) T cell-dependent expansion of pathogen-specific CD8(+) cytotoxic T cells.

Routine detection of Epstein-Barr virus specific T-cells in the peripheral blood by flow cytometry

NASA Technical Reports Server (NTRS)

Crucian, B. E.; Stowe, R. P.; Pierson, D. L.; Sams, C. F.

2001-01-01

The ability to detect cytomegalovirus-specific T-cells (CD4(+)) in the peripheral blood by flow cytometry has been recently described by Picker et al. In this method, cells are incubated with viral antigen and responding (cytokine producing) T-cells are then identified by flow cytometry. To date, this technique has not been reliably used to detect Epstein-Barr virus (EBV)-specific T-cells primarily due to the superantigen/mitogenic properties of the virus which non-specifically activate T-cells. By modifying culture conditions under which the antigens are presented, we have overcome this limitation and developed an assay to detect and quantitate EBV-specific T-cells. The detection of cytokine producing T-cells by flow cytometry requires an extremely strong signal (such as culture in the presence of PMA and ionomycin). Our data indicate that in modified culture conditions (early removal of viral antigen) the non-specific activation of T-cells by EBV is reduced, but antigen presentation will continue uninhibited. Using this method, EBV-specific T-cells may be legitimately detected using flow cytometry. No reduction in the numbers of antigen-specific T-cells was observed by the early removal of target antigen when verified using cytomegalovirus antigen (a virus with no non-specific T-cell activation properties). In EBV-seropositive individuals, the phenotype of the EBV-specific cytokine producing T-cells was evaluated using four-color flow cytometry and found to be CD45(+), CD3(+), CD4(+), CD45RA(-), CD69(+), CD25(-). This phenotype indicates the stimulation of circulating previously unactivated memory T-cells. No cytokine production was observed in CD4(+) T-cells from EBV-seronegative individuals, confirming the specificity of this assay. In addition, the use of four color cytometry (CD45, CD3, CD69, IFNgamma/IL-2) allows the total quantitative assessment of EBV-specific T-cells while monitoring the interference of EBV non-specific mitogenic activity. This method may have significant utility for the monitoring of the immune response to latent virus infection/reactivation.

Long-lived tissue resident HIV-1 specific memory CD8+ T cells are generated by skin immunization with live virus vectored microneedle arrays.

PubMed

Zaric, Marija; Becker, Pablo Daniel; Hervouet, Catherine; Kalcheva, Petya; Ibarzo Yus, Barbara; Cocita, Clement; O'Neill, Lauren Alexandra; Kwon, Sung-Yun; Klavinskis, Linda Sylvia

2017-12-28

The generation of tissue resident memory (T RM ) cells at the body surfaces to provide a front line defence against invading pathogens represents an important goal in vaccine development for a wide variety of pathogens. It has been widely assumed that local vaccine delivery to the mucosae is necessary to achieve that aim. Here we characterise a novel micro-needle array (MA) delivery system fabricated to deliver a live recombinant human adenovirus type 5 vaccine vector (AdHu5) encoding HIV-1 gag. We demonstrate rapid dissolution kinetics of the microneedles in skin. Moreover, a consequence of MA vaccine cargo release was the generation of long-lived antigen-specific CD8 + T cells that accumulate in mucosal tissues, including the female genital and respiratory tract. The memory CD8 + T cell population maintained in the peripheral mucosal tissues was attributable to a MA delivered AdHu5 vaccine instructing CD8 + T cell expression of CXCR3 + , CD103 +, CD49a + , CD69 + , CD127 + homing, retention and survival markers. Furthermore, memory CD8 + T cells generated by MA immunization significantly expanded upon locally administered antigenic challenge and showed a predominant poly-functional profile producing high levels of IFNγ and Granzyme B. These data demonstrate that skin vaccine delivery using microneedle technology induces mobilization of long lived, poly-functional CD8 + T cells to peripheral tissues, phenotypically displaying hallmarks of residency and yields new insights into how to design and deliver effective vaccine candidates with properties to exert local immunosurveillance at the mucosal surfaces. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

PubMed Central

Streng-Ouwehand, Ingeborg; Ho, Nataschja I; Litjens, Manja; Kalay, Hakan; Boks, Martine Annemarie; Cornelissen, Lenneke AM; Kaur Singh, Satwinder; Saeland, Eirikur; Garcia-Vallejo, Juan J; Ossendorp, Ferry A; Unger, Wendy WJ; van Kooyk, Yvette

2016-01-01

Antigen uptake by dendritic cells and intracellular routing of antigens to specific compartments is regulated by C-type lectin receptors that recognize glycan structures. We show that the modification of Ovalbumin (OVA) with the glycan-structure LewisX (LeX) re-directs OVA to the C-type lectin receptor MGL1. LeX-modification of OVA favored Th1 skewing of CD4+ T cells and enhanced cross-priming of CD8+ T cells. While cross-presentation of native OVA requires high antigen dose and TLR stimuli, LeX modification reduces the required amount 100-fold and obviates its dependence on TLR signaling. The OVA-LeX-induced enhancement of T cell cross-priming is MGL1-dependent as shown by reduced CD8+ effector T cell frequencies in MGL1-deficient mice. Moreover, MGL1-mediated cross-presentation of OVA-LeX neither required TAP-transporters nor Cathepsin-S and was still observed after prolonged intracellular storage of antigen in Rab11+LAMP1+ compartments. We conclude that controlled neo-glycosylation of antigens can crucially influence intracellular routing of antigens, the nature and strength of immune responses and should be considered for optimizing current vaccination strategies. DOI: http://dx.doi.org/10.7554/eLife.11765.001 PMID:26999763

Single-cell multiplexed cytokine profiling of CD19 CAR-T cells reveals a diverse landscape of polyfunctional antigen-specific response.

PubMed

Xue, Qiong; Bettini, Emily; Paczkowski, Patrick; Ng, Colin; Kaiser, Alaina; McConnell, Timothy; Kodrasi, Olja; Quigley, Máire F; Heath, James; Fan, Rong; Mackay, Sean; Dudley, Mark E; Kassim, Sadik H; Zhou, Jing

2017-11-21

It remains challenging to characterize the functional attributes of chimeric antigen receptor (CAR)-engineered T cell product targeting CD19 related to potency and immunotoxicity ex vivo, despite promising in vivo efficacy in patients with B cell malignancies. We employed a single-cell, 16-plex cytokine microfluidics device and new analysis techniques to evaluate the functional profile of CD19 CAR-T cells upon antigen-specific stimulation. CAR-T cells were manufactured from human PBMCs transfected with the lentivirus encoding the CD19-BB-z transgene and expanded with anti-CD3/anti-CD28 coated beads. The enriched CAR-T cells were stimulated with anti-CAR or control IgG beads, stained with anti-CD4 RPE and anti-CD8 Alexa Fluor 647 antibodies, and incubated for 16 h in a single-cell barcode chip (SCBC). Each SCBC contains ~12,000 microchambers, covered with a glass slide that was pre-patterned with a complete copy of a 16-plex antibody array. Protein secretions from single CAR-T cells were captured and subsequently analyzed using proprietary software and new visualization methods. We demonstrate a new method for single-cell profiling of CD19 CAR-T pre-infusion products prepared from 4 healthy donors. CAR-T single cells exhibited a marked heterogeneity of cytokine secretions and polyfunctional (2+ cytokine) subsets specific to anti-CAR bead stimulation. The breadth of responses includes anti-tumor effector (Granzyme B, IFN-γ, MIP-1α, TNF-α), stimulatory (GM-CSF, IL-2, IL-8), regulatory (IL-4, IL-13, IL-22), and inflammatory (IL-6, IL-17A) functions. Furthermore, we developed two new bioinformatics tools for more effective polyfunctional subset visualization and comparison between donors. Single-cell, multiplexed, proteomic profiling of CD19 CAR-T product reveals a diverse landscape of immune effector response of CD19 CAR-T cells to antigen-specific challenge, providing a new platform for capturing CAR-T product data for correlative analysis. Additionally, such high dimensional data requires new visualization methods to further define precise polyfunctional response differences in these products. The presented biomarker capture and analysis system provides a more sensitive and comprehensive functional assessment of CAR-T pre-infusion products and may provide insights into the safety and efficacy of CAR-T cell therapy.

Empty conformers of HLA-B preferentially bind CD8 and regulate CD8+ T cell function.

PubMed

Geng, Jie; Altman, John D; Krishnakumar, Sujatha; Raghavan, Malini

2018-05-09

When complexed with antigenic peptides, human leukocyte antigen (HLA) class I (HLA-I) molecules initiate CD8 + T cell responses via interaction with the T cell receptor (TCR) and co-receptor CD8. Peptides are generally critical for the stable cell surface expression of HLA-I molecules. However, for HLA-I alleles such as HLA-B*35:01, peptide-deficient (empty) heterodimers are thermostable and detectable on the cell surface. Additionally, peptide-deficient HLA-B*35:01 tetramers preferentially bind CD8 and to a majority of blood-derived CD8 + T cells via a CD8-dependent binding mode. Further functional studies reveal that peptide-deficient conformers of HLA-B*35:01 do not directly activate CD8 + T cells, but accumulate at the immunological synapse in antigen-induced responses, and enhance cognate peptide-induced cell adhesion and CD8 + T cell activation. Together, these findings indicate that HLA-I peptide occupancy influences CD8 binding affinity, and reveal a new set of regulators of CD8 + T cell activation, mediated by the binding of empty HLA-I to CD8. © 2018, Geng et al.

Multi-scale modeling of the CD8 immune response

NASA Astrophysics Data System (ADS)

Barbarroux, Loic; Michel, Philippe; Adimy, Mostafa; Crauste, Fabien

2016-06-01

During the primary CD8 T-Cell immune response to an intracellular pathogen, CD8 T-Cells undergo exponential proliferation and continuous differentiation, acquiring cytotoxic capabilities to address the infection and memorize the corresponding antigen. After cleaning the organism, the only CD8 T-Cells left are antigen-specific memory cells whose role is to respond stronger and faster in case they are presented this very same antigen again. That is how vaccines work: a small quantity of a weakened pathogen is introduced in the organism to trigger the primary response, generating corresponding memory cells in the process, giving the organism a way to defend himself in case it encounters the same pathogen again. To investigate this process, we propose a non linear, multi-scale mathematical model of the CD8 T-Cells immune response due to vaccination using a maturity structured partial differential equation. At the intracellular scale, the level of expression of key proteins is modeled by a delay differential equation system, which gives the speeds of maturation for each cell. The population of cells is modeled by a maturity structured equation whose speeds are given by the intracellular model. We focus here on building the model, as well as its asymptotic study. Finally, we display numerical simulations showing the model can reproduce the biological dynamics of the cell population for both the primary response and the secondary responses.

Multi-scale modeling of the CD8 immune response

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barbarroux, Loic, E-mail: loic.barbarroux@doctorant.ec-lyon.fr; Ecole Centrale de Lyon, 36 avenue Guy de Collongue, 69134 Ecully; Michel, Philippe, E-mail: philippe.michel@ec-lyon.fr

During the primary CD8 T-Cell immune response to an intracellular pathogen, CD8 T-Cells undergo exponential proliferation and continuous differentiation, acquiring cytotoxic capabilities to address the infection and memorize the corresponding antigen. After cleaning the organism, the only CD8 T-Cells left are antigen-specific memory cells whose role is to respond stronger and faster in case they are presented this very same antigen again. That is how vaccines work: a small quantity of a weakened pathogen is introduced in the organism to trigger the primary response, generating corresponding memory cells in the process, giving the organism a way to defend himself inmore » case it encounters the same pathogen again. To investigate this process, we propose a non linear, multi-scale mathematical model of the CD8 T-Cells immune response due to vaccination using a maturity structured partial differential equation. At the intracellular scale, the level of expression of key proteins is modeled by a delay differential equation system, which gives the speeds of maturation for each cell. The population of cells is modeled by a maturity structured equation whose speeds are given by the intracellular model. We focus here on building the model, as well as its asymptotic study. Finally, we display numerical simulations showing the model can reproduce the biological dynamics of the cell population for both the primary response and the secondary responses.« less

Flow Cytometric and Cytokine ELISpot Approaches To Characterize the Cell-Mediated Immune Response in Ferrets following Influenza Virus Infection

PubMed Central

DiPiazza, Anthony; Richards, Katherine; Batarse, Frances; Lockard, Laura; Zeng, Hui; García-Sastre, Adolfo; Albrecht, Randy A.

2016-01-01

ABSTRACT Influenza virus infections represent a significant socioeconomic and public health burden worldwide. Although ferrets are considered by many to be ideal for modeling human responses to influenza infection and vaccination, efforts to understand the cellular immune response have been severely hampered by a paucity of standardized procedures and reagents. In this study, we developed flow cytometric and T cell enzyme-linked immunosorbent spot (ELISpot) approaches to characterize the leukocyte composition and antigen-specific T cell response within key lymphoid tissues following influenza virus infection in ferrets. Through a newly designed and implemented set of serological reagents, we used multiparameter flow cytometry to directly quantify the frequency of CD4+ and CD8+ T cells, Ig+ B cells, CD11b+ myeloid-derived cells, and major histocompatibility complex (MHC) class II-positive antigen-presenting cells (APCs) both prior to and after intranasal infection with A/California/04/09 (H1N1). We found that the leukocyte composition was altered at 10 days postinfection, with notable gains in the frequency of T cells and myeloid cells within the draining lymph node. Furthermore, these studies revealed that the antigen specificity of influenza virus-reactive CD4 and CD8 T cells was very broad, with recognition of the viral HA, NA, M1, NS1, and NP proteins, and that total reactivity to influenza virus postinfection represented approximately 0.1% of the circulating peripheral blood mononuclear cells (PBMC). Finally, we observed distinct patterns of reactivity between individual animals, suggesting heterogeneity at the MHC locus in ferrets within commercial populations, a finding of considerable interest in efforts to move the ferret model forward for influenza vaccine and challenge studies. IMPORTANCE Ferrets are an ideal animal model to study transmission, diseases, and vaccine efficacies of respiratory viruses because of their close anatomical and physiological resemblances to humans. However, a lack of reagents has limited our understanding of the cell-mediated immune response following infection and vaccination. In this study, we used cross-reactive and ferret-specific antibodies to study the leukocyte composition and antigen-specific CD4 and CD8 T cell responses following influenza A/California/04/09 (H1N1) virus infection. These studies revealed strikingly distinct patterns of reactivity between CD4 and CD8 T cells, which were overlaid with differences in protein-specific responses between individual animals. Our results provide a first, in-depth look at the T cell repertoire in response to influenza infection and suggest that there is considerable heterogeneity at the MHC locus, which is akin to that in humans and an area of intense research interest. PMID:27356897

Flow Cytometric and Cytokine ELISpot Approaches To Characterize the Cell-Mediated Immune Response in Ferrets following Influenza Virus Infection.

PubMed

DiPiazza, Anthony; Richards, Katherine; Batarse, Frances; Lockard, Laura; Zeng, Hui; García-Sastre, Adolfo; Albrecht, Randy A; Sant, Andrea J

2016-09-01

Influenza virus infections represent a significant socioeconomic and public health burden worldwide. Although ferrets are considered by many to be ideal for modeling human responses to influenza infection and vaccination, efforts to understand the cellular immune response have been severely hampered by a paucity of standardized procedures and reagents. In this study, we developed flow cytometric and T cell enzyme-linked immunosorbent spot (ELISpot) approaches to characterize the leukocyte composition and antigen-specific T cell response within key lymphoid tissues following influenza virus infection in ferrets. Through a newly designed and implemented set of serological reagents, we used multiparameter flow cytometry to directly quantify the frequency of CD4(+) and CD8(+) T cells, Ig(+) B cells, CD11b(+) myeloid-derived cells, and major histocompatibility complex (MHC) class II-positive antigen-presenting cells (APCs) both prior to and after intranasal infection with A/California/04/09 (H1N1). We found that the leukocyte composition was altered at 10 days postinfection, with notable gains in the frequency of T cells and myeloid cells within the draining lymph node. Furthermore, these studies revealed that the antigen specificity of influenza virus-reactive CD4 and CD8 T cells was very broad, with recognition of the viral HA, NA, M1, NS1, and NP proteins, and that total reactivity to influenza virus postinfection represented approximately 0.1% of the circulating peripheral blood mononuclear cells (PBMC). Finally, we observed distinct patterns of reactivity between individual animals, suggesting heterogeneity at the MHC locus in ferrets within commercial populations, a finding of considerable interest in efforts to move the ferret model forward for influenza vaccine and challenge studies. Ferrets are an ideal animal model to study transmission, diseases, and vaccine efficacies of respiratory viruses because of their close anatomical and physiological resemblances to humans. However, a lack of reagents has limited our understanding of the cell-mediated immune response following infection and vaccination. In this study, we used cross-reactive and ferret-specific antibodies to study the leukocyte composition and antigen-specific CD4 and CD8 T cell responses following influenza A/California/04/09 (H1N1) virus infection. These studies revealed strikingly distinct patterns of reactivity between CD4 and CD8 T cells, which were overlaid with differences in protein-specific responses between individual animals. Our results provide a first, in-depth look at the T cell repertoire in response to influenza infection and suggest that there is considerable heterogeneity at the MHC locus, which is akin to that in humans and an area of intense research interest. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Genome-wide RNA profiling of long-lasting stem cell-like memory CD8 T cells induced by Yellow Fever vaccination in humans.

PubMed

Fuertes Marraco, Silvia A; Soneson, Charlotte; Delorenzi, Mauro; Speiser, Daniel E

2015-09-01

The live-attenuated Yellow Fever (YF) vaccine YF-17D induces a broad and polyfunctional CD8 T cell response in humans. Recently, we identified a population of stem cell-like memory CD8 T cells induced by YF-17D that persists at stable frequency for at least 25 years after vaccination. The YF-17D is thus a model system of human CD8 T cell biology that furthermore allows to track and study long-lasting and antigen-specific human memory CD8 T cells. Here, we describe in detail the sample characteristics and preparation of a microarray dataset acquired for genome-wide gene expression profiling of long-lasting YF-specific stem cell-like memory CD8 T cells, compared to the reference CD8 T cell differentiation subsets from total CD8 T cells. We also describe the quality controls, annotations and exploratory analyses of the dataset. The microarray data is available from the Gene Expression Omnibus (GEO) public repository with accession number GSE65804.

In vivo imaging of CD8+ T cell-mediated elimination of malaria liver stages

PubMed Central

Cockburn, Ian A.; Amino, Rogerio; Kelemen, Reka K.; Kuo, Scot C.; Tse, Sze-Wah; Radtke, Andrea; Mac-Daniel, Laura; Ganusov, Vitaly V.; Zavala, Fidel; Ménard, Robert

2013-01-01

CD8+ T cells are specialized cells of the adaptive immune system capable of finding and eliminating pathogen-infected cells. To date it has not been possible to observe the destruction of any pathogen by CD8+ T cells in vivo. Here we demonstrate a technique for imaging the killing of liver-stage malaria parasites by CD8+ T cells bearing a transgenic T cell receptor specific for a parasite epitope. We report several features that have not been described by in vitro analysis of the process, chiefly the formation of large clusters of effector CD8+ T cells around infected hepatocytes. The formation of clusters requires antigen-specific CD8+ T cells and signaling by G protein-coupled receptors, although CD8+ T cells of unrelated specificity are also recruited to clusters. By combining mathematical modeling and data analysis, we suggest that formation of clusters is mainly driven by enhanced recruitment of T cells into larger clusters. We further show various death phenotypes of the parasite, which typically follow prolonged interactions between infected hepatocytes and CD8+ T cells. These findings stress the need for intravital imaging for dissecting the fine mechanisms of pathogen recognition and killing by CD8+ T cells. PMID:23674673

TLR1/2 activation during heterologous prime-boost vaccination (DNA-MVA) enhances CD8+ T Cell responses providing protection against Leishmania (Viannia).

PubMed

Jayakumar, Asha; Castilho, Tiago M; Park, Esther; Goldsmith-Pestana, Karen; Blackwell, Jenefer M; McMahon-Pratt, Diane

2011-06-01

Leishmania (Viannia) parasites present particular challenges, as human and murine immune responses to infection are distinct from other Leishmania species, indicating a unique interaction with the host. Further, vaccination studies utilizing small animal models indicate that modalities and antigens that prevent infection by other Leishmania species are generally not protective. Using a newly developed mouse model of chronic L. (Viannia) panamensis infection and the heterologous DNA prime - modified vaccinia virus Ankara (MVA) boost vaccination modality, we examined whether the conserved vaccine candidate antigen tryparedoxin peroxidase (TRYP) could provide protection against infection/disease. Heterologous prime - boost (DNA/MVA) vaccination utilizing TRYP antigen can provide protection against disease caused by L. (V.) panamensis. However, protection is dependent on modulating the innate immune response using the TLR1/2 agonist Pam3CSK4 during DNA priming. Prime-boost vaccination using DNA alone fails to protect. Prior to infection protectively vaccinated mice exhibit augmented CD4 and CD8 IFNγ and memory responses as well as decreased IL-10 and IL-13 responses. IL-13 and IL-10 have been shown to be independently critical for disease in this model. CD8 T cells have an essential role in mediating host defense, as CD8 depletion reversed protection in the vaccinated mice; vaccinated mice depleted of CD4 T cells remained protected. Hence, vaccine-induced protection is dependent upon TLR1/2 activation instructing the generation of antigen specific CD8 cells and restricting IL-13 and IL-10 responses. Given the general effectiveness of prime-boost vaccination, the recalcitrance of Leishmania (Viannia) to vaccine approaches effective against other species of Leishmania is again evident. However, prime-boost vaccination modality can with modulation induce protective responses, indicating that the delivery system is critical. Moreover, these results suggest that CD8 T cells should be targeted for the development of a vaccine against infection caused by Leishmania (Viannia) parasites. Further, TLR1/2 modulation may be useful in vaccines where CD8 T cell responses are critical.

Delayed BCG vaccination results in minimal alterations in T cell immunogenicity of acellular pertussis and tetanus immunizations in HIV-exposed infants.

PubMed

Blakney, Anna K; Tchakoute, Christophe Toukam; Hesseling, Anneke C; Kidzeru, Elvis B; Jones, Christine E; Passmore, Jo-Ann S; Sodora, Donald L; Gray, Clive M; Jaspan, Heather B

2015-09-11

Bacille Calmette-Guerin (BCG) is effective in preventing disseminated tuberculosis (TB) in children but may also have non-specific benefits, and is thought to improve immunity to unrelated antigens through trained innate immunity. In HIV-infected infants, there is a risk of BCG-associated adverse events. We aimed to explore whether delaying BCG vaccination by 8 weeks, in utero or perinatal HIV infection is excluded, affected T-cell responses to B. pertussis (BP) and tetanus toxoid (TT), in HIV-exposed, uninfected infants. Infants were randomized to receive BCG vaccination at birth or 8 weeks of age. At 8 and 14 weeks, T cell proliferation and intracellular cytokine (IL-2, IL-13, IL-17, and IFN-γ) expression was analyzed in response to BP, TT and Staphylococcal enterotoxin B (SEB) antigens. Delaying BCG vaccination did not alter T-cell proliferation to BP or TT antigens. Infants immunized with BCG at birth had higher CD4+ T cell proliferation to SEB at 14 weeks of age (p=0.018). Birth-vaccinated infants had increased CD8+ IL-2 expression in response to BP, but not TT or SEB, at 8 weeks. Infants vaccinated with BCG at 8 weeks had significantly lower IL-13 expression by BP-specific CD4+ and CD8+ T cells at 14 weeks (p=0.032 and p=0.0035, respectively). There were no observed differences in multifunctional cytokine response to TT, BP or SEB between infants vaccinated with BCG at birth versus 8 weeks of age. Delaying BCG vaccination until 8 weeks of age results in robust T-cellular responses to BP and TT in HIV-exposed infants. NCT02062580. Copyright © 2015 Elsevier Ltd. All rights reserved.

Identification of HLA-A2–restricted CD8+ Cytotoxic T Cell Responses in Primary Biliary Cirrhosis

PubMed Central

Kita, Hiroto; Lian, Zhe-Xiong; Van de Water, Judy; He, Xiao-Song; Matsumura, Shuji; Kaplan, Marshall; Luketic, Velimir; Coppel, Ross L.; Ansari, Aftab A.; Gershwin, M. Eric

2002-01-01

Primary biliary cirrhosis (PBC) is characterized by an intense biliary inflammatory CD4+ and CD8+ T cell response. Very limited information on autoantigen-specific cytotoxic T lymphocyte (CTL) responses is available compared with autoreactive CD4+ T cell responses. Using peripheral blood mononuclear cells (PBMCs) from PBC, we identified an HLA-A2–restricted CTL epitope of the E2 component of pyruvate dehydrogenase (PDC-E2), the immunodominant mitochondrial autoantigen. This peptide, amino acids 159–167 of PDC-E2, induces specific MHC class I–restricted CD8+ CTL lines from 10/12 HLA-A2+ PBC patients, but not controls, after in vitro stimulation with antigen-pulsed dendritic cells (DCs). PDC-E2–specific CTLs could also be generated by pulsing DCs with full-length recombinant PDC-E2 protein. Furthermore, using soluble PDC-E2 complexed with either PDC-E2–specific human monoclonal antibody or affinity-purified autoantibodies against PDC-E2, the generation of PDC-E2–specific CTLs, occurred at 100-fold and 10-fold less concentration, respectively, compared with soluble antigen alone. Collectively, these data demonstrate that autoantibody, helper, and CTL epitopes all contain a shared peptide sequence. The finding that autoantigen–immune complexes can not only cross-present but also that presentation of the autoantigen is of a higher relative efficiency, for the first time defines a unique role for autoantibodies in the pathogenesis of an autoimmune disease. PMID:11781370

Detection of Virus-Specific CD8+ T Cells With Cross-Reactivity Against Alloantigens: Potency and Flaws of Present Experimental Methods

PubMed Central

van den Heuvel, Heleen; Heutinck, Kirstin M.; van der Meer-Prins, Ellen P.M.W.; Yong, Si La; Claas, Frans H.J.; ten Berge, Ineke J.M.

2015-01-01

Background Virus-specific T cells have the intrinsic capacity to cross-react against allogeneic HLA antigens, a phenomenon known as heterologous immunity. In transplantation, these cells may contribute to the alloimmune response and negatively impact graft outcome. This study describes the various techniques that can be used to detect heterologous immune responses of virus-specific CD8+ T cells against allogeneic HLA antigens. The strengths and weaknesses of the different approaches are discussed and illustrated by experimental data. Methods Mixed-lymphocyte reactions (MLRs) were performed to detect allo-HLA cross-reactivity of virus-specific CD8+ T cells in total peripheral blood mononuclear cells. T-cell lines and clones were generated to confirm allo-HLA cross-reactivity by IFNγ production and cytotoxicity. In addition, the conventional MLR protocol was adjusted by introducing a 3-day resting phase and subsequent short restimulation with alloantigen or viral peptide, whereupon the expression of IFNγ, IL-2, CD107a, and CD137 was determined. Results The accuracy of conventional MLR is challenged by potential bystander activation. T-cell lines and clones can circumvent this issue, yet their generation is laborious and time-consuming. Using the adjusted MLR and restimulation protocol, we found that only truly cross-reactive T cells responded to re-encounter of alloantigen and viral peptide, whereas bystander-activated cells did not. Conclusions The introduction of a restimulation phase improved the accuracy of the MLR as a screening tool for the detection of allo-HLA cross-reactivity by virus-specific CD8+ T cells at bulk level. For detailed characterization of cross-reactive cells, T-cell lines and clones remain the golden standard. PMID:27500209

Toward the Clonotype Analysis of Alopecia Areata-Specific, Intralesional Human CD8+ T Lymphocytes.

PubMed

Bertolini, Marta; Uchida, Youhei; Paus, Ralf

2015-11-01

Alopecia areata (AA) is an organ-restricted autoimmune disease that mainly affects the hair follicle (HF). Several findings support a key primary effector role of CD8+ T cells in the disease pathogenesis. Autoreactive CD8+ T cells are not only present in the characteristic peribulbar inflammatory cell infiltrate of lesional AA HFs but are also found to be infiltrating in lesional HF epithelium where they are thought to recognize major histocompatibility complex class I-presented (auto-)antigens. However, the latter still remain unidentified. Therefore, one key aim in AA research is to identify the clonotypes of autoaggressive, intralesional CD8+ T cells. Therapeutically, this is important (a) so that these lymphocytes can be selectively eliminated or inhibited, (b) to identify the-as yet elusive-key (auto-)antigens in AA, and/or (c) to induce peripheral tolerance against the latter. Therefore, we have recently embarked on a National Alopecia Areata Foundation-supported project that attempts to isolate disease-specific, intralesional CD8+ T cells from AA skin in order to determine their TCR clonotype, using two complementary strategies. The first method is based on the enzymatic skin digestion from lesional AA skin, followed by either MACS technology and single-cell picking or FACS cell sorting, while the second method on laser microdissection. The identification of disease-specific TCRs can serve as a basis for specific AA immunotherapy along the lines sketched above and may possibly also provide prognostic biomarkers. If successful, this research strategy promises to permit, at long last, the causal therapy of AA.

Development of Augmented Leukemia/Lymphoma-Specific T-Cell Immunotherapy for Deployment with Haploidentical Hematopoietic Progenitor-Cell Transplant

DTIC Science & Technology

2009-05-01

adoptive therapy using CD19-specific chimeric antigen receptor re-directed T cells for recurrent/refrctory follicular lymphoma...Beauty (SB) transposon/transposase system to express a CD19-specific chimeric antigen receptor (CAR). T cells that have undergone transposition...accomplished using genetic engineering to express a chimeric antigen receptor (CAR) to redirect the specificity of T cells for CD19 on malignant B cells

Differential CD4+ versus CD8+ T-cell responses elicited by different poxvirus-based human immunodeficiency virus type 1 vaccine candidates provide comparable efficacies in primates.

PubMed

Mooij, Petra; Balla-Jhagjhoorsingh, Sunita S; Koopman, Gerrit; Beenhakker, Niels; van Haaften, Patricia; Baak, Ilona; Nieuwenhuis, Ivonne G; Kondova, Ivanela; Wagner, Ralf; Wolf, Hans; Gómez, Carmen E; Nájera, José L; Jiménez, Victoria; Esteban, Mariano; Heeney, Jonathan L

2008-03-01

Poxvirus vectors have proven to be highly effective for boosting immune responses in diverse vaccine settings. Recent reports reveal marked differences in the gene expression of human dendritic cells infected with two leading poxvirus-based human immunodeficiency virus (HIV) vaccine candidates, New York vaccinia virus (NYVAC) and modified vaccinia virus Ankara (MVA). To understand how complex genomic changes in these two vaccine vectors translate into antigen-specific systemic immune responses, we undertook a head-to-head vaccine immunogenicity and efficacy study in the pathogenic HIV type 1 (HIV-1) model of AIDS in Indian rhesus macaques. Differences in the immune responses in outbred animals were not distinguished by enzyme-linked immunospot assays, but differences were distinguished by multiparameter fluorescence-activated cell sorter analysis, revealing a difference between the number of animals with both CD4(+) and CD8(+) T-cell responses to vaccine inserts (MVA) and those that elicit a dominant CD4(+) T-cell response (NYVAC). Remarkably, vector-induced differences in CD4(+)/CD8(+) T-cell immune responses persisted for more than a year after challenge and even accompanied antigenic modulation throughout the control of chronic infection. Importantly, strong preexposure HIV-1/simian immunodeficiency virus-specific CD4(+) T-cell responses did not prove deleterious with respect to accelerated disease progression. In contrast, in this setting, animals with strong vaccine-induced polyfunctional CD4(+) T-cell responses showed efficacies similar to those with stronger CD8(+) T-cell responses.

T-cell help permits memory CD8(+) T-cell inflation during cytomegalovirus latency.

PubMed

Walton, Senta M; Torti, Nicole; Mandaric, Sanja; Oxenius, Annette

2011-08-01

CD4(+) T cells are implied to sustain CD8(+) T-cell responses during persistent infections. As CD4(+) T cells are often themselves antiviral effectors, they might shape CD8(+) T-cell responses via help or via controlling antigen load. We used persistent murine CMV (MCMV) infection to dissect the impact of CD4(+) T cells on virus-specific CD8(+) T cells, distinguishing between increased viral load in the absence of CD4(+) T cells and CD4(+) T-cell-mediated helper mechanisms. Absence of T-helper cells was associated with sustained lytic MCMV replication and led to a slow and gradual reduction of the size and function of the MCMV-specific CD8(+) T-cell pool. However, when virus replication was controlled in the absence of CD4(+) T cells, CD8(+) T-cell function was comparably impaired, but in addition CD8(+) T-cell inflation, a hallmark of CMV infection, was completely abolished. Thus, CD8(+) T-cell inflation during latent CMV infection is strongly dependent on CD4(+) T-cell helper functions, which can partially be compensated by ongoing lytic viral replication in the absence of CD4(+) T cells. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

T LYMPHOCYTES TARGETING NATIVE RECEPTORS

PubMed Central

Rooney, Cliona M; Leen, Ann M; Vera, Juan F; Heslop, Helen E

2013-01-01

Summary The adoptive transfer of T cells specific for native tumor antigens (TAs) is an increasingly popular cancer treatment option because of the ability of these cells to discriminate between normal and tumor tissues and corresponding lack of short or long-term toxicities. Infusions of antigen-specific CD4+ and CD8+ T cells targeting viral antigens derived from Epstein Barr virus (EBV) induce sustained complete tumor remissions in patients with highly immunogenic tumor’s such as post-transplant lymphoproliferative disease, although resistance occurred when the infused T-cell population had restricted antigen specificity. T cells specific for EBV antigens have also produced complete remissions of EBV-positive nasopharyngeal carcinomas and lymphomas developing in immunocompetent individuals, even though in these patients tumor survival is dependent on their ability to evade T-cell immunity. Adapting this strategy to non-viral tumors is more challenging, as the target antigens expressed are less immunogenic and the tumors lack the potent danger signals that are characteristic of viruses. The goals of current studies are to define conditions that promote expansion of antigen-specific T cells ex vivo and to ensure their in vivo persistence and survival by combining with maneuvers such as lymphodepletion, checkpoint inhibition, cytokine infusions, or genetic manipulations. More pragmatic goals are to streamline manufacturing to facilitate the transition of these therapies to late phase trials and to evaluate closely histocompatibility antigen (HLA)-matched banked antigen-specific T-cells so that T-cell therapies can be made more broadly available. PMID:24329788

Conserved region C functions to regulate PD-1 expression and subsequent CD8 T cell memory1

PubMed Central

Bally, Alexander P. R.; Tang, Yan; Lee, Joshua T.; Barwick, Benjamin G.; Martinez, Ryan; Evavold, Brian D.; Boss, Jeremy M.

2016-01-01

Expression of programmed death 1 (PD-1) on CD8 T cells promotes T cell exhaustion during chronic antigen exposure. During acute infections, PD-1 is transiently expressed and has the potential to modulate CD8 T cell memory formation. Conserved Region C (CR-C), a promoter proximal cis-regulatory element that is critical to PD-1 expression in vitro, responds to NFATc1, FoxO1, and/or NF-κB signaling pathways. Here, a CR-C knockout mouse (CRC−) was established to determine its role on PD-1 expression and corresponding effects on T cell function in vivo. Deletion of CR-C decreased PD-1 expression on CD4 T cells and antigen-specific CD8 T cells during acute and chronic lymphocytic choriomeningitis virus (LCMV) challenges, but did not affect the ability to clear an infection. Following acute LCMV infection, memory CD8 T cells in the CRC− mouse were formed in greater numbers, were more functional, and were more effective at responding to a melanoma tumor than wild-type memory cells. These data implicate a critical role for CR-C in governing PD-1 expression, and a subsequent role in guiding CD8 T cell differentiation. The data suggest the possibility that titrating PD-1 expression during CD8 T cell activation could have important ramifications in vaccine development and clinical care. PMID:27895178

Engineered outer membrane vesicle is potent to elicit HPV16E7-specific cellular immunity in a mouse model of TC-1 graft tumor.

PubMed

Wang, Shijie; Huang, Weiwei; Li, Kui; Yao, Yufeng; Yang, Xu; Bai, Hongmei; Sun, Wenjia; Liu, Cunbao; Ma, Yanbing

2017-01-01

Currently, therapeutic tumor vaccines under development generally lack significant effects in human clinical trials. Exploring a powerful antigen delivery system is a potential approach to improve vaccine efficacy. We sought to explore engineered bacterial outer membrane vesicles (OMVs) as a new vaccine carrier for efficiently delivering tumor antigens and provoking robust antitumor immune responses. First, the tumoral antigen human papillomavirus type 16 early protein E7 (HPV16E7) was presented on Escherichia coli -derived OMVs by genetic engineering methods, acquiring the recombinant OMV vaccine. Second, the ability of recombinant OMVs delivering their components and the model antigen green fluorescent protein to antigen-presenting cells was investigated in the macrophage Raw264.7 cells and in bone marrow-derived dendritic cells in vitro. Third, it was evaluated in TC-1 graft tumor model in mice that the recombinant OMVs displaying HPV16E7 stimulated specific cellular immune response and intervened the growth of established tumor. E. coli DH5α-derived OMVs could be taken up rapidly by dendritic cells, for which vesicle structure has been proven to be important. OMVs significantly stimulated the expression of dendritic cellmaturation markers CD80, CD86, CD83 and CD40. The HPV16E7 was successfully embedded in engineered OMVs through gene recombinant techniques. Subcutaneous immunization with the engineered OMVs induced E7 antigen-specific cellular immune responses, as shown by the increased numbers of interferon-gamma-expressing splenocytes by enzyme-linked immunospot assay and interferon-gamma-expressing CD4 + and CD8 + cells by flow cytometry analyses. Furthermore, the growth of grafted TC-1 tumors in mice was significantly suppressed by therapeutic vaccination. The recombinant E7 proteins presented by OMVs were more potent than those mixed with wild-type OMVs or administered alone for inducing specific cellular immunity and suppressing tumor growth. The results indicated that the nano-grade OMVs might be a useful vaccine platform for antigen delivery in cancer immunotherapy.

In vitro cytotoxicity of CD8+ T cells in multi-drug-resistant tuberculosis. A preliminary report.

PubMed

Sada-Ovalle, Isabel; Torre-Bouscoulet, Luis; Valdez-Vázquez, Rafael; Lascurain, Ricardo

2009-05-01

Specific CD8+ T-cell cytotoxicity has been recognized as being involved in the elimination of drug-susceptible tuberculosis (DS-TB). Given that there is currently no information on the cytotoxic effector functions of CD8+ T cells in multi-drug-resistant tuberculosis (MDR-TB), our objective was to analyse the cytotoxic activity, both basal and stimulated, of CD8+ T cells from MDR-TB patients and compare it with that of DS-TB patients, as well as purified protein derivative (PPD)+ and PPD- subjects. Cytotoxic activity of CD8+ T cells from MDR-TB patients, DS-TB patients, PPD+ and PPD- subjects was measured by a colorimetric assay, using H37Rv culture filtrate protein as the antigenic stimulus. Twenty-eight subjects were studied (7 MDR-TB patients, 7 DS-TB patients, 7 PPD+ subjects and 7 PPD- subjects). In the presence of the antigenic stimulus, the cytotoxic activity of CD8+ T cells from MDR-TB patients (% lysis) increased from 6.7% to 59.6% (P < 0.001). In DS-TB patients lysis increased from 3.2% to 22.5% (P < 0.001), whereas in PPD+ subjects it increased from 2.7% to 12.0% (P < 0.001) and in PPD- subjects from 1.3% to 3.2% (P < 0.001). Basal cytotoxic activity was significantly higher for MDR-TB patients than PPD+ and PPD- subjects (P = 0.003), but not compared with that for DS-TB patients (P = 0.05). Stimulated cytotoxic activity was highest for MDR-TB patients. CD8+ T cells from MDR-TB patients showed an exaggerated cytotoxic activity after antigenic stimulation. Further studies are required to elucidate the role of this response in the immunopathogenesis of MDR-TB.

Intrinsic and extrinsic contributors to defective CD8+ T cell responses with aging.

PubMed

Jergović, Mladen; Smithey, Megan J; Nikolich-Žugich, Janko

2018-05-01

Aging has a profound effect on the immune system, and both innate and adaptive arms of the immune system show functional decline with age. In response to infection with intracellular microorganisms, old animals mobilize decreased numbers of antigen-specific CD8+ T cells with reduced production of effector molecules and impaired cytolytic activity. However, the CD8+ T cell-intrinsic contribution to, and molecular mechanisms behind, these defects remain unclear. In this review we will discuss the mechanistic contributions of age related changes in the CD8+ T cell pool and the relative roles of intrinsic functional defects in aged CD8+ T cells vs. defects in the aged environment initiating the CD8+ T cell response. Copyright © 2018 Elsevier Inc. All rights reserved.

Expression of Tac antigen component of bovine interleukin-2 receptor in different leukocyte populations infected with Theileria parva or Theileria annulata.

PubMed Central

Dobbelaere, D A; Prospero, T D; Roditi, I J; Kelke, C; Baumann, I; Eichhorn, M; Williams, R O; Ahmed, J S; Baldwin, C L; Clevers, H

1990-01-01

The Tac antigen component of the bovine interleukin-2 receptor was expressed as a Cro-beta-galactosidase fusion protein in Escherichia coli and used to raise antibodies in rabbits. These antibodies were used for flow cytofluorimetric analysis to investigate the expression of Tac antigen in a variety of Theileria parva-infected cell lines and also in three Theileria annulata-infected cell lines. Cells expressing Tac antigen on their surface were found in all T. parva-infected cell lines tested whether these were of T- or B-cell origin. T cells expressing Tac antigen could be CD4- CD8-, CD4+ CD8-, CD4- CD8+, or CD4+ CD8+. Tac antigen expression was observed both in cultures which had been maintained in the laboratory for several years and in transformed cell lines which had recently been established by infection of lymphocytes in vitro with T. parva. Northern (RNA) blot analysis demonstrated Tac antigen transcripts in RNA isolated from all T. parva-infected cell lines. Three T. annulata-infected cell lines which were not of T-cell origin were also tested. Two of them expressed Tac antigen on their surface. Abundant Tac antigen mRNA was detected in these T. annulata-infected cell lines, but only trace amounts were demonstrated in the third cell line, which contained very few Tac antigen-expressing cells. In all cell lines tested, whether cloned or uncloned, a proportion of the cells did not express detectable levels of Tac antigen on their surface. This was also the case for a number of other leukocyte surface markers. In addition, we showed that the interleukin-2 receptors were biologically functional, because addition of recombinant interleukin-2 to cultures stimulated cell proliferation. Recombinant interleukin-2 treatment also resulted in increased amounts of steady-state Tac antigen mRNA. The relevance of interleukin-2 receptor expression on Theileria-infected cells is discussed. Images PMID:1979317

Germline bias dictates cross-serotype reactivity in a common dengue-virus-specific CD8+ T cell response.

PubMed

Culshaw, Abigail; Ladell, Kristin; Gras, Stephanie; McLaren, James E; Miners, Kelly L; Farenc, Carine; van den Heuvel, Heleen; Gostick, Emma; Dejnirattisai, Wanwisa; Wangteeraprasert, Apirath; Duangchinda, Thaneeya; Chotiyarnwong, Pojchong; Limpitikul, Wannee; Vasanawathana, Sirijitt; Malasit, Prida; Dong, Tao; Rossjohn, Jamie; Mongkolsapaya, Juthathip; Price, David A; Screaton, Gavin R

2017-11-01

Adaptive immune responses protect against infection with dengue virus (DENV), yet cross-reactivity with distinct serotypes can precipitate life-threatening clinical disease. We found that clonotypes expressing the T cell antigen receptor (TCR) β-chain variable region 11 (TRBV11-2) were 'preferentially' activated and mobilized within immunodominant human-leukocyte-antigen-(HLA)-A*11:01-restricted CD8 + T cell populations specific for variants of the nonstructural protein epitope NS3 133 that characterize the serotypes DENV1, DENV3 and DENV4. In contrast, the NS3 133 -DENV2-specific repertoire was largely devoid of such TCRs. Structural analysis of a representative TRBV11-2 + TCR demonstrated that cross-serotype reactivity was governed by unique interplay between the variable antigenic determinant and germline-encoded residues in the second β-chain complementarity-determining region (CDR2β). Extensive mutagenesis studies of three distinct TRBV11-2 + TCRs further confirmed that antigen recognition was dependent on key contacts between the serotype-defined peptide and discrete residues in the CDR2β loop. Collectively, these data reveal an innate-like mode of epitope recognition with potential implications for the outcome of sequential exposure to heterologous DENVs.

Genes encoding two Theileria parva antigens recognized by CD8+ T-cells exhibit sequence diversity in South Sudanese cattle populations but the majority of alleles are similar to the Muguga component of the live vaccine cocktail

PubMed Central

Pelle, Roger; Mwacharo, Joram M.; Njahira, Moses N.; Marcellino, Wani L.; Kiara, Henry; Malak, Agol K.; EL Hussein, Abdel Rahim M.; Bishop, Richard; Skilton, Robert A.

2017-01-01

East Coast fever (ECF), caused by Theileria parva infection, is a frequently fatal disease of cattle in eastern, central and southern Africa, and an emerging disease in South Sudan. Immunization using the infection and treatment method (ITM) is increasingly being used for control in countries affected by ECF, but not yet in South Sudan. It has been reported that CD8+ T-cell lymphocytes specific for parasitized cells play a central role in the immunity induced by ITM and a number of T. parva antigens recognized by parasite-specific CD8+ T-cells have been identified. In this study we determined the sequence diversity among two of these antigens, Tp1 and Tp2, which are under evaluation as candidates for inclusion in a sub-unit vaccine. T. parva samples (n = 81) obtained from cattle in four geographical regions of South Sudan were studied for sequence polymorphism in partial sequences of the Tp1 and Tp2 genes. Eight positions (1.97%) in Tp1 and 78 positions (15.48%) in Tp2 were shown to be polymorphic, giving rise to four and 14 antigen variants in Tp1 and Tp2, respectively. The overall nucleotide diversity in the Tp1 and Tp2 genes was π = 1.65% and π = 4.76%, respectively. The parasites were sampled from regions approximately 300 km apart, but there was limited evidence for genetic differentiation between populations. Analyses of the sequences revealed limited numbers of amino acid polymorphisms both overall and in residues within the mapped CD8+ T-cell epitopes. Although novel epitopes were identified in the samples from South Sudan, a large number of the samples harboured several epitopes in both antigens that were similar to those in the T. parva Muguga reference stock, which is a key component in the widely used live vaccine cocktail. PMID:28231338

Memory T cells maintain protracted protection against malaria.

PubMed

Krzych, Urszula; Zarling, Stasya; Pichugin, Alexander

2014-10-01

Immunologic memory is one of the cardinal features of antigen-specific immune responses, and the persistence of memory cells contributes to prophylactic immunizations against infectious agents. Adequately maintained memory T and B cell pools assure a fast, effective and specific response against re-infections. However, many aspects of immunologic memory are still poorly understood, particularly immunologic memory inducible by parasites, for example, Plasmodium spp., the causative agents of malaria. For example, memory responses to Plasmodium antigens amongst residents of malaria endemic areas appear to be either inadequately developed or maintained, because persons who survive episodes of childhood malaria remain vulnerable to intermittent malaria infections. By contrast, multiple exposures of humans and laboratory rodents to radiation-attenuated Plasmodium sporozoites (γ-spz) induce sterile and long-lasting protection against experimental sporozoite challenge. Multifactorial immune mechanisms maintain this protracted and sterile protection. While the presence of memory CD4 T cell subsets has been associated with lasting protection in humans exposed to multiple bites from Anopheles mosquitoes infected with attenuated Plasmodium falciparum, memory CD8 T cells maintain protection induced with Plasmodium yoelii and Plasmodium berghei γ-spz in murine models. In this review, we discuss our observations that show memory CD8 T cells specific for antigens expressed by P. berghei liver stage parasites as an indispensable component for the maintenance of protracted protective immunity against experimental malaria infection; moreover, the provision of an Ag-depot assures a quick recall of memory T cells as IFN-γ-producing effector CD8 T cells and IL-4- producing CD4 T cells that collaborate with B cells for an effective antibody response. Published by Elsevier B.V.

Discovery of a protective Rickettsia prowazekii antigen recognized by CD8+ T cells, RP884, using an in vivo screening platform.

PubMed

Gazi, Michal; Caro-Gomez, Erika; Goez, Yenny; Cespedes, Maria A; Hidalgo, Marylin; Correa, Paula; Valbuena, Gustavo

2013-01-01

Rickettsia prowazekii has been tested for biological warfare due to the high mortality that it produces after aerosol transmission of very low numbers of rickettsiae. Epidemic typhus, the infection caused by these obligately intracellular bacteria, continues to be a threat because it is difficult to diagnose due to initial non-specific symptoms and the lack of commercial diagnostic tests that are sensitive and specific during the initial clinical presentation. A vaccine to prevent epidemic typhus would constitute an effective deterrent to the weaponization of R. prowazekii; however, an effective and safe vaccine is not currently available. Due to the cytoplasmic niche of Rickettsia, CD8(+) T-cells are critical effectors of immunity; however, the identification of antigens recognized by these cells has not been systematically addressed. To help close this gap, we designed an antigen discovery strategy that uses cell-based vaccination with antigen presenting cells expressing microbe's proteins targeted to the MHC class I presentation pathway. We report the use of this method to discover a protective T-cell rickettsial antigen, RP884, among a test subset of rickettsial proteins.

Manipulating the tumor microenvironment ex vivo for enhanced expansion of tumor-infiltrating lymphocytes for adoptive cell therapy

PubMed Central

Chacon, Jessica Ann; Sarnaik, Amod A; Chen, Jie Qing; Creasy, Caitlin; Kale, Charuta; Robinson, John; Weber, Jeffrey; Hwu, Patrick; Pilon-Thomas, Shari; Radvanyi, Laszlo

2014-01-01

Purpose Cultured tumor fragments from melanoma metastases have been used for years as a source of tumor-infiltrating lymphocytes (TIL) for adoptive cell therapy. The expansion of tumor-reactive CD8+ T cells with IL-2 in these early cultures is critical in generating clinically active TIL infusion products, with a population of activated 4-1BB CD8+ T cells recently found to constitute the majority of tumor-specific T cells. Experimental Design We used an agonistic anti-4-1BB antibody added during the initial tumor fragment cultures to provide in situ 4-1BB co-stimulation. Results We found that addition of an agonistic anti-4-1BB antibody could activate 4-1BB signaling within early cultured tumor fragments and accelerated the rate of memory CD8+ TIL outgrowth that were highly enriched for melanoma antigen specificity. This was associated with NFκB activation and the induction of T-cell survival and memory genes, as well as enhanced IL-2 responsiveness, in the CD8+ T cells in the fragments and emerging from the fragments. Early provision of 4-1BB co-stimulation also affected the dendritic cells (DC) by activating NFκB in DC and promoting their maturation inside the tumor fragments. Blocking HLA class I prevented the enhanced outgrowth of CD8+ T cells with anti-4-1BB, suggesting that an ongoing HLA class I-mediated antigen presentation in early tumor fragment cultures plays a role in mediating tumor-specific CD8+ TIL outgrowth. Conclusions Our results highlight a previously unrecognized concept in TIL adoptive cell therapy that the tumor microenvironment can be dynamically regulated in the initial tumor fragment cultures to regulate the types of T cells expanded and their functional characteristics. PMID:25472998

Manipulating the tumor microenvironment ex vivo for enhanced expansion of tumor-infiltrating lymphocytes for adoptive cell therapy.

PubMed

Chacon, Jessica Ann; Sarnaik, Amod A; Chen, Jie Qing; Creasy, Caitlin; Kale, Charuta; Robinson, John; Weber, Jeffrey; Hwu, Patrick; Pilon-Thomas, Shari; Radvanyi, Laszlo

2015-02-01

Cultured tumor fragments from melanoma metastases have been used for years as a source of tumor-infiltrating lymphocytes (TIL) for adoptive cell therapy (ACT). The expansion of tumor-reactive CD8(+) T cells with interleukin-2 (IL2) in these early cultures is critical in generating clinically active TIL infusion products, with a population of activated 4-1BB CD8(+) T cells recently found to constitute the majority of tumor-specific T cells. We used an agonistic anti-4-1BB antibody added during the initial tumor fragment cultures to provide in situ 4-1BB costimulation. We found that addition of an agonistic anti-4-1BB antibody could activate 4-1BB signaling within early cultured tumor fragments and accelerated the rate of memory CD8(+) TIL outgrowth that were highly enriched for melanoma antigen specificity. This was associated with NFκB activation and the induction of T-cell survival and memory genes, as well as enhanced IL2 responsiveness, in the CD8(+) T cells in the fragments and emerging from the fragments. Early provision of 4-1BB costimulation also affected the dendritic cells (DC) by activating NFκB in DC and promoting their maturation inside the tumor fragments. Blocking HLA class I prevented the enhanced outgrowth of CD8(+) T cells with anti-4-1BB, suggesting that an ongoing HLA class I-mediated antigen presentation in early tumor fragment cultures plays a role in mediating tumor-specific CD8(+) TIL outgrowth. Our results highlight a previously unrecognized concept in TIL ACT that the tumor microenvironment can be dynamically regulated in the initial tumor fragment cultures to regulate the types of T cells expanded and their functional characteristics. ©2014 American Association for Cancer Research.

How Do CD4+ T Cells Detect and Eliminate Tumor Cells That Either Lack or Express MHC Class II Molecules?

PubMed Central

Haabeth, Ole Audun Werner; Tveita, Anders Aune; Fauskanger, Marte; Schjesvold, Fredrik; Lorvik, Kristina Berg; Hofgaard, Peter O.; Omholt, Hilde; Munthe, Ludvig A.; Dembic, Zlatko; Corthay, Alexandre; Bogen, Bjarne

2014-01-01

CD4+ T cells contribute to tumor eradication, even in the absence of CD8+ T cells. Cytotoxic CD4+ T cells can directly kill MHC class II positive tumor cells. More surprisingly, CD4+ T cells can indirectly eliminate tumor cells that lack MHC class II expression. Here, we review the mechanisms of direct and indirect CD4+ T cell-mediated elimination of tumor cells. An emphasis is put on T cell receptor (TCR) transgenic models, where anti-tumor responses of naïve CD4+ T cells of defined specificity can be tracked. Some generalizations can tentatively be made. For both MHCIIPOS and MHCIINEG tumors, presentation of tumor-specific antigen by host antigen-presenting cells (APCs) appears to be required for CD4+ T cell priming. This has been extensively studied in a myeloma model (MOPC315), where host APCs in tumor-draining lymph nodes are primed with secreted tumor antigen. Upon antigen recognition, naïve CD4+ T cells differentiate into Th1 cells and migrate to the tumor. At the tumor site, the mechanisms for elimination of MHCIIPOS and MHCIINEG tumor cells differ. In a TCR-transgenic B16 melanoma model, MHCIIPOS melanoma cells are directly killed by cytotoxic CD4+ T cells in a perforin/granzyme B-dependent manner. By contrast, MHCIINEG myeloma cells are killed by IFN-γ stimulated M1-like macrophages. In summary, while the priming phase of CD4+ T cells appears similar for MHCIIPOS and MHCIINEG tumors, the killing mechanisms are different. Unresolved issues and directions for future research are addressed. PMID:24782871

Just-in-time vaccines: Biomineralized calcium phosphate core-immunogen shell nanoparticles induce long-lasting CD8(+) T cell responses in mice.

PubMed

Zhou, Weibin; Moguche, Albanus O; Chiu, David; Murali-Krishna, Kaja; Baneyx, François

2014-04-01

Distributed and on-demand vaccine production could be game-changing for infectious disease treatment in the developing world by providing new therapeutic opportunities and breaking the refrigeration "cold chain". Here, we show that a fusion protein between a calcium phosphate binding domain and the model antigen ovalbumin can mineralize a biocompatible adjuvant in a single step. The resulting 50 nm calcium phosphate core-immunogen shell particles are comparable to soluble protein in inducing ovalbumin-specific antibody response and class switch recombination in mice. However, single dose vaccination with nanoparticles leads to higher expansion of ovalbumin-specific CD8(+) T cells upon challenge with an influenza virus bearing the ovalbumin-derived SIINFEKL peptide, and these cells produce high levels of IFN-γ. Furthermore, mice exhibit a robust antigen-specific CD8(+) T cell recall response when challenged with virus 8 months post-immunization. These results underscore the promise of immunogen-controlled adjuvant mineralization for just-in-time manufacturing of effective T cell vaccines. This paper reports that a fusion protein between a calcium phosphate binding domain and the model antigen ovalbumin can mineralize into a biocompatible adjuvant in a single step, enabling distributed and on-demand vaccine production and eliminating the need for refrigeration of vaccines. The findings highlight the possibility of immunogen-controlled adjuvant mineralization for just-in-time manufacturing of effective T cell vaccines. Copyright © 2014 Elsevier Inc. All rights reserved.

Infusion of donor-derived CD19-redirected virus-specific T cells for B-cell malignancies relapsed after allogeneic stem cell transplant: a phase 1 study.

PubMed

Cruz, Conrad Russell Y; Micklethwaite, Kenneth P; Savoldo, Barbara; Ramos, Carlos A; Lam, Sharon; Ku, Stephanie; Diouf, Oumar; Liu, Enli; Barrett, A John; Ito, Sawa; Shpall, Elizabeth J; Krance, Robert A; Kamble, Rammurti T; Carrum, George; Hosing, Chitra M; Gee, Adrian P; Mei, Zhuyong; Grilley, Bambi J; Heslop, Helen E; Rooney, Cliona M; Brenner, Malcolm K; Bollard, Catherine M; Dotti, Gianpietro

2013-10-24

Autologous T cells expressing a CD19-specific chimeric antigen receptor (CD19.CAR) are active against B-cell malignancies, but it is unknown whether allogeneic CD19.CAR T cells are safe or effective. After allogeneic hematopoietic stem cell transplantation (HSCT), infused donor-derived virus-specific T cells (VSTs) expand in vivo, persist long term, and display antiviral activity without inducing graft-vs-host disease; therefore, we determined whether donor VSTs, engineered to express CD19.CAR, retained the characteristics of nonmanipulated allogeneic VSTs while gaining antitumor activity. We treated 8 patients with allogeneic (donor-derived) CD19.CAR-VSTs 3 months to 13 years after HSCT. There were no infusion-related toxicities. VSTs persisted for a median of 8 weeks in blood and up to 9 weeks at disease sites. Objective antitumor activity was evident in 2 of 6 patients with relapsed disease during the period of CD19.CAR-VST persistence, whereas 2 patients who received cells while in remission remain disease free. In 2 of 3 patients with viral reactivation, donor CD19.CAR-VSTs expanded concomitantly with VSTs. Hence CD19.CAR-VSTs display antitumor activity and, because their number may be increased in the presence of viral stimuli, earlier treatment post-HSCT (when lymphodepletion is greater and the incidence of viral infection is higher) or planned vaccination with viral antigens may enhance disease control.

Blocking of PDL-1 interaction enhances primary and secondary CD8 T cell response to herpes simplex virus-1 infection.

PubMed

Channappanavar, Rudragouda; Twardy, Brandon S; Suvas, Susmit

2012-01-01

The blocking of programmed death ligand-1 (PDL-1) has been shown to enhance virus-specific CD8 T cell function during chronic viral infections. Though, how PDL-1 blocking at the time of priming affects the quality of CD8 T cell response to acute infections is not well understood and remains controversial. This report demonstrates that the magnitude of the primary and secondary CD8 T cell responses to herpes simplex virus-1 (HSV-1) infection is subject to control by PDL-1. Our results showed that after footpad HSV-1 infection, PD-1 expression increases on immunodominant SSIEFARL peptide specific CD8 T cells. Additionally, post-infection, the level of PDL-1 expression also increases on CD11c+ dendritic cells. Intraperitoneal administration of anti-PDL-1 monoclonal antibody given one day prior to and three days after cutaneous HSV-1 infection, resulted in a marked increase in effector and memory CD8 T cell response to SSIEFARL peptide. This was shown by measuring the quantity and quality of SSIEFARL-specific CD8 T cells by making use of ex-vivo assays that determine antigen specific CD8 T cell function, such as intracellular cytokine assay, degranulation assay to measure cytotoxicity and viral clearance. Our results are discussed in terms of the beneficial effects of blocking PDL-1 interactions, while giving prophylactic vaccines, to generate a more effective CD8 T cell response to viral infection.

A fusion protein of HCMV IE1 exon4 and IE2 exon5 stimulates potent cellular immunity in an MVA vaccine vector

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Z.; Zhou, W.; Srivastava, T.

2008-08-01

A therapeutic CMV vaccine incorporating an antigenic repertoire capable of eliciting a cellular immune response has yet to be successfully implemented for patients who already have acquired an infection. To address this problem, we have developed a vaccine candidate derived from modified vaccinia Ankara (MVA) that expresses three immunodominant antigens (pp65, IE1, IE2) from CMV. The novelty of this vaccine is the fusion of two adjacent exons from the immediate-early region of CMV, their successful expression in MVA, and robust immunogenicity in both primary and memory response models. Evaluation of the immunogenicity of the viral vaccine in mouse models showsmore » that it can stimulate primary immunity against all three antigens in both the CD4{sup +} and CD8{sup +} T cell subsets. Evaluation of human PBMC from healthy CMV-positive donors or patients within 6 months of receiving hematopoietic cell transplant shows robust stimulation of existing CMV-specific CD4{sup +} and CD8{sup +} T cell subsets.« less

Augmented lymphocyte expansion from solid tumors with engineered cells for costimulatory enhancement

PubMed Central

Friedman, Kevin M; DeVillier, Laura E; Feldman, Steven A; Rosenberg, Steven A; Dudley, Mark E

2011-01-01

Treatment of patients with adoptive T cell therapy requires expansion of unique tumor-infiltrating lymphocyte (TIL) cultures from single cell suspensions processed from melanoma biopsies. Strategies which increase the expansion and reliability of TIL generation from tumor digests are necessary to improve access to TIL therapy. Prior work evaluated artificial antigen presenting cells (aAPCs) for their antigen-specific and costimulatory properties. We investigated engineered cells for co-stimulatory enhancement (ECCE) consisting of K562 cells which express 4-1BBL in the absence of artificial antigen stimulation. ECCE accelerated TIL expansion and significantly improved TIL numbers (p=0.001) from single cell melanoma suspensions. TIL generated with ECCE contain significantly more CD8+CD62L+ and CD8+CD27+ T cells then comparable IL-2-expanded TIL and maintained anti-tumor reactivity. Moreover, ECCE improved TIL expansion from non-melanoma cell suspensions similar to that seen with melanoma tumors. These data demonstrate that ECCE addition to TIL production will enable treatment of patients ineligible using current methods. PMID:21989413

Dynamics of Dengue Virus (DENV)-Specific B Cells in the Response to DENV Serotype 1 Infections, Using Flow Cytometry With Labeled Virions.

PubMed

Woda, Marcia; Friberg, Heather; Currier, Jeffrey R; Srikiatkhachorn, Anon; Macareo, Louis R; Green, Sharone; Jarman, Richard G; Rothman, Alan L; Mathew, Anuja

2016-10-01

The development of reagents to identify and characterize antigen-specific B cells has been challenging. We recently developed Alexa Fluor-labeled dengue viruses (AF DENVs) to characterize antigen-specific B cells in the peripheral blood of DENV-immune individuals. In this study, we used AF DENV serotype 1 (AF DENV-1) together with AF DENV-2 on peripheral blood mononuclear cells (PBMCs) from children in Thailand with acute primary or secondary DENV-1 infections to analyze the phenotypes of antigen-specific B cells that reflected their exposure or clinical diagnosis. DENV serotype-specific and cross-reactive B cells were identified in PBMCs from all subjects. Frequencies of AF DENV(+) class-switched memory B cells (IgD(-)CD27(+) CD19(+) cells) reached up to 8% during acute infection and early convalescence. AF DENV-labeled B cells expressed high levels of CD27 and CD38 during acute infection, characteristic of plasmablasts, and transitioned into memory B cells (CD38(-)CD27(+)) at the early convalescent time point. There was higher activation of memory B cells early during acute secondary infection, suggesting reactivation from a previous DENV infection. AF DENVs reveal changes in the phenotype of DENV serotype-specific and cross-reactive B cells during and after natural DENV infection and could be useful in analysis of the response to DENV vaccination. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Processing of urushiol (poison ivy) hapten by both endogenous and exogenous pathways for presentation to T cells in vitro.

PubMed

Kalish, R S; Wood, J A; LaPorte, A

1994-05-01

The antigen processing requirements for urushiol, the immunogen of poison ivy (Toxicodendron radicans), were tested by presentation of urushiol to cultured human urushiol-responsive T cells. Urushiol was added to antigen-presenting cells (APC) either before or after fixation with paraformaldehyde. Three distinct routes of antigen processing were detected. CD8+ and CD4+ T cells, which were dependent upon processing, proliferated if urushiol was added to APC before fixation, but did not proliferate when urushiol was added to APC after fixation. Processing of urushiol for presentation to CD8+ T cells was inhibited by azide, monensin, and brefeldin A. This suggests that urushiol was processed by the endogenous pathway. In contrast, presentation of urushiol to CD4+ T cells was inhibited by monensin but not by brefeldin A. This was compatible with antigen processing by the endosomal (exogenous) pathway. Finally, certain CD8+ T cells recognized urushiol in the absence of processing. These cells proliferated in response to APC incubated with urushiol after fixation. Classification of contact allergens by antigen processing pathway may predict the relative roles of CD4+ and CD8+ cells in the immunopathogensis of allergic contact dermatitis.

Processing of urushiol (poison ivy) hapten by both endogenous and exogenous pathways for presentation to T cells in vitro.

PubMed Central

Kalish, R S; Wood, J A; LaPorte, A

1994-01-01

The antigen processing requirements for urushiol, the immunogen of poison ivy (Toxicodendron radicans), were tested by presentation of urushiol to cultured human urushiol-responsive T cells. Urushiol was added to antigen-presenting cells (APC) either before or after fixation with paraformaldehyde. Three distinct routes of antigen processing were detected. CD8+ and CD4+ T cells, which were dependent upon processing, proliferated if urushiol was added to APC before fixation, but did not proliferate when urushiol was added to APC after fixation. Processing of urushiol for presentation to CD8+ T cells was inhibited by azide, monensin, and brefeldin A. This suggests that urushiol was processed by the endogenous pathway. In contrast, presentation of urushiol to CD4+ T cells was inhibited by monensin but not by brefeldin A. This was compatible with antigen processing by the endosomal (exogenous) pathway. Finally, certain CD8+ T cells recognized urushiol in the absence of processing. These cells proliferated in response to APC incubated with urushiol after fixation. Classification of contact allergens by antigen processing pathway may predict the relative roles of CD4+ and CD8+ cells in the immunopathogensis of allergic contact dermatitis. Images PMID:7910172

BTLA interaction with HVEM expressed on CD8(+) T cells promotes survival and memory generation in response to a bacterial infection.

PubMed

Steinberg, Marcos W; Huang, Yujun; Wang-Zhu, Yiran; Ware, Carl F; Cheroutre, Hilde; Kronenberg, Mitchell

2013-01-01

The B and T lymphocyte attenuator (BTLA) is an Ig super family member that binds to the herpes virus entry mediator (HVEM), a TNF receptor super family (TNFRSF) member. Engagement of BTLA by HVEM triggers inhibitory signals, although recent evidence indicates that BTLA also may act as an activating ligand for HVEM. In this study, we reveal a novel role for the BTLA-HVEM pathway in promoting the survival of activated CD8(+) T cells in the response to an oral microbial infection. Our data show that both BTLA- and HVEM-deficient mice infected with Listeria monocytogenes had significantly reduced numbers of primary effector and memory CD8(+) T cells, despite normal proliferation and expansion compared to controls. In addition, blockade of the BTLA-HVEM interaction early in the response led to significantly reduced numbers of antigen-specific CD8(+) T cells. HVEM expression on the CD8(+) T cells as well as BTLA expression on a cell type other than CD8(+) T lymphocytes, was required. Collectively, our data demonstrate that the function of the BTLA-HVEM pathway is not limited to inhibitory signaling in T lymphocytes, and instead, that BTLA can provide crucial, HVEM-dependent signals that promote survival of antigen activated CD8(+) T cell during bacterial infection.

Direct visualization of antigen-specific T cells: HTLV-1 Tax11-19- specific CD8(+) T cells are activated in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients.

PubMed

Greten, T F; Slansky, J E; Kubota, R; Soldan, S S; Jaffee, E M; Leist, T P; Pardoll, D M; Jacobson, S; Schneck, J P

1998-06-23

Human T lymphotropic virus type 1 (HTLV-1) -associated myelopathy/tropic spastic paraparesis is a demyelinating inflammatory neurologic disease associated with HTLV-1 infection. HTLV-1 Tax11-19-specific cytotoxic T cells have been isolated from HLA-A2-positive patients. We have used a peptide-loaded soluble HLA-A2-Ig complex to directly visualize HTLV-1 Tax11-19-specific T cells from peripheral blood and cerebrospinal fluid without in vitro stimulation. Five of six HTLV-1-associated myelopathy/tropic spastic paraparesis patients carried a significant number (up to 13.87%) of CD8(+) lymphocytes specific for the HTLV-1 Tax11-19 peptide in their peripheral blood, which were not found in healthy controls. Simultaneous comparison of peripheral blood and cerebrospinal fluid from one patient revealed 2.5-fold more Tax11-19-specific T cells in the cerebrospinal fluid (23.7% vs. 9.4% in peripheral blood lymphocyte). Tax11-19-specific T cells were seen consistently over a 9-yr time course in one patient as far as 19 yrs after the onset of clinical symptoms. Further analysis of HTLV-1 Tax11-19-specific CD8(+) T lymphocytes in HAM/TSP patients showed different expression patterns of activation markers, intracellular TNF-alpha and gamma-interferon depending on the severity of the disease. Thus, visualization of antigen-specific T cells demonstrates that HTLV-1 Tax11-19-specific CD8(+) T cells are activated, persist during the chronic phase of the disease, and accumulate in cerebrospinal fluid, showing their pivotal role in the pathogenesis of this neurologic disease.

An inducible transgenic mouse breast cancer model for the analysis of tumor antigen specific CD8+ T-cell responses

PubMed Central

Bruns, Michael; Wanger, Jara; Utermöhlen, Olaf; Deppert, Wolfgang

2015-01-01

In Simian virus 40 (SV40) transgenic BALB/c WAP-T mice tumor development and progression is driven by SV40 tumor antigens encoded by inducible transgenes. WAP-T mice constitute a well characterized mouse model for breast cancer with strong similarities to the corresponding human disease. BALB/c mice mount only a weak cellular immune response against SV40 T-antigen (T-Ag). For studying tumor antigen specific CD8+ T-cell responses against transgene expressing cells, we created WAP-TNP mice, in which the transgene additionally codes for the NP118–126-epitope contained within the nucleoprotein of lymphocytic choriomeningitis virus (LCMV), the immune-dominant T-cell epitope in BALB/c mice. We then investigated in WAP-TNP mice the immune responses against SV40 tumor antigens and the NP-epitope within the chimeric T-Ag/NP protein (T-AgNP). Analysis of the immune-reactivity against T-Ag in WAP-T and of T-AgNP in WAP-TNP mice revealed that, in contrast to wild type (wt) BALB/c mice, WAP-T and WAP-TNP mice were non-reactive against T-Ag. However, like wtBALB/c mice, WAP-T as well as WAP-TNP mice were highly reactive against the immune-dominant LCMV NP-epitope, thereby allowing the analysis of NP-epitope specific cellular immune responses in WAP-TNP mice. LCMV infection of WAP-TNP mice induced a strong, LCMV NP-epitope specific CD8+ T-cell response, which was able to specifically eliminate T-AgNP expressing mammary epithelial cells both prior to tumor formation (i.e. in cells of lactating mammary glands), as well as in invasive tumors. Elimination of tumor cells, however, was only transient, even after repeated LCMV infections. Further studies showed that already non-infected WAP-TNP tumor mice contained LCMV NP-epitope specific CD8+ T-cells, albeit with strongly reduced, though measurable activity. Functional impairment of these ‘endogenous’ NP-epitope specific T-cells seems to be caused by expression of the programmed death-1 protein (PD1), as anti-PD1 treatment of splenocytes from WAP-TNP tumor mice restored their activity. These characteristics are similar to those found in many tumor patients and render WAP-TNP mice a suitable model for analyzing parameters to overcome the blockade of immune checkpoints in tumor patients. PMID:26513294

Human CD1c+ dendritic cells drive the differentiation of CD103+ CD8+ mucosal effector T cells via the cytokine TGF-β

PubMed Central

Yu, Chun I; Becker, Christian; Wang, Yuanyuan; Marches, Florentina; Helft, Julie; Leboeuf, Marylene; Anguiano, Esperanza; Pourpe, Stephane; Goller, Kristina; Pascual, Virginia; Banchereau, Jacques; Merad, Miriam; Palucka, Karolina

2013-01-01

Summary In comparison to murine dendritic cells (DCs), less is known about the function of human DCs in tissues. Here, we analyzed, using lung tissues from humans and humanized mice, the role of human CD1c+ and CD141+ DCs in determining the type of CD8+ T cell immunity generated to live-attenuated influenza virus (LAIV) vaccine. We found that both lung DC subsets acquired influenza antigens in vivo and expanded specific cytotoxic CD8+ T cells in vitro. However, lung-tissue-resident CD1c+ DCs but not CD141+ DCs were able to drive CD103 expression on CD8+ T cells and promoted CD8+ T cell accumulation in lung epithelia in vitro and in vivo. CD1c+ DCs induction of CD103 expression was dependent on membrane-bound cytokine TGF-β1. Thus, CD1c+ and CD141+ DCs generate CD8+ T cells with different properties, and CD1c+ DCs specialize in the regulation of mucosal CD8+ T cells. PMID:23562160

Optimization of a gene electrotransfer procedure for efficient intradermal immunization with an hTERT-based DNA vaccine in mice

PubMed Central

Calvet, Christophe Y; Thalmensi, Jessie; Liard, Christelle; Pliquet, Elodie; Bestetti, Thomas; Huet, Thierry; Langlade-Demoyen, Pierre; Mir, Lluis M

2014-01-01

DNA vaccination consists in administering an antigen-encoding plasmid in order to trigger a specific immune response. This specific vaccine strategy is of particular interest to fight against various infectious diseases and cancer. Gene electrotransfer is the most efficient and safest non-viral gene transfer procedure and specific electrical parameters have been developed for several target tissues. Here, a gene electrotransfer protocol into the skin has been optimized in mice for efficient intradermal immunization against the well-known telomerase tumor antigen. First, the luciferase reporter gene was used to evaluate gene electrotransfer efficiency into the skin as a function of the electrical parameters and electrodes, either non-invasive or invasive. In a second time, these parameters were tested for their potency to generate specific cellular CD8 immune responses against telomerase epitopes. These CD8 T-cells were fully functional as they secreted IFNγ and were endowed with specific cytotoxic activity towards target cells. This simple and optimized procedure for efficient gene electrotransfer into the skin using the telomerase antigen is to be used in cancer patients for the phase 1 clinical evaluation of a therapeutic cancer DNA vaccine called INVAC-1. PMID:26015983

Functional characterization of mouse spinal cord infiltrating CD8+ lymphocytes

PubMed Central

Deb, Chandra; Howe, Charles L

2011-01-01

Understanding the immunopathogenesis of neuroimmunological diseases of the CNS requires a robust method for isolating and characterizing the immune effector cells that infiltrate the spinal cord in animal models. We have developed a simple and rapid isolation method that produces high yields of spinal cord infiltrating leukocytes from a single demyelinated spinal cord and which maintains high surface expression of key immunophenotyping antigens. Using this method and the Theiler’s virus model of chronic demyelination, we report the presence of spinal cord infiltrating acute effector CD8+ lymphocytes that are CD45hiCD44loCD62L− and a population of spinal cord infiltrating target effector memory CD8+ lymphocytes that are CD45hiCD44hiCD62L−. These cells respond robustly to ex vivo stimulation by producing interferon γ but do not exhibit specificity for Theiler’s virus in a cytotoxicity assay. We conclude that target-derived lymphocytes in a mouse model of chronic spinal cord demyelination may have unique functional specificities. PMID:19596449

Intravacuolar Membranes Regulate CD8 T Cell Recognition of Membrane-Bound Toxoplasma gondii Protective Antigen.

PubMed

Lopez, Jodie; Bittame, Amina; Massera, Céline; Vasseur, Virginie; Effantin, Grégory; Valat, Anne; Buaillon, Célia; Allart, Sophie; Fox, Barbara A; Rommereim, Leah M; Bzik, David J; Schoehn, Guy; Weissenhorn, Winfried; Dubremetz, Jean-François; Gagnon, Jean; Mercier, Corinne; Cesbron-Delauw, Marie-France; Blanchard, Nicolas

2015-12-15

Apicomplexa parasites such as Toxoplasma gondii target effectors to and across the boundary of their parasitophorous vacuole (PV), resulting in host cell subversion and potential presentation by MHC class I molecules for CD8 T cell recognition. The host-parasite interface comprises the PV limiting membrane and a highly curved, membranous intravacuolar network (IVN) of uncertain function. Here, using a cell-free minimal system, we dissect how membrane tubules are shaped by the parasite effectors GRA2 and GRA6. We show that membrane association regulates access of the GRA6 protective antigen to the MHC I pathway in infected cells. Although insertion of GRA6 in the PV membrane is key for immunogenicity, association of GRA6 with the IVN limits presentation and curtails GRA6-specific CD8 responses in mice. Thus, membrane deformations of the PV regulate access of antigens to the MHC class I pathway, and the IVN may play a role in immune modulation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Elderly dendritic cells respond to LPS/IFN-γ and CD40L stimulation despite incomplete maturation

PubMed Central

Musk, Arthur W.; Alvarez, John; Mamotte, Cyril D. S.; Jackaman, Connie; Nowak, Anna K.; Nelson, Delia J.

2018-01-01

There is evidence that dendritic cells (DCs) undergo age-related changes that modulate their function with their key role being priming antigen-specific effector T cells. This occurs once DCs develop into antigen-presenting cells in response to stimuli/danger signals. However, the effects of aging on DC responses to bacterial lipopolysaccharide (LPS), the pro-inflammatory cytokine interferon (IFN)-γ and CD40 ligand (CD40L) have not yet been systematically evaluated. We examined responses of blood myeloid (m)DC1s, mDC2s, plasmacytoid (p)DCs, and monocyte-derived DCs (MoDCs) from young (21–40 years) and elderly (60–84 years) healthy human volunteers to LPS/IFN-γ or CD40L stimulation. All elderly DC subsets demonstrated comparable up-regulation of co-stimulatory molecules (CD40, CD80 and/or CD86), intracellular pro-inflammatory cytokine levels (IFN-γ, tumour necrosis factor (TNF)-α, IL-6 and/or IL-12), and/or secreted cytokine levels (IFN-α, IFN-γ, TNF-α, and IL-12) to their younger counterparts. Furthermore, elderly-derived LPS/IFN-γ or CD40L-activated MoDCs induced similar or increased levels of CD8+ and CD4+ T cell proliferation, and similar T cell functional phenotypes, to their younger counterparts. However, elderly LPS/IFN-γ-activated MoDCs were unreliable in their ability to up-regulate chemokine (IL-8 and monocyte chemoattractant protein (MCP)-1) and IL-6 secretion, implying an inability to dependably induce an inflammatory response. A key age-related difference was that, unlike young-derived MoDCs that completely lost their ability to process antigen, elderly-derived MoDCs maintained their antigen processing ability after LPS/IFN-γ maturation, measured using the DQ-ovalbumin assay; this response implies incomplete maturation that may enable elderly DCs to continuously present antigen. These differences may impact on the efficacy of anti-pathogen and anti-tumour immune responses in the elderly. PMID:29652910

Comparative analysis of activation induced marker (AIM) assays for sensitive identification of antigen-specific CD4 T cells

PubMed Central

Cirelli, Kimberly M.; Dan, Jennifer M.; Morou, Antigoni; Daigneault, Audrey; Brassard, Nathalie; Silvestri, Guido; Routy, Jean-Pierre; Havenar-Daughton, Colin; Crotty, Shane

2017-01-01

The identification and study of antigen-specific CD4 T cells, both in peripheral blood and in tissues, is key for a broad range of immunological research, including vaccine responses and infectious diseases. Detection of these cells is hampered by both their rarity and their heterogeneity, in particular with regards to cytokine secretion profiles. These factors prevent the identification of the total pool of antigen-specific CD4 T cells by classical methods. We have developed assays for the highly sensitive detection of such cells by measuring the upregulation of surface activation induced markers (AIM). Here, we compare two such assays based on concurrent expression of CD69 plus CD40L (CD154) or expression of OX40 plus CD25, and we develop additional AIM assays based on OX40 plus PD-L1 or 4-1BB. We compare the relative sensitivity of these assays for detection of vaccine and natural infection-induced CD4 T cell responses and show that these assays identify distinct, but overlapping populations of antigen-specific CD4 T cells, a subpopulation of which can also be detected on the basis of cytokine synthesis. Bystander activation had minimal effect on AIM markers. However, some T regulatory cells upregulate CD25 upon antigen stimulation. We therefore validated AIM assays designed to exclude most T regulatory cells, for both human and non-human primate (NHP, Macaca mulatta) studies. Overall, through head-to-head comparisons and methodological improvements, we show that AIM assays represent a sensitive and valuable method for the detection of antigen-specific CD4 T cells. PMID:29065175

Comparative analysis of activation induced marker (AIM) assays for sensitive identification of antigen-specific CD4 T cells.

PubMed

Reiss, Samantha; Baxter, Amy E; Cirelli, Kimberly M; Dan, Jennifer M; Morou, Antigoni; Daigneault, Audrey; Brassard, Nathalie; Silvestri, Guido; Routy, Jean-Pierre; Havenar-Daughton, Colin; Crotty, Shane; Kaufmann, Daniel E

2017-01-01

The identification and study of antigen-specific CD4 T cells, both in peripheral blood and in tissues, is key for a broad range of immunological research, including vaccine responses and infectious diseases. Detection of these cells is hampered by both their rarity and their heterogeneity, in particular with regards to cytokine secretion profiles. These factors prevent the identification of the total pool of antigen-specific CD4 T cells by classical methods. We have developed assays for the highly sensitive detection of such cells by measuring the upregulation of surface activation induced markers (AIM). Here, we compare two such assays based on concurrent expression of CD69 plus CD40L (CD154) or expression of OX40 plus CD25, and we develop additional AIM assays based on OX40 plus PD-L1 or 4-1BB. We compare the relative sensitivity of these assays for detection of vaccine and natural infection-induced CD4 T cell responses and show that these assays identify distinct, but overlapping populations of antigen-specific CD4 T cells, a subpopulation of which can also be detected on the basis of cytokine synthesis. Bystander activation had minimal effect on AIM markers. However, some T regulatory cells upregulate CD25 upon antigen stimulation. We therefore validated AIM assays designed to exclude most T regulatory cells, for both human and non-human primate (NHP, Macaca mulatta) studies. Overall, through head-to-head comparisons and methodological improvements, we show that AIM assays represent a sensitive and valuable method for the detection of antigen-specific CD4 T cells.

LBSapSal-vaccinated dogs exhibit increased circulating T-lymphocyte subsets (CD4+ and CD8+) as well as a reduction of parasitism after challenge with Leishmania infantum plus salivary gland of Lutzomyia longipalpis

PubMed Central

2014-01-01

Background The development of a protective vaccine against canine visceral leishmaniasis (CVL) is an alternative approach for interrupting the domestic cycle of Leishmania infantum. Given the importance of sand fly salivary proteins as potent immunogens obligatorily co-deposited during transmission of Leishmania parasites, their inclusion in an anti-Leishmania vaccine has been investigated in the last few decades. In this context, we previously immunized dogs with a vaccine composed of L. braziliensis antigens plus saponin as the adjuvant and sand fly salivary gland extract (LBSapSal vaccine). This vaccine elicited an increase in both anti-saliva and anti-Leishmania IgG isotypes, higher counts of specific circulating CD8+ T cells, and high NO production. Methods We investigated the immunogenicity and protective effect of LBSapSal vaccination after intradermal challenge with 1 × 107 late-log-phase L. infantum promastigotes in the presence of sand fly saliva of Lutzomyia longipalpis. The dogs were followed for up to 885 days after challenge. Results The LBSapSal vaccine presents extensive antigenic diversity with persistent humoral and cellular immune responses, indicating resistance against CVL is triggered by high levels of total IgG and its subtypes (IgG1 and IgG2); expansion of circulating CD5+, CD4+, and CD8+ T lymphocytes and is Leishmania-specific; and reduction of splenic parasite load. Conclusions These results encourage further study of vaccine strategies addressing Leishmania antigens in combination with proteins present in the saliva of the vector. PMID:24507702

Abundant cytomegalovirus (CMV) reactive clonotypes in the CD8(+) T cell receptor alpha repertoire following allogeneic transplantation.

PubMed

Link, C S; Eugster, A; Heidenreich, F; Rücker-Braun, E; Schmiedgen, M; Oelschlägel, U; Kühn, D; Dietz, S; Fuchs, Y; Dahl, A; Domingues, A M J; Klesse, C; Schmitz, M; Ehninger, G; Bornhäuser, M; Schetelig, J; Bonifacio, E

2016-06-01

Allogeneic stem cell transplantation is potentially curative, but associated with post-transplantation complications, including cytomegalovirus (CMV) infections. An effective immune response requires T cells recognizing CMV epitopes via their T cell receptors (TCRs). Little is known about the TCR repertoire, in particular the TCR-α repertoire and its clinical relevance in patients following stem cell transplantation. Using next-generation sequencing we examined the TCR-α repertoire of CD8(+) T cells and CMV-specific CD8(+) T cells in four patients. Additionally, we performed single-cell TCR-αβ sequencing of CMV-specific CD8(+) T cells. The TCR-α composition of human leucocyte antigen (HLA)-A*0201 CMVpp65- and CMVIE -specific T cells was oligoclonal and defined by few dominant clonotypes. Frequencies of single clonotypes reached up to 11% of all CD8(+) T cells and half of the total CD8(+) T cell repertoire was dominated by few CMV-reactive clonotypes. Some TCR-α clonotypes were shared between patients. Gene expression of the circulating CMV-specific CD8(+) T cells was consistent with chronically activated effector memory T cells. The CD8(+) T cell response to CMV reactivation resulted in an expansion of a few TCR-α clonotypes to dominate the CD8(+) repertoires. These results warrant further larger studies to define the ability of oligoclonally expanded T cell clones to achieve an effective anti-viral T cell response in this setting. © 2016 British Society for Immunology.

New Perspective on Dextran Sodium Sulfate Colitis: Antigen-Specific T Cell Development during Intestinal Inflammation

PubMed Central

Morgan, Mary E.; Zheng, Bin; Koelink, Pim J.; van de Kant, Hendrick J. G.; Haazen, Lizette C. J. M.; van Roest, Manon; Garssen, Johan; Folkerts, Gert; Kraneveld, Aletta D.

2013-01-01

CD4+ T cell responses against oral antigens can develop in inflammatory bowel disease (IBD) patients, which may modulate disease. Dextran sodium sulfate (DSS) colitis is commonly used to study IBD, however, it is not considered the best model in which to study T cell involvement in intestinal disease. Our aim was to determine if antigen-specific T cells could be induced during DSS colitis and if they could be detected after disease resolution. To induce antigen-specific T cells, the tracking antigen, ovalbumin (OVA), was administered orally during colitis initiation. Disease severity was monitored, and the antigen-reactivity of CD4+ T cells examined using CD69 expression. While OVA-directed, CD4+ Foxp3+ regulatory T cells could be detected in the spleens of both OVA-treated control and DSS mice, OVA-reactive, CD4+ Foxp3-T cells were only found in the OVA and DSS-treated mice. These results indicate that during DSS colitis T cells develop that are specific against oral antigens, and they are found systemically after colitis resolution. This gives added depth and utility to the DSS model as well as a way to track T cells that are primed against luminal antigens. PMID:23936123

Antigen sensitivity of CD22-specific chimeric T cell receptors is modulated by target epitope distance from the cell membrane

PubMed Central

James, Scott E.; Greenberg, Philip D.; Jensen, Michael C.; Lin, Yukang; Wang, Jinjuan; Till, Brian G.; Raubitschek, Andrew A.; Forman, Stephen J.; Press, Oliver W.

2008-01-01

We have targeted CD22 as a novel tumor-associated antigen for recognition by human CTL genetically modified to express chimeric T cell receptors (cTCR) recognizing this surface molecule. CD22-specifc cTCR targeting different epitopes of the CD22 molecule promoted efficient lysis of target cells expressing high levels of CD22 with a maximum lytic potential that appeared to decrease as the distance of the target epitope from the target cell membrane increased. Targeting membrane-distal CD22 epitopes with cTCR+ CTL revealed defects in both degranulation and lytic granule targeting. CD22-specific cTCR+ CTL exhibited lower levels of maximum lysis and lower antigen sensitivity than CTL targeting CD20, which has a shorter extracellular domain than CD22. This diminished sensitivity was not a result of reduced avidity of antigen engagement, but instead reflected weaker signaling per triggered cTCR molecule when targeting membrane-distal epitopes of CD22. Both of these parameters were restored by targeting a ligand expressing the same epitope but constructed as a truncated CD22 molecule to approximate the length of a TCR:pMHC complex. The reduced sensitivity of CD22-specific cTCR+ CTL for antigen-induced triggering of effector functions has potential therapeutic applications, as such cells selectively lysed B cell lymphoma lines expressing high levels of CD22 but demonstrated minimal activity against autologous normal B cells, which express lower levels of CD22. Thus, our results demonstrate that cTCR signal strength – and consequently antigen sensitivity – can be modulated by differential choice of target epitopes with respect to distance from the cell membrane, allowing discrimination between targets with disparate antigen density. PMID:18453625

CD8(+) T-cell Cytotoxic Capacity Associated with Human Immunodeficiency Virus-1 Control Can Be Mediated through Various Epitopes and Human Leukocyte Antigen Types.

PubMed

Migueles, Stephen A; Mendoza, Daniel; Zimmerman, Matthew G; Martins, Kelly M; Toulmin, Sushila A; Kelly, Elizabeth P; Peterson, Bennett A; Johnson, Sarah A; Galson, Eric; Poropatich, Kate O; Patamawenu, Andy; Imamichi, Hiromi; Ober, Alexander; Rehm, Catherine A; Jones, Sara; Hallahan, Claire W; Follmann, Dean A; Connors, Mark

2015-01-01

Understanding natural immunologic control over Human Immunodeficiency Virus (HIV)-1 replication, as occurs in rare long-term nonprogressors/elite controllers (LTNP/EC), should inform the design of efficacious HIV vaccines and immunotherapies. Durable control in LTNP/EC is likely mediated by highly functional virus-specific CD8(+) T-cells. Protective Human Leukocyte Antigen (HLA) class I alleles, like B*27 and B*57, are present in most, but not all LTNP/EC, providing an opportunity to investigate features shared by their HIV-specific immune responses. To better understand the contribution of epitope targeting and conservation to immune control, we compared the CD8(+) T-cell specificity and function of B*27/57(neg) LTNP/EC (n = 23), B*27/57(pos) LTNP/EC (n = 23) and B*27/57(neg) progressors (n = 13). Fine mapping revealed 11 previously unreported immunodominant responses. Although B*27/57(neg) LTNP/EC did not target more highly conserved epitopes, their CD8(+) T-cell cytotoxic capacity was significantly higher than progressors. Similar to B*27/57(pos) LTNP/EC, this superior cytotoxicity was mediated by preferential expansion of immunodominant responses and lysis through the predicted HLA. These findings suggest that increased CD8(+) T-cell cytotoxic capacity is a common mechanism of control in most LTNP/EC regardless of HLA type. They also suggest that potent cytotoxicity can be mediated through various epitopes and HLA molecules and could, in theory, be induced in most people.

CD8+ T-cell Cytotoxic Capacity Associated with Human Immunodeficiency Virus-1 Control Can Be Mediated through Various Epitopes and Human Leukocyte Antigen Types

PubMed Central

Migueles, Stephen A.; Mendoza, Daniel; Zimmerman, Matthew G.; Martins, Kelly M.; Toulmin, Sushila A.; Kelly, Elizabeth P.; Peterson, Bennett A.; Johnson, Sarah A.; Galson, Eric; Poropatich, Kate O.; Patamawenu, Andy; Imamichi, Hiromi; Ober, Alexander; Rehm, Catherine A.; Jones, Sara; Hallahan, Claire W.; Follmann, Dean A.; Connors, Mark

2014-01-01

Understanding natural immunologic control over Human Immunodeficiency Virus (HIV)-1 replication, as occurs in rare long-term nonprogressors/elite controllers (LTNP/EC), should inform the design of efficacious HIV vaccines and immunotherapies. Durable control in LTNP/EC is likely mediated by highly functional virus-specific CD8+ T-cells. Protective Human Leukocyte Antigen (HLA) class I alleles, like B*27 and B*57, are present in most, but not all LTNP/EC, providing an opportunity to investigate features shared by their HIV-specific immune responses. To better understand the contribution of epitope targeting and conservation to immune control, we compared the CD8+ T-cell specificity and function of B*27/57neg LTNP/EC (n = 23), B*27/57pos LTNP/EC (n = 23) and B*27/57neg progressors (n = 13). Fine mapping revealed 11 previously unreported immunodominant responses. Although B*27/57neg LTNP/EC did not target more highly conserved epitopes, their CD8+ T-cell cytotoxic capacity was significantly higher than progressors. Similar to B*27/57pos LTNP/EC, this superior cytotoxicity was mediated by preferential expansion of immunodominant responses and lysis through the predicted HLA. These findings suggest that increased CD8+ T-cell cytotoxic capacity is a common mechanism of control in most LTNP/EC regardless of HLA type. They also suggest that potent cytotoxicity can be mediated through various epitopes and HLA molecules and could, in theory, be induced in most people. PMID:26137533

T cell-depleted splenocytes from mice pre-immunized with neuroantigen in incomplete Freund's adjuvant involved in protection from experimental autoimmune encephalomyelitis.

PubMed

Zheng, Hui; Zhang, Han; Liu, Feng; Qi, Yuanyuan; Jiang, Hong

2014-01-01

Mice immunized with neuroantigens in incomplete Freund's adjuvant (IFA) are resistant to subsequent induction of experimental autoimmune encephalomyelitis (EAE). The mechanisms involved in this protection are complex. Studies on relevant CD4(+) or CD8(+) T cells, including effective and regulatory T cells, have been performed by others. In this work, the effects of CD4(-)-, CD8(-)- splenocytes on protection from EAE in C57BL/6 mice which were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG)35-55 in IFA were evaluated. We observed that MOG-reactive CD4(+) T cells failed to be activated and proliferate when CD4(-)-, CD8(-)- splenocytes from MOG/IFA-immunized mice were regarded as antigen-presenting cells (APC). It was shown that these APC expressed lower levels of major histocompatibility complex class II (MHC-II), CD80, and CD86 than naïve cells. In addition, CD4(-)-, CD8(-)- splenocytes from MOG/IFA-immunized mice showed significantly higher levels of IL-10 mRNA expression. When the immunized-mice were induced to develop EAE, these cells secreted significantly higher levels of IL-10 and produced lower levels of IL-6, leading to decreased secretion of IL-17 and IFN-γ from MOG-specific CD4(+) T cells. The transfer of CD4(-)-, CD8(-)- splenocytes from MOG/IFA-immunized mice was able to ameliorate the subsequent induction of EAE in recipient mice. Thus, MOG/IFA immunization can modulate CD4(-)-, CD8(-)- splenocytes by reducing the expression of antigen-presenting molecules and altering the levels of secreted cytokines. Our study reveals an additional mechanism involved in the protective effects of MOG/IFA pre-immunization in an EAE model. Copyright © 2013 Elsevier B.V. All rights reserved.

T-cell Responses to HSV-1 in Persons Who Have Survived Childhood Herpes Simplex Encephalitis.

PubMed

Ott, Mariliis; Jing, Lichen; Lorenzo, Lazaro; Casanova, Jean-Laurent; Zhang, Shen-Ying; Koelle, David M

2017-08-01

Herpes simplex encephalitis (HSE) after primary herpes simplex virus (HSV)-1 infection can occur in children due to inborn errors of cell-intrinsic immunity in the central nervous system. Paradoxically, symptomatic mucocutaneous HSV-1 recurrences are rare survivors of childhood HSE. T-cell-acquired immunity is thought to be involved in control of recurrent mucocutaneous HSV infection. We thus tested HSV-1-specific immunity in HSE survivors. We obtained serum and peripheral blood mononuclear cells (PBMCs) from participants a median of 13.5 years after HSE. HSV-1 and HSV-2 IgG was detected by type-specific immunoblot. PBMCs from subjects passing quality control criteria were tested using enzyme-linked immunospot assay for CD4 interferon-γ responses with an HSV-1 lysate and for CD8 responses using pooled synthetic HSV-1 peptide CD8 T-cell epitopes. Healthy adult PBMCs were used to standardize assays and as comparators. All participants were HSV-1 seropositive. Most (23/24) HSE survivors had human leukocyte antigen class I types matching the human leukocyte antigen restriction of the pooled peptides. We detected HSV-specific CD8 T-cell responses in 14 of 24 (58%) HSE survivors and in 9 of 9 healthy HSV-1 seropositive adults. HSV-specific CD4 T-cell responses were present in all 5 HSE subjects tested and in 8 of 9 healthy adults. Response magnitudes were overlapping between subject groups. The defects in cell-intrinsic immunity leading to failure to control primary central nervous system HSV-1 infection do not preclude the acquisition of specific immunity or the control of recurrent mucocutaneous HSV infections. The rarity and lack of severe or recurrent mucocutaneous HSV infection in survivors of childhood HSE corresponds with intact adaptive T-cell immunity.

Profiling MHC II immunopeptidome of blood-stage malaria reveals that cDC1 control the functionality of parasite-specific CD4 T cells.

PubMed

Draheim, Marion; Wlodarczyk, Myriam F; Crozat, Karine; Saliou, Jean-Michel; Alayi, Tchilabalo Dilezitoko; Tomavo, Stanislas; Hassan, Ali; Salvioni, Anna; Demarta-Gatsi, Claudia; Sidney, John; Sette, Alessandro; Dalod, Marc; Berry, Antoine; Silvie, Olivier; Blanchard, Nicolas

2017-11-01

In malaria, CD4 Th1 and T follicular helper (T FH ) cells are important for controlling parasite growth, but Th1 cells also contribute to immunopathology. Moreover, various regulatory CD4 T-cell subsets are critical to hamper pathology. Yet the antigen-presenting cells controlling Th functionality, as well as the antigens recognized by CD4 T cells, are largely unknown. Here, we characterize the MHC II immunopeptidome presented by DC during blood-stage malaria in mice. We establish the immunodominance hierarchy of 14 MHC II ligands derived from conserved parasite proteins. Immunodominance is shaped differently whether blood stage is preceded or not by liver stage, but the same ETRAMP-specific dominant response develops in both contexts. In naïve mice and at the onset of cerebral malaria, CD8α + dendritic cells (cDC1) are superior to other DC subsets for MHC II presentation of the ETRAMP epitope. Using in vivo depletion of cDC1, we show that cDC1 promote parasite-specific Th1 cells and inhibit the development of IL-10 + CD4 T cells. This work profiles the P. berghei blood-stage MHC II immunopeptidome, highlights the potency of cDC1 to present malaria antigens on MHC II, and reveals a major role for cDC1 in regulating malaria-specific CD4 T-cell responses. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

In vivo induction of a high-avidity, high-frequency cytotoxic T-lymphocyte response is associated with antiviral protective immunity.

PubMed

Sedlik, C; Dadaglio, G; Saron, M F; Deriaud, E; Rojas, M; Casal, S I; Leclerc, C

2000-07-01

Many approaches are currently being developed to deliver exogenous antigen into the major histocompatibility complex class I-restricted antigen pathway, leading to in vivo priming of CD8(+) cytotoxic T cells. One attractive possibility consists of targeting the antigen to phagocytic or macropinocytic antigen-presenting cells. In this study, we demonstrate that strong CD8(+) class I-restricted cytotoxic responses are induced upon intraperitoneal immunization of mice with different peptides, characterized as CD8(+) T-cell epitopes, bound to 1-microm synthetic latex microspheres and injected in the absence of adjuvant. The cytotoxic response induced against a lymphocytic choriomeningitis virus (LCMV) peptide linked to these microspheres was compared to the cytotoxic T-lymphocyte (CTL) response obtained upon immunization with the nonreplicative porcine parvovirus-like particles (PPV:VLP) carrying the same peptide (PPV:VLP-LCMV) previously described (C. Sedlik, M. F. Saron, J. Sarraseca, I. Casal, and C. Leclerc, Proc. Natl. Acad. Sci. USA 94:7503-7508, 1997). We show that the induction of specific CTL activity by peptides bound to microspheres requires CD4(+) T-cell help in contrast to the CTL response obtained with the peptide delivered by viral pseudoparticles. Furthermore, PPV:VLP are 100-fold more efficient than microspheres in generating a strong CTL response characterized by a high frequency of specific T cells of high avidity. Moreover, PPV:VLP-LCMV are able to protect mice against a lethal LCMV challenge whereas microspheres carrying the LCMV epitope fail to confer such protection. This study demonstrates the crucial involvement of the frequency and avidity of CTLs in conferring antiviral protective immunity and highlights the importance of considering these parameters when developing new vaccine strategies.

In Vivo Induction of a High-Avidity, High-Frequency Cytotoxic T-Lymphocyte Response Is Associated with Antiviral Protective Immunity†

PubMed Central

Sedlik, C.; Dadaglio, G.; Saron, M. F.; Deriaud, E.; Rojas, M.; Casal, S. I.; Leclerc, C.

2000-01-01

Many approaches are currently being developed to deliver exogenous antigen into the major histocompatibility complex class I-restricted antigen pathway, leading to in vivo priming of CD8+ cytotoxic T cells. One attractive possibility consists of targeting the antigen to phagocytic or macropinocytic antigen-presenting cells. In this study, we demonstrate that strong CD8+ class I-restricted cytotoxic responses are induced upon intraperitoneal immunization of mice with different peptides, characterized as CD8+ T-cell epitopes, bound to 1-μm synthetic latex microspheres and injected in the absence of adjuvant. The cytotoxic response induced against a lymphocytic choriomeningitis virus (LCMV) peptide linked to these microspheres was compared to the cytotoxic T-lymphocyte (CTL) response obtained upon immunization with the nonreplicative porcine parvovirus-like particles (PPV:VLP) carrying the same peptide (PPV:VLP-LCMV) previously described (C. Sedlik, M. F. Saron, J. Sarraseca, I. Casal, and C. Leclerc, Proc. Natl. Acad. Sci. USA 94:7503–7508, 1997). We show that the induction of specific CTL activity by peptides bound to microspheres requires CD4+ T-cell help in contrast to the CTL response obtained with the peptide delivered by viral pseudoparticles. Furthermore, PPV:VLP are 100-fold more efficient than microspheres in generating a strong CTL response characterized by a high frequency of specific T cells of high avidity. Moreover, PPV:VLP-LCMV are able to protect mice against a lethal LCMV challenge whereas microspheres carrying the LCMV epitope fail to confer such protection. This study demonstrates the crucial involvement of the frequency and avidity of CTLs in conferring antiviral protective immunity and highlights the importance of considering these parameters when developing new vaccine strategies. PMID:10846055

Antigen Presenting Properties of a Myeloid Dendritic-Like Cell in Murine Spleen.

PubMed

Hey, Ying-Ying; O'Neill, Helen C

This paper distinguishes a rare subset of myeloid dendritic-like cells found in mouse spleen from conventional (c) dendritic cells (DC) in terms of phenotype, function and gene expression. These cells are tentatively named "L-DC" since they resemble dendritic-like cells produced in longterm cultures of spleen. L-DC can be distinguished on the basis of their unique phenotype as CD11bhiCD11cloMHCII-CD43+Ly6C-Ly6G-Siglec-F- cells. They demonstrate similar ability as cDC to uptake and retain complex antigens like mannan via mannose receptors, but much lower ability to endocytose and retain soluble antigen. While L-DC differ from cDC by their inability to activate CD4+ T cells, they are capable of antigen cross-presentation for activation of CD8+ T cells, although less effectively so than the cDC subsets. In terms of gene expression, CD8- cDC and CD8+ cDC are quite distinct from L-DC. CD8+ cDC are distinguishable from the other two subsets by expression of CD24a, Clec9a, Xcr1 and Tlr11, while CD8- cDC are distinguished by expression of Ccnd1 and H-2Eb2. L-DC are distinct from the two cDC subsets through upregulated expression of Clec4a3, Emr4, Itgam, Csf1r and CD300ld. The L-DC gene profile is quite distinct from that of cDC, confirming a myeloid cell type with distinct antigen presenting properties.

Toll-like receptor agonist imiquimod facilitates antigen-specific CD8+ T-cell accumulation in the genital tract leading to tumor control through IFNγ.

PubMed

Soong, Ruey-Shyang; Song, Liwen; Trieu, Janson; Knoff, Jayne; He, Liangmei; Tsai, Ya-Chea; Huh, Warner; Chang, Yung-Nien; Cheng, Wen-Fang; Roden, Richard B S; Wu, T-C; Trimble, Cornelia L; Hung, Chien-Fu

2014-11-01

Imiquimod is a Toll-like receptor 7 agonist used topically to treat external genital warts and basal cell carcinoma. We examined the combination of topical imiquimod with intramuscular administration of CRT/E7, a therapeutic human papillomavirus (HPV) vaccine comprised of a naked DNA vector expressing calreticulin fused to HPV16 E7. Using an orthotopic HPV16 E6/E7(+) syngeneic tumor, TC-1, as a model of high-grade cervical/vaginal/vulvar intraepithelial neoplasia, we assessed if combining CRT/E7 vaccination with cervicovaginal deposition of imiquimod could result in synergistic activities promoting immune-mediated tumor clearance. Imiquimod induced cervicovaginal accumulation of activated E7-specific CD8(+) T cells elicited by CRT/E7 vaccination. Recruitment was not dependent upon the specificity of the activated CD8(+) T cells, but was significantly reduced in mice lacking the IFNγ receptor. Intravaginal imiquimod deposition induced upregulation of CXCL9 and CXCL10 mRNA expression in the genital tract, which are produced in response to IFNγ receptor signaling and attract cells expressing their ligand, CXCR3. The T cells attracted by imiquimod to the cervicovaginal tract expressed CXCR3 as well as CD49a, an integrin involved in homing and retention of CD8(+) T cells at mucosal sites. Our results indicate that intramuscular CRT/E7 vaccination in conjunction with intravaginal imiquimod deposition recruits antigen-specific CXCR3(+) CD8(+) T cells to the genital tract. Several therapeutic HPV vaccination clinical trials using a spectrum of DNA vaccines, including vaccination in concert with cervical imiquimod, are ongoing. Our study identifies a mechanism by which these strategies could provide therapeutic benefit. Our findings support accumulating evidence that manipulation of the tumor microenvironment can enhance the therapeutic efficacy of strategies that induce tumor-specific T cells. ©2014 American Association for Cancer Research.

CD4 T-Cell Memory Generation and Maintenance

PubMed Central

Gasper, David J.; Tejera, Melba Marie; Suresh, M.

2014-01-01

Immunologic memory is the adaptive immune system's powerful ability to remember a previous antigen encounter and react with accelerated vigor upon antigen re-exposure. It provides durable protection against reinfection with pathogens and is the foundation for vaccine-induced immunity. Unlike the relatively restricted immunologic purview of memory B cells and CD8 T cells, the field of CD4 T-cell memory must account for multiple distinct lineages with diverse effector functions, the issue of lineage commitment and plasticity, and the variable distribution of memory cells within each lineage. Here, we discuss the evidence for lineage-specific CD4 T-cell memory and summarize the known factors contributing to memory-cell generation, plasticity, and long-term maintenance. PMID:24940912

Non-replicating adenovirus vectors expressing avian influenza virus hemagglutinin and nucleocapsid proteins induce chicken specific effector, memory and effector memory CD8+ T lymphocytes

PubMed Central

Singh, Shailbala; Toro, Haroldo; Tang, De-Chu; Briles, Worthie E.; Yates, Linda M.; Kopulos, Renee T.; Collisson, Ellen W.

2010-01-01

Avian influenza virus (AIV) specific CD8+ T lymphocyte responses stimulated by intramuscular administration of an adenovirus (Ad) vector expressing either HA or NP were evaluated in chickens following ex vivo stimulation by non-professional antigen presenting cells. The CD8+ T lymphocyte responses were AIV specific, MHC-I restricted, and cross-reacted with heterologousH7N2 AIV strain. Specific effector responses, at 10 days post-inoculation (p.i.), were undetectable at 2 weeks p.i., and memory responses were detected from 3 to 8 weeks p.i. Effector memory responses, detected 1 week following a booster inoculation, were significantly greater than the primary responses and, within 7 days, declined to undetectable levels. Inoculation of an Ad-vector expressing human NP resulted in significantly greater MHC restricted, activation of CD8+ T cell responses specific for AIV. Decreases in all responses with time were most dramatic with maximum activation of T cells as observed following effector and effector memory responses. PMID:20557918

Immunological Tolerance to Muscle Autoantigens Involves Peripheral Deletion of Autoreactive CD8+ T Cells

PubMed Central

Franck, Emilie; Bonneau, Carole; Jean, Laetitia; Henry, Jean-Paul; Lacoume, Yann; Salvetti, Anna; Boyer, Olivier; Adriouch, Sahil

2012-01-01

Muscle potentially represents the most abundant source of autoantigens of the body and can be targeted by a variety of severe autoimmune diseases. Yet, the mechanisms of immunological tolerance toward muscle autoantigens remain mostly unknown. We investigated this issue in transgenic SM-Ova mice that express an ovalbumin (Ova) neo-autoantigen specifically in skeletal muscle. We previously reported that antigen specific CD4+ T cell are immunologically ignorant to endogenous Ova in this model but can be stimulated upon immunization. In contrast, Ova-specific CD8+ T cells were suspected to be either unresponsive to Ova challenge or functionally defective. We now extend our investigations on the mechanisms governing CD8+ tolerance in SM-Ova mice. We show herein that Ova-specific CD8+ T cells are not detected upon challenge with strongly immunogenic Ova vaccines even after depletion of regulatory T cells. Ova-specific CD8+ T cells from OT-I mice adoptively transferred to SM-Ova mice started to proliferate in vivo, acquired CD69 and PD-1 but subsequently down-regulated Bcl-2 and disappeared from the periphery, suggesting a mechanism of peripheral deletion. Peripheral deletion of endogenous Ova-specific cells was formally demonstrated in chimeric SM-Ova mice engrafted with bone marrow cells containing T cell precursors from OT-I TCR-transgenic mice. Thus, the present findings demonstrate that immunological tolerance to muscle autoantigens involves peripheral deletion of autoreactive CD8+ T cells. PMID:22570714

Chimeric Antigen Receptor (CAR)-Specific Monoclonal Antibody to Detect CD19-Specific T Cells in Clinical Trials

PubMed Central

Jena, Bipulendu; Maiti, Sourindra; Huls, Helen; Singh, Harjeet; Lee, Dean A.; Champlin, Richard E.; Cooper, Laurence J. N.

2013-01-01

Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). We describe a novel anti-idiotype monoclonal antibody (mAb) to detect CD19-specific CAR+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1) was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19+ tumor targets. This clone can be used to detect CD19-specific CAR+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy. PMID:23469246

General and Virus-Specific Immune Cell Reconstitution Following Double Cord Blood Transplantation

PubMed Central

Saliba, Rima M.; Rezvani, Katayoun; Leen, Ann; Jorgensen, Jeffrey; Shah, Nina; Hosing, Chitra; Parmar, Simrit; Oran, Betul; Olson, Amanda; Mehta, Rohtesh S.; Chemaly, Roy F.; Saunders, Ila M.; Bollard, Catherine M.; Shpall, Elizabeth J.

2015-01-01

Cord blood transplantation (CBT) is curative for many patients with hematologic malignancies but is associated with delayed immune recovery and an increased risk of viral infections compared to human leukocyte antigen (HLA) matched bone marrow or peripheral blood progenitor cell transplantation. In this study we evaluated the significance of lymphocyte recovery in 125 consecutive patients with hematologic malignancies who underwent double-unit CBT (DUCBT) with an anti-thymocyte globulin-containing regimen at our institution. A subset of 65 patients were prospectively evaluated for recovery of T, natural killer (NK) and B cells and in 46 patients we also examined viral-specific T cell recovery against Adenovirus, Epstein-Barr virus, cytomegalovirus, BK virus, respiratory syncytial virus and Influenza antigen. Our results indicate that in recipients of DUCBT, the day 30 absolute lymphocyte count is highly predictive of non-relapse mortality (NRM) and overall survival (OS). Immune recovery post-DUCBT was characterized by prolonged CD8+ and CD4+ T lymphopenia associated with preferential expansion of B and NK cells. We also observed profound delays in quantitative and functional recovery of viral-specific CD4+ and CD8+ T-cell responses for the first year post-CBT. Taken together, our data support efforts aimed at optimizing viral-specific T cell recovery to improve outcomes post-CBT. PMID:25708219

Depletion of CD8+ cells in human thymic medulla results in selective immune deficiency

PubMed Central

1989-01-01

CD8 molecules expressed on the surface of a subset of T cells participate in the selection of class I MHC antigen-restricted T cells in the thymus, and in MHC-restricted immune responses of mature class I MHC antigen-restricted T cells. Here we describe an immune-deficient patient with lack of CD8+ peripheral blood cells. The patient presented with Pneumocystis carinii pneumonia and was unable to reject an allogeneic skin graft, but had normal primary and secondary antibody responses. Examination of the patient's thymus revealed that the loss of CD8+ cells occurred during intrathymic differentiation: the patient's immature cortical thymocytes included both CD4+ and CD8+ cells while the mature medullary cells expressed the CD4 but not the CD8 protein on their surface. Northern blot and polymerase chain reaction analyses revealed the presence of CD8 alpha and beta mRNA in the patient's thymus but not in the peripheral blood. Both class I MHC antigen expression and the expressed TCR V beta repertoire are normal in this patient. These data are consistent with an impaired selection of CD8+ cells in the patient's thymus and support the role of the CD8 surface protein in thymic selection previously characterized in genetically manipulated and inbred mice. PMID:2511270

Detection and Tracking of NY-ESO-1-Specific CD8+ T Cells by High-Throughput T Cell Receptor β (TCRB) Gene Rearrangements Sequencing in a Peptide-Vaccinated Patient.

PubMed

Miyai, Manami; Eikawa, Shingo; Hosoi, Akihiro; Iino, Tamaki; Matsushita, Hirokazu; Isobe, Midori; Uenaka, Akiko; Udono, Heiichiro; Nakajima, Jun; Nakayama, Eiichi; Kakimi, Kazuhiro

2015-01-01

Comprehensive immunological evaluation is crucial for monitoring patients undergoing antigen-specific cancer immunotherapy. The identification and quantification of T cell responses is most important for the further development of such therapies. Using well-characterized clinical samples from a high responder patient (TK-f01) in an NY-ESO-1f peptide vaccine study, we performed high-throughput T cell receptor β-chain (TCRB) gene next generation sequencing (NGS) to monitor the frequency of NY-ESO-1-specific CD8+ T cells. We compared these results with those of conventional immunological assays, such as IFN-γ capture, tetramer binding and limiting dilution clonality assays. We sequenced human TCRB complementarity-determining region 3 (CDR3) rearrangements of two NY-ESO-1f-specific CD8+ T cell clones, 6-8L and 2F6, as well as PBMCs over the course of peptide vaccination. Clone 6-8L possessed the TCRB CDR3 gene TCRBV11-03*01 and BJ02-01*01 with amino acid sequence CASSLRGNEQFF, whereas 2F6 possessed TCRBV05-08*01 and BJ02-04*01 (CASSLVGTNIQYF). Using these two sequences as models, we evaluated the frequency of NY-ESO-1-specific CD8+ T cells in PBMCs ex vivo. The 6-8L CDR3 sequence was the second most frequent in PBMC and was present at high frequency (0.7133%) even prior to vaccination, and sustained over the course of vaccination. Despite a marked expansion of NY-ESO-1-specific CD8+ T cells detected from the first through 6th vaccination by tetramer staining and IFN-γ capture assays, as evaluated by CDR3 sequencing the frequency did not increase with increasing rounds of peptide vaccination. By clonal analysis using 12 day in vitro stimulation, the frequency of B*52:01-restricted NY-ESO-1f peptide-specific CD8+ T cells in PBMCs was estimated as only 0.0023%, far below the 0.7133% by NGS sequencing. Thus, assays requiring in vitro stimulation might be underestimating the frequency of clones with lower proliferation potential. High-throughput TCRB sequencing using NGS can potentially better estimate the actual frequency of antigen-specific T cells and thus provide more accurate patient monitoring.

Detection and Tracking of NY-ESO-1-Specific CD8+ T Cells by High-Throughput T Cell Receptor β (TCRB) Gene Rearrangements Sequencing in a Peptide-Vaccinated Patient

PubMed Central

Miyai, Manami; Eikawa, Shingo; Hosoi, Akihiro; Iino, Tamaki; Matsushita, Hirokazu; Isobe, Midori; Uenaka, Akiko; Udono, Heiichiro; Nakajima, Jun; Nakayama, Eiichi; Kakimi, Kazuhiro

2015-01-01

Comprehensive immunological evaluation is crucial for monitoring patients undergoing antigen-specific cancer immunotherapy. The identification and quantification of T cell responses is most important for the further development of such therapies. Using well-characterized clinical samples from a high responder patient (TK-f01) in an NY-ESO-1f peptide vaccine study, we performed high-throughput T cell receptor β-chain (TCRB) gene next generation sequencing (NGS) to monitor the frequency of NY-ESO-1-specific CD8+ T cells. We compared these results with those of conventional immunological assays, such as IFN-γ capture, tetramer binding and limiting dilution clonality assays. We sequenced human TCRB complementarity-determining region 3 (CDR3) rearrangements of two NY-ESO-1f-specific CD8+ T cell clones, 6-8L and 2F6, as well as PBMCs over the course of peptide vaccination. Clone 6-8L possessed the TCRB CDR3 gene TCRBV11-03*01 and BJ02-01*01 with amino acid sequence CASSLRGNEQFF, whereas 2F6 possessed TCRBV05-08*01 and BJ02-04*01 (CASSLVGTNIQYF). Using these two sequences as models, we evaluated the frequency of NY-ESO-1-specific CD8+ T cells in PBMCs ex vivo. The 6-8L CDR3 sequence was the second most frequent in PBMC and was present at high frequency (0.7133%) even prior to vaccination, and sustained over the course of vaccination. Despite a marked expansion of NY-ESO-1-specific CD8+ T cells detected from the first through 6th vaccination by tetramer staining and IFN-γ capture assays, as evaluated by CDR3 sequencing the frequency did not increase with increasing rounds of peptide vaccination. By clonal analysis using 12 day in vitro stimulation, the frequency of B*52:01-restricted NY-ESO-1f peptide-specific CD8+ T cells in PBMCs was estimated as only 0.0023%, far below the 0.7133% by NGS sequencing. Thus, assays requiring in vitro stimulation might be underestimating the frequency of clones with lower proliferation potential. High-throughput TCRB sequencing using NGS can potentially better estimate the actual frequency of antigen-specific T cells and thus provide more accurate patient monitoring. PMID:26291626

Regulatory CD8{sup +} T cells induced by exposure to all-trans retinoic acid and TGF-{beta} suppress autoimmune diabetes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kishi, Minoru; Yasuda, Hisafumi, E-mail: yasuda@med.kobe-u.ac.jp; Abe, Yasuhisa

Antigen-specific regulatory CD4{sup +} T cells have been described but there are few reports on regulatory CD8{sup +} T cells. We generated islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific regulatory CD8{sup +} T cells from 8.3-NOD transgenic mice. CD8{sup +} T cells from 8.3-NOD splenocytes were cultured with IGRP, splenic dendritic cells (SpDCs), TGF-{beta}, and all-trans retinoic acid (ATRA) for 5 days. CD8{sup +} T cells cultured with either IGRP alone or IGRP and SpDCs in the absence of TGF-{beta} and ATRA had low Foxp3{sup +} expression (1.7 {+-} 0.9% and 3.2 {+-} 4.5%, respectively). In contrast, CD8{sup +} T cellsmore » induced by exposure to IGRP, SpDCs, TGF-{beta}, and ATRA showed the highest expression of Foxp3{sup +} in IGRP-reactive CD8{sup +} T cells (36.1 {+-} 10.6%), which was approximately 40-fold increase compared with that before induction culture. CD25 expression on CD8{sup +} T cells cultured with IGRP, SpDCs, TGF-{beta}, and ATRA was only 7.42%, whereas CD103 expression was greater than 90%. These CD8{sup +} T cells suppressed the proliferation of diabetogenic CD8{sup +} T cells from 8.3-NOD splenocytes in vitro and completely prevented diabetes onset in NOD-scid mice in cotransfer experiments with diabetogenic splenocytes from NOD mice in vivo. Here we show that exposure to ATRA and TGF-{beta} induces CD8{sup +}Foxp3{sup +} T cells ex vivo, which suppress diabetogenic T cells in vitro and in vivo.« less

The contribution of Chlamydia-specific CD8⁺ T cells to upper genital tract pathology.

PubMed

Vlcek, Kelly R; Li, Weidang; Manam, Srikanth; Zanotti, Brian; Nicholson, Bruce J; Ramsey, Kyle H; Murthy, Ashlesh K

2016-02-01

Genital chlamydial infections lead to severe upper reproductive tract pathology in a subset of untreated women. We demonstrated previously that tumor necrosis factor (TNF)-α-producing CD8(+) T cells contribute significantly to chlamydial upper genital tract pathology in female mice. In addition, we observed that minimal chlamydial oviduct pathology develops in OT-1 transgenic (OT-1) mice, wherein the CD8(+) T-cell repertoire is restricted to recognition of the ovalbumin peptide Ova(257-264), suggesting that non-Chlamydia-specific CD8(+) T cells may not be responsible for chlamydial pathogenesis. In the current study, we evaluated whether antigen-specific CD8(+) T cells mediate chlamydial pathology. Groups of wild-type (WT) C57BL/6J, OT-1 mice, and OT-1 mice replete with WT CD8(+) T cells (1 × 10(6) cells per mouse intravenously) were infected intravaginally with C. muridarum (5 × 10(4) IFU/mouse). Serum total anti-Chlamydia antibody and total splenic anti-Chlamydia interferon (IFN)-γ and TNF-α responses were comparable among the three groups of animals. However, Chlamydia-specific IFN-γ and TNF-α production from purified splenic CD8(+) T cells of OT-1 mice was minimal, whereas responses in OT-1 mice replete with WT CD8(+) T cells were comparable to those in WT animals. Vaginal chlamydial clearance was comparable between the three groups of mice. Importantly, the incidence and severity of oviduct and uterine horn pathology was significantly reduced in OT-1 mice but reverted to WT levels in OT-1 mice replete with WT CD8(+) T cells. Collectively, these results demonstrate that Chlamydia-specific CD8(+) T cells contribute significantly to upper genital tract pathology.

Characterization of amoxicillin- and clavulanic acid-specific T cells in patients with amoxicillin-clavulanate-induced liver injury.

PubMed

Kim, Seung-Hyun; Saide, Katy; Farrell, John; Faulkner, Lee; Tailor, Arun; Ogese, Monday; Daly, Ann K; Pirmohamed, Munir; Park, B Kevin; Naisbitt, Dean J

2015-09-01

Drug-induced liver injury (DILI) frequently has a delayed onset with several human leukocyte antigen (HLA) genotypes affecting susceptibility, indicating a potential role for the adaptive immune system in the disease. The aim of this study was to investigate whether drug-responsive T lymphocytes are detectable in patients who developed DILI with the combination, antimicrobial amoxicillin-clavulanate. Lymphocytes from 6 of 7 patients were found to proliferate and/or secrete interferon-gamma (IFN-γ) when cultured with amoxicillin and/or clavulanic acid. Amoxicillin (n = 105) and clavulanic acid (n = 16) responsive CD4(+) and CD8(+) T-cell clones expressing CCR, chemokine (C-C motif) receptor 4, CCR9, and chemokine (C-X-C motif) receptor 3 were generated from patients with and without HLA risk alleles; no cross-reactivity was observed between the two drug antigens. Amoxicillin clones were found to secrete a heterogeneous panel of mediators, including IFN-γ, interleukin-22 and cytolytic molecules. In contrast, cytokine secretion by the clavulanic acid clones was more restricted. CD4(+) and CD8(+) clones were major histocompatability complex class II and I restricted, respectively, with the drug antigen being presented to CD4(+) clones in the context of HLA-DR molecules. Several pieces of evidence indicate that the clones were activated by a hapten mechanism: First, professional antigen-presenting cells (APCs) were required for optimal activation; second, pulsing APCs for 4-16 hours activated the clones; and third, inhibition of processing abrogated the proliferative response and cytokine release. Both amoxicillin- and clavulanic acid-specific T cells participate in the liver injury that develops in certain patients exposed to amoxicillin-clavulanate. © 2015 by the American Association for the Study of Liver Diseases.

The Presence of HLA-E-Restricted, CMV-Specific CD8+ T Cells in the Blood of Lung Transplant Recipients Correlates with Chronic Allograft Rejection.

PubMed

Sullivan, Lucy C; Westall, Glen P; Widjaja, Jacqueline M L; Mifsud, Nicole A; Nguyen, Thi H O; Meehan, Aislin C; Kotsimbos, Tom C; Brooks, Andrew G

2015-01-01

The human cytomegalovirus (CMV) immune evasion protein, UL40, shares an identical peptide sequence with that found in the leader sequence of many human leukocyte antigen (HLA)-C alleles and when complexed with HLA-E, can modulate NK cell functions via interactions with the CD94-NKG2 receptors. However the UL40-derived sequence can also be immunogenic, eliciting robust CD8+ T cell responses. In the setting of solid organ transplantation these T cells may not only be involved in antiviral immunity but also can potentially contribute to allograft rejection when the UL40 epitope is also present in allograft-encoded HLA. Here we assessed 15 bilateral lung transplant recipients for the presence of HLA-E-restricted UL40 specific T cells by tetramer staining of peripheral blood mononuclear cells (PBMC). UL40-specific T cells were observed in 7 patients post-transplant however the magnitude of the response varied significantly between patients. Moreover, unlike healthy CMV seropositive individuals, longitudinal analyses revealed that proportions of such T cells fluctuated markedly. Nine patients experienced low-grade acute cellular rejection, of which 6 also demonstrated UL40-specific T cells. Furthermore, the presence of UL40-specific CD8+ T cells in the blood was significantly associated with allograft dysfunction, which manifested as Bronchiolitis Obliterans Syndrome (BOS). Therefore, this study suggests that minor histocompatibility antigens presented by HLA-E can represent an additional risk factor following lung transplantation.

Increased peripheral blood CD4+ T cell responses to deamidated but not to native gliadin in children with coeliac disease

PubMed Central

Lammi, A; Arikoski, P; Vaarala, O; Kinnunen, T; Ilonen, J

2012-01-01

T cell recognition of gliadin from dietary gluten is essential for the pathogenesis of coeliac disease (CD). The aim of the present study was to analyse whether gliadin-specific T cells are detectable in the circulation of children with newly diagnosed coeliac disease by using a sensitive carboxfluorescein diacetate succinimidyl ester (CFSE) dilution method. Peripheral blood CD4+ T cell responses were analysed in 20 children at diagnosis of CD and compared to those in 64 healthy control children carrying the CD-associated human leucocyte antigen (HLA)-DQ2 or -DQ8 alleles. Deamidated gliadin (gTG)-specific T cells were detectable in the peripheral blood of more than half the children with CD (11 of 20, 55%) compared to 15 of 64 (23·4%) of the control children (P = 0·008). Proliferative responses to gTG were also significantly stronger in children with CD than in controls (P = 0·01). In contrast, T cells specific to native gliadin were detectable at comparable frequencies in children with CD (two of 19, 10·5%) and controls (13 of 64, 20·3%). gTG-specific T cells had a memory phenotype more often than those specific to native gliadin in children with CD (P = 0·02), whereas controls had similar percentages of memory cells in both stimulations. Finally, gTG-specific CD4+ T cells had a higher expression of the gut-homing molecule β7 integrin than those specific to the control antigen tetanus toxoid. Collectively, our current results demonstrate that the frequency of circulating memory CD4+ T cells specific to gTG but not native gliadin is increased in children with newly diagnosed CD. PMID:22471282

Increased peripheral blood CD4+ T cell responses to deamidated but not to native gliadin in children with coeliac disease.

PubMed

Lammi, A; Arikoski, P; Vaarala, O; Kinnunen, T; Ilonen, J

2012-05-01

T cell recognition of gliadin from dietary gluten is essential for the pathogenesis of coeliac disease (CD). The aim of the present study was to analyse whether gliadin-specific T cells are detectable in the circulation of children with newly diagnosed coeliac disease by using a sensitive carboxfluorescein diacetate succinimidyl ester (CFSE) dilution method. Peripheral blood CD4(+) T cell responses were analysed in 20 children at diagnosis of CD and compared to those in 64 healthy control children carrying the CD-associated human leucocyte antigen (HLA)-DQ2 or -DQ8 alleles. Deamidated gliadin (gTG)-specific T cells were detectable in the peripheral blood of more than half the children with CD (11 of 20, 55%) compared to 15 of 64 (23.4%) of the control children (P = 0.008). Proliferative responses to gTG were also significantly stronger in children with CD than in controls (P = 0.01). In contrast, T cells specific to native gliadin were detectable at comparable frequencies in children with CD (two of 19, 10.5%) and controls (13 of 64, 20.3%). gTG-specific T cells had a memory phenotype more often than those specific to native gliadin in children with CD (P = 0.02), whereas controls had similar percentages of memory cells in both stimulations. Finally, gTG-specific CD4(+) T cells had a higher expression of the gut-homing molecule β7 integrin than those specific to the control antigen tetanus toxoid. Collectively, our current results demonstrate that the frequency of circulating memory CD4(+) T cells specific to gTG but not native gliadin is increased in children with newly diagnosed CD. © 2012 The Authors;Clinical and Experimental Immunology © 2012 British Society for Immunology.

Antigen-specific, CD4+CD25+ regulatory T cell clones induced in Peyer's patches.

PubMed

Tsuji, Noriko M; Mizumachi, Koko; Kurisaki, Jun-Ichi

2003-04-01

Since intestine is exposed to numerous exogenous antigens such as food and commensal bacteria, the organ bears efficient mechanisms for establishment of tolerance and induction of regulatory T cells (T(reg)). Intestinal and inducible T(reg) include T(r)1-like and T(h)3 cells whose major effector molecules are IL-10 and transforming growth factor (TGF)-beta. These antigen-specific T(reg) are expected to become clinical targets to modify the inflammatory immune response associated with allergy, autoimmune diseases and transplantation. In the present study, we characterized the antigen-specific T(reg) induced in the intestine by orally administering high-dose beta-lactoglobulin (BLG) to BALB/c mice. Seven days after feeding, only Peyer's patch (PP) cells among different organs exerted significant suppressive effect on antibody production upon in vitro BLG stimulation. This suppressive effect was also prominent in six BLG-specific CD4(+) T cell clones (OPP1-6) established from PP from mice orally administered with high doses of BLG and was partially reversed by antibodies to TGF-beta. Intravenous transfer of OPP2 efficiently suppressed BLG-specific IgG1 production in serum following immunization, indicating the role of such T(reg) in the systemic tolerance after oral administration of antigen (oral tolerance). OPP clones secrete TGF-beta, IFN-gamma and low levels of IL-10, a cytokine pattern similar to that secreted by anergic T cells. OPP clones bear a CD4(+)CD25(+) phenotype and show significantly lower proliferative response compared to T(h)0 clones. This lower response is recovered by the addition of IL-2. Thus, antigen-specific CD4(+)CD25(+) T(reg), which have characteristics of anergic cells and actively suppress antibody production are induced in PP upon oral administration of protein antigen.

Positive Selection of γδ CTL by TL Antigen Expressed in the Thymus

PubMed Central

Tsujimura, Kunio; Takahashi, Toshitada; Morita, Akimichi; Hasegawa-Nishiwaki, Hitomi; Iwase, Shigeru; Obata, Yuichi

1996-01-01

To elucidate the function of the mouse TL antigen in the thymus, we have derived two TL transgenic mouse strains by introducing Tla a -3 of A strain origin with its own promoter onto a C3H background with no expression of TL in the thymus. These transgenic mouse strains, both of which express high levels of Tlaa-3-TL antigen in their thymus, were analyzed for their T cell function with emphasis on cytotoxic T lymphocyte (CTL) generation. A T cell response against TL was induced in Tg.Tlaa-3-1, Tg.Tlaa-3-2, and control C3H mice by skin grafts from H-2K b/T3 b transgenic mice, Tg.Con.3-1, expressing T3b-TL ubiquitously. Spleen cells from mice that had rejected the T3b-TL positive skin grafts were restimulated in vitro with Tg.Con.3-1 irradiated spleen cells. In mixed lymphocyte cultures (MLC), approximately 20% and 15% of Thy-1+ T cells derived from Tg.Tlaa-3-1 and Tg.Tlaa-3-2, respectively, expressed TCRγδ, whereas almost all those from C3H expressed TCRαβ. The MLC from Tg.Tlaa-3-2 and C3H demonstrated high CTL activity against TL, while those from Tg.Tlaa-3-1 had little or none. The generation of γδ CTL recognizing TL in Tg.Tlaa-3-2, but not C3H mice, was confirmed by the establishment of CTL clones. A total of 14 γδ CTL clones were established from Tg.Tlaa-3-2, whereas none were obtained from C3H. Of the 14 γδ CTL clones, 8 were CD8+ and 6 were CD4−CD8− double negative. The CTL activity of all these clones was TL specific and inhibited by anti-TL, but not by anti-H-2 antibodies, demonstrating that they recognize TL directly without antigen presentation by H-2. The CTL activity was blocked by antibodies to TCRγδ and CD3, and also by antibodies to CD8α and CD8β in CD8+ clones, showing that the activity was mediated by TCRγδ and coreceptors. The thymic origin of these γδ CTL clones was indicated by the expression of Thy-1 and Ly-1 (CD5), and also CD8αβ heterodimers in CD8+ clones on their surfaces and by the usage of TCR Vγ4 chains in 12 of the 14 clones. Taken together, these results suggest that Tlaa-3-TL antigen expressed in the thymus engages in positive selection of a sizable population of γδ T cells. PMID:8976173

Mycobacterium tuberculosis genome-wide screen exposes multiple CD8+ T cell epitopes

PubMed Central

Hammond, A S; Klein, M R; Corrah, T; Fox, A; Jaye, A; McAdam, K P; Brookes, R H

2005-01-01

Mounting evidence suggests human leucocyte antigen (HLA) class I-restricted CD8+ T cells play a role in protective immunity against tuberculosis yet relatively few epitopes specific for the causative organism, Mycobacterium tuberculosis, are reported. Here a total genome-wide screen of M. tuberculosis was used to identify putative HLA-B*3501 T cell epitopes. Of 479 predicted epitopes, 13 with the highest score were synthesized and used to restimulate lymphocytes from naturally exposed HLA-B*3501 healthy individuals in cultured and ex vivo enzyme-linked immunospot (ELISPOT) assays for interferon (IFN)-γ. All 13 peptides elicited a response that varied considerably between individuals. For three peptides CD8+ T cell lines were expanded and four of the 13 were recognized permissively through the HLA-B7 supertype family. Although further testing is required we show the genome-wide screen to be feasible for the identification of unknown mycobacterial antigens involved in immunity against natural infection. While the mechanisms of protective immunity against M. tuberculosis infection remain unclear, conventional class I-restricted CD8+ T cell responses appear to be widespread throughout the genome. PMID:15762882

Antigen expression level threshold tunes the fate of CD8 T cells during primary hepatic immune responses.

PubMed

Tay, Szun Szun; Wong, Yik Chun; McDonald, David M; Wood, Nicole A W; Roediger, Ben; Sierro, Frederic; Mcguffog, Claire; Alexander, Ian E; Bishop, G Alex; Gamble, Jennifer R; Weninger, Wolfgang; McCaughan, Geoffrey W; Bertolino, Patrick; Bowen, David G

2014-06-24

CD8 T-cell responses to liver-expressed antigens range from deletional tolerance to full effector differentiation resulting in overt hepatotoxicity. The reasons for these heterogeneous outcomes are not well understood. To identify factors that govern the fate of CD8 T cells activated by hepatocyte-expressed antigen, we exploited recombinant adenoassociated viral vectors that enabled us to vary potential parameters determining these outcomes in vivo. Our findings reveal a threshold of antigen expression within the liver as the dominant factor determining T-cell fate, irrespective of T-cell receptor affinity or antigen cross-presentation. Thus, when a low percentage of hepatocytes expressed cognate antigen, high-affinity T cells developed and maintained effector function, whereas, at a high percentage, they became functionally exhausted and silenced. Exhaustion was not irreversibly determined by initial activation, but was maintained by high intrahepatic antigen load during the early phase of the response; cytolytic function was restored when T cells primed under high antigen load conditions were transferred into an environment of low-level antigen expression. Our study reveals a hierarchy of factors dictating the fate of CD8 T cells during hepatic immune responses, and provides an explanation for the different immune outcomes observed in a variety of immune-mediated liver pathologic conditions.

Early T Cell Recognition of B Cells following Epstein-Barr Virus Infection: Identifying Potential Targets for Prophylactic Vaccination

PubMed Central

Brooks, Jill M.; Long, Heather M.; Tierney, Rose J.; Shannon-Lowe, Claire; Leese, Alison M.; Fitzpatrick, Martin; Taylor, Graham S.; Rickinson, Alan B.

2016-01-01

Epstein-Barr virus, a B-lymphotropic herpesvirus, is the cause of infectious mononucleosis, has strong aetiologic links with several malignancies and has been implicated in certain autoimmune diseases. Efforts to develop a prophylactic vaccine to prevent or reduce EBV-associated disease have, to date, focused on the induction of neutralising antibody responses. However, such vaccines might be further improved by inducing T cell responses capable of recognising and killing recently-infected B cells. In that context, EBNA2, EBNA-LP and BHRF1 are the first viral antigens expressed during the initial stage of B cell growth transformation, yet have been poorly characterised as CD8+ T cell targets. Here we describe CD8+ T cell responses against each of these three “first wave” proteins, identifying target epitopes and HLA restricting alleles. While EBNA-LP and BHRF1 each contained one strong CD8 epitope, epitopes within EBNA2 induced immunodominant responses through several less common HLA class I alleles (e.g. B*3801 and B*5501), as well as subdominant responses through common class I alleles (e.g. B7 and C*0304). Importantly, such EBNA2-specific CD8+ T cells recognised B cells within the first day post-infection, prior to CD8+ T cells against well-characterised latent target antigens such as EBNA3B or LMP2, and effectively inhibited outgrowth of EBV-transformed B cell lines. We infer that “first wave” antigens of the growth-transforming infection, especially EBNA2, constitute potential CD8+ T cell immunogens for inclusion in prophylactic EBV vaccine design. PMID:27096949

Adoptive Transfer of IL13Rα2-Specific Chimeric Antigen Receptor T Cells Creates a Pro-inflammatory Environment in Glioblastoma.

PubMed

Pituch, Katarzyna C; Miska, Jason; Krenciute, Giedre; Panek, Wojciech K; Li, Gina; Rodriguez-Cruz, Tania; Wu, Meijing; Han, Yu; Lesniak, Maciej S; Gottschalk, Stephen; Balyasnikova, Irina V

2018-04-04

In order to fully harness the potential of immunotherapy with chimeric antigen receptor (CAR)-modified T cells, pre-clinical studies must be conducted in immunocompetent animal models that closely mimic the immunosuppressive malignant glioma (MG) microenvironment. Thus, the goal of this project was to study the in vivo fate of T cells expressing CARs specific for the MG antigen IL13Rα2 (IL13Rα2-CARs) in immunocompetent MG models. Murine T cells expressing IL13Rα2-CARs with a CD28.ζ (IL13Rα2-CAR.CD28.ζ) or truncated signaling domain (IL13Rα2-CAR.Δ) were generated by retroviral transduction, and their effector function was evaluated both in vitro and in vivo. IL13Rα2-CAR.CD28.ζ T cells' specificity toward IL13Rα2 was confirmed through cytokine production and cytolytic activity. In vivo, a single intratumoral injection of IL13Rα2-CAR.CD28.ζ T cells significantly extended the survival of IL13Rα2-expressing GL261 and SMA560 glioma-bearing mice; long-term survivors were resistant to re-challenge with IL13Rα2-negative and IL13Rα2-positive tumors. IL13Rα2-CAR.CD28.ζ T cells proliferated, produced cytokines (IFNγ, TNF-α), and promoted a phenotypically pro-inflammatory glioma microenvironment by inducing a significant increase in the number of CD4 + and CD8 + T cells and CD8α + dendritic cells and a decrease in Ly6G + myeloid-derived suppressor cells (MDSCs). Our data underline the significance of CAR T cell studies in immunocompetent hosts and further validate IL13Rα2-CAR T cells as an efficacious therapeutic strategy for MG. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Antigen-Specific CD8 Lytic Phenotype Induced by Sipuleucel-T in Hormone-Sensitive or Castration-Resistant Prostate Cancer and Association With Overall Survival.

PubMed

Antonarakis, Emmanuel S; Small, Eric J; Petrylak, Daniel; Quinn, David I; Kibel, Adam S; Chang, Nancy; Dearstyne, Erica; Harmon, Matthew; Campogan, Dwayne; Haynes, Heather; Vu, Tuyen; Sheikh, Nadeem A; Drake, Charles G

2018-06-01

Sipuleucel-T is FDA-approved for the treatment of metastatic castration-resistant prostate cancer (mCRPC) based on the IMPACT trial showing a 4.1-month benefit in median overall survival (OS) for patients receiving sipuleucel-T vs control. Although efficacy of sipuleucel-T is well-established, its mechanism remains incompletely understood. Patient samples from three sipuleucel-T trials were assessed for peripheral cellular immune responses to the immunogen PA2024 and the target antigen prostatic acid phosphatase (PAP). PAP- and PA2024-specific proliferative and cytolytic responses were characterized to delineate sipuleucel-T-induced immune responses. To quantify potential cytotoxic T lymphocyte (CTL) activity, cell-surface CD107a expression on PAP- or PA2024-specific CD8+ T cells was measured in sipuleucel-T-treated patient and healthy volunteer samples. Increased PA2024-specific CD4+ (p=0.030) and CD8+ (p=0.052) T-cell proliferation from baseline to week 6 was observed (N=14) post-sipuleucel-T, with greater magnitude of PA2024-specific responses compared to PAP. PAP- and PA2024-CTL activity (CD107a positivity) significantly increased at weeks 6 and 26 after sipuleucel-T treatment (p<0.0001; N=22). At 26 weeks post-sipuleucel-T, OS correlated with the magnitude of PAP (Pearson's R, 0.52; p=0.013) or PA2024 (Pearson's R, 0.67; p=0.0006) CTL activity. Higher PA2024-CTL activity at week 26 was significantly associated with longer OS using tertile analysis (p=0.0005; N=22), with PA2024 responses correlating with PAP responses at week 26 (R=0.90; p=1.53E -08 ). This study is the first to report PAP-specific CD8+ T-cell responses elicited by sipuleucel-T treatment. Increased and persistent potential PA2024-specific CTL activity correlated with PAP-specific CTL activity and associated with improved OS following sipuleucel-T treatment. Copyright ©2018, American Association for Cancer Research.

A novel CD4-CD8alpha+CD205+CD11b- murine spleen dendritic cell line: establishment, characterization and functional analysis in a model of vaccination to toxoplasmosis.

PubMed

Ruiz, Sophie; Beauvillain, Céline; Mévélec, Marie-Noëlle; Roingeard, Philippe; Breton, Pascal; Bout, Daniel; Dimier-Poisson, Isabelle

2005-11-01

Dendritic cells (DCs) play an essential role in the induction of immune responses to pathogen infections. Native DCs are difficult to obtain in large numbers and consequently the vast majority of DCs employed in all experiments are derived from bone marrow progenitors. In an attempt to solve this problem, we have established a novel CD8alpha(+) DC line (H-2(k)) from spleen, which we have named SRDC line, and which is easy to culture in vitro. These cells display similar morphology, phenotype and activity to CD4(-)CD8alpha(+)CD205(+)CD11b(-) DCs purified ex vivo. Toxoplasma gondii antigen was shown to be taken up by these cells and to increase class I and class II major histocompatibility complex (MHC), CD40, CD80 and CD86 surface expression. We report that vaccination with T. gondii antigen-pulsed SRDCs, which synthesize large amounts of interleukin-12, induced protective immune responses against this intracellular pathogen in syngeneic CBA/J mice. This protection was associated with strong cellular and humoral immune responses at systemic and intestinal levels. Spleen and mesenteric lymph node cell proliferations were correlated with a Th1/Th2-type response and a specific SRDC homing to spleen and intestine was observed. The SRDC or CD4(-)CD8alpha(+)CD205(+)CD11b(-) DC line can be expected to be a very useful tool for immunobiology studies of DC.

Persistent viral infection in humans can drive high frequency low-affinity T-cell expansions

PubMed Central

Khan, Naeem; Cobbold, Mark; Cummerson, Joanne; Moss, Paul A H

2010-01-01

CD8 T cells that recognize cytomegalovirus (CMV) -encoded peptides can be readily detected by staining with human leucocyte antigen (HLA) –peptide tetramers. These cells are invariably highly differentiated effector memory cells with high avidity T-cell receptors (TCR). In this report we demonstrate an HLA-A*0201 restricted CMV-specific CD8 T-cell response (designated YVL) that represents several percent of the CD8 T-cell subset, yet fails to bind tetrameric major histocompatibility complex (MHC) ligands. However, these tetramer-negative cells are both phenotypically and functionally similar to other CMV-specific CD8 T cells. YVL peptide-specific CD8 T-cell clones were generated and found to be of high avidity in both cytotoxicity and interferon-γ (IFN-γ) assays, and comparable with other CMV peptide-specific CD8 T-cell clones. However, under conditions of CD8 blockade, the response was almost nullified even at very high ligand concentrations. This was also the case in IFN-γ experiments using peripheral blood mononuclear cells stimulated with peptide ex vivo. In contrast, all other CMV specificities (tetramer-positive) displayed minimal or only partial CD8 dependence. This suggests that YVL-specific responses depict a low-affinity TCR–MHC–peptide interaction, that is compensated by substantial CD8 involvement for functional purposes, yet cannot engage multivalent soluble ligands for ex vivo analysis. It is interesting that such a phenomenon is apparent in the face of a persistent virus infection such as CMV, where the responding cells represent an immunodominant response in that individual and may present a highly differentiated effector phenotype. PMID:20722762

Using Antigen-Specific B Cells to Combine Antibody and T Cell-Based Cancer Immunotherapy.

PubMed

Wennhold, Kerstin; Thelen, Martin; Schlößer, Hans Anton; Haustein, Natalie; Reuter, Sabrina; Garcia-Marquez, Maria; Lechner, Axel; Kobold, Sebastian; Rataj, Felicitas; Utermöhlen, Olaf; Chakupurakal, Geothy; Theurich, Sebastian; Hallek, Michael; Abken, Hinrich; Shimabukuro-Vornhagen, Alexander; von Bergwelt-Baildon, Michael

2017-09-01

Cancer immunotherapy by therapeutic activation of T cells has demonstrated clinical potential. Approaches include checkpoint inhibitors and chimeric antigen receptor T cells. Here, we report the development of an alternative strategy for cellular immunotherapy that combines induction of a tumor-directed T-cell response and antibody secretion without the need for genetic engineering. CD40 ligand stimulation of murine tumor antigen-specific B cells, isolated by antigen-biotin tetramers, resulted in the development of an antigen-presenting phenotype and the induction of a tumor antigen-specific T-cell response. Differentiation of antigen-specific B cells into antibody-secreting plasma cells was achieved by stimulation with IL21, IL4, anti-CD40, and the specific antigen. Combined treatment of tumor-bearing mice with antigen-specific CD40-activated B cells and antigen-specific plasma cells induced a therapeutic antitumor immune response resulting in remission of established tumors. Human CEA or NY-ESO-1-specific B cells were detected in tumor-draining lymph nodes and were able to induce antigen-specific T-cell responses in vitro, indicating that this approach could be translated into clinical applications. Our results describe a technique for the exploitation of B-cell effector functions and provide the rationale for their use in combinatorial cancer immunotherapy. Cancer Immunol Res; 5(9); 730-43. ©2017 AACR . ©2017 American Association for Cancer Research.

Autophagy-related protein Vps34 controls the homeostasis and function of antigen cross-presenting CD8α+ dendritic cells.

PubMed

Parekh, Vrajesh V; Pabbisetty, Sudheer K; Wu, Lan; Sebzda, Eric; Martinez, Jennifer; Zhang, Jianhua; Van Kaer, Luc

2017-08-01

The class III PI3K Vacuolar protein sorting 34 (Vps34) plays a role in both canonical and noncanonical autophagy, key processes that control the presentation of antigens by dendritic cells (DCs) to naive T lymphocytes. We generated DC-specific Vps34 -deficient mice to assess the contribution of Vps34 to DC functions. We found that DCs from these animals have a partially activated phenotype, spontaneously produce cytokines, and exhibit enhanced activity of the classic MHC class I and class II antigen-presentation pathways. Surprisingly, these animals displayed a defect in the homeostatic maintenance of splenic CD8α + DCs and in the capacity of these cells to cross-present cell corpse-associated antigens to MHC class I-restricted T cells, a property that was associated with defective expression of the T-cell Ig mucin (TIM)-4 receptor. Importantly, mice deficient in the Vps34-associated protein Rubicon, which is critical for a noncanonical form of autophagy called "Light-chain 3 (LC3)-associated phagocytosis" (LAP), lacked such defects. Finally, consistent with their defect in the cross-presentation of apoptotic cells, DC-specific Vps34 -deficient animals developed increased metastases in response to challenge with B16 melanoma cells. Collectively, our studies have revealed a critical role of Vps34 in the regulation of CD8α + DC homeostasis and in the capacity of these cells to process and present antigens associated with apoptotic cells to MHC class I-restricted T cells. Our findings also have important implications for the development of small-molecule inhibitors of Vps34 for therapeutic purposes.

Infusion of donor-derived CD19-redirected virus-specific T cells for B-cell malignancies relapsed after allogeneic stem cell transplant: a phase 1 study

PubMed Central

Cruz, Conrad Russell Y.; Micklethwaite, Kenneth P.; Savoldo, Barbara; Ramos, Carlos A.; Lam, Sharon; Ku, Stephanie; Diouf, Oumar; Liu, Enli; Barrett, A. John; Ito, Sawa; Shpall, Elizabeth J.; Krance, Robert A.; Kamble, Rammurti T.; Carrum, George; Hosing, Chitra M.; Gee, Adrian P.; Mei, Zhuyong; Grilley, Bambi J.; Heslop, Helen E.; Rooney, Cliona M.; Brenner, Malcolm K.; Bollard, Catherine M.

2013-01-01

Autologous T cells expressing a CD19-specific chimeric antigen receptor (CD19.CAR) are active against B-cell malignancies, but it is unknown whether allogeneic CD19.CAR T cells are safe or effective. After allogeneic hematopoietic stem cell transplantation (HSCT), infused donor-derived virus-specific T cells (VSTs) expand in vivo, persist long term, and display antiviral activity without inducing graft-vs-host disease; therefore, we determined whether donor VSTs, engineered to express CD19.CAR, retained the characteristics of nonmanipulated allogeneic VSTs while gaining antitumor activity. We treated 8 patients with allogeneic (donor-derived) CD19.CAR-VSTs 3 months to 13 years after HSCT. There were no infusion-related toxicities. VSTs persisted for a median of 8 weeks in blood and up to 9 weeks at disease sites. Objective antitumor activity was evident in 2 of 6 patients with relapsed disease during the period of CD19.CAR-VST persistence, whereas 2 patients who received cells while in remission remain disease free. In 2 of 3 patients with viral reactivation, donor CD19.CAR-VSTs expanded concomitantly with VSTs. Hence CD19.CAR-VSTs display antitumor activity and, because their number may be increased in the presence of viral stimuli, earlier treatment post-HSCT (when lymphodepletion is greater and the incidence of viral infection is higher) or planned vaccination with viral antigens may enhance disease control. This study is registered at clinicaltrials.gov as #NCT00840853. PMID:24030379

Effective Respiratory CD8 T-Cell Immunity to Influenza Virus Induced by Intranasal Carbomer-Lecithin-Adjuvanted Non-replicating Vaccines

PubMed Central

Gasper, David J.; Neldner, Brandon; Plisch, Erin H.; Rustom, Hani; Imai, Hirotaka; Kawaoka, Yoshihiro; Suresh, M.

2016-01-01

CD8+ cytotoxic T lymphocytes (CTLs) are critical for clearing many viral infections, and protective CTL memory can be induced by vaccination with attenuated viruses and vectors. Non-replicating vaccines are typically potentiated by the addition of adjuvants that enhance humoral responses, however few are capable of generating CTL responses. Adjuplex is a carbomer-lecithin-based adjuvant demonstrated to elicit robust humoral immunity to non-replicating antigens. We report that mice immunized with non-replicating Adjuplex-adjuvanted vaccines generated robust antigen-specific CTL responses. Vaccination by the subcutaneous or the intranasal route stimulated systemic and mucosal CTL memory respectively. However, only CTL memory induced by intranasal vaccination was protective against influenza viral challenge, and correlated with an enhancement of memory CTLs in the airways and CD103+ CD69+ CXCR3+ resident memory-like CTLs in the lungs. Mechanistically, Myd88-deficient mice mounted primary CTL responses to Adjuplex vaccines that were similar in magnitude to wild-type mice, but exhibited altered differentiation of effector cell subsets. Immune potentiating effects of Adjuplex entailed alterations in the frequency of antigen-presenting-cell subsets in vaccine draining lymph nodes, and in the lungs and airways following intranasal vaccination. Further, Adjuplex enhanced the ability of dendritic cells to promote antigen-induced proliferation of naïve CD8 T cells by modulating antigen uptake, its intracellular localization, and rate of processing. Taken together, we have identified an adjuvant that elicits both systemic and mucosal CTL memory to non-replicating antigens, and engenders protective CTL-based heterosubtypic immunity to influenza A virus in the respiratory tract. Further, findings presented in this manuscript have provided key insights into the mechanisms and factors that govern the induction and programming of systemic and protective memory CTLs in the respiratory tract. PMID:27997610

Effective Respiratory CD8 T-Cell Immunity to Influenza Virus Induced by Intranasal Carbomer-Lecithin-Adjuvanted Non-replicating Vaccines.

PubMed

Gasper, David J; Neldner, Brandon; Plisch, Erin H; Rustom, Hani; Carrow, Emily; Imai, Hirotaka; Kawaoka, Yoshihiro; Suresh, M

2016-12-01

CD8+ cytotoxic T lymphocytes (CTLs) are critical for clearing many viral infections, and protective CTL memory can be induced by vaccination with attenuated viruses and vectors. Non-replicating vaccines are typically potentiated by the addition of adjuvants that enhance humoral responses, however few are capable of generating CTL responses. Adjuplex is a carbomer-lecithin-based adjuvant demonstrated to elicit robust humoral immunity to non-replicating antigens. We report that mice immunized with non-replicating Adjuplex-adjuvanted vaccines generated robust antigen-specific CTL responses. Vaccination by the subcutaneous or the intranasal route stimulated systemic and mucosal CTL memory respectively. However, only CTL memory induced by intranasal vaccination was protective against influenza viral challenge, and correlated with an enhancement of memory CTLs in the airways and CD103+ CD69+ CXCR3+ resident memory-like CTLs in the lungs. Mechanistically, Myd88-deficient mice mounted primary CTL responses to Adjuplex vaccines that were similar in magnitude to wild-type mice, but exhibited altered differentiation of effector cell subsets. Immune potentiating effects of Adjuplex entailed alterations in the frequency of antigen-presenting-cell subsets in vaccine draining lymph nodes, and in the lungs and airways following intranasal vaccination. Further, Adjuplex enhanced the ability of dendritic cells to promote antigen-induced proliferation of naïve CD8 T cells by modulating antigen uptake, its intracellular localization, and rate of processing. Taken together, we have identified an adjuvant that elicits both systemic and mucosal CTL memory to non-replicating antigens, and engenders protective CTL-based heterosubtypic immunity to influenza A virus in the respiratory tract. Further, findings presented in this manuscript have provided key insights into the mechanisms and factors that govern the induction and programming of systemic and protective memory CTLs in the respiratory tract.

The XC chemokine receptor 1 is a conserved selective marker of mammalian cells homologous to mouse CD8α+ dendritic cells

PubMed Central

Crozat, Karine; Guiton, Rachel; Contreras, Vanessa; Feuillet, Vincent; Dutertre, Charles-Antoine; Ventre, Erwan; Vu Manh, Thien-Phong; Baranek, Thomas; Storset, Anne K.; Marvel, Jacqueline; Boudinot, Pierre; Hosmalin, Anne; Schwartz-Cornil, Isabelle

2010-01-01

Human BDCA3+ dendritic cells (DCs) were suggested to be homologous to mouse CD8α+ DCs. We demonstrate that human BDCA3+ DCs are more efficient than their BDCA1+ counterparts or plasmacytoid DCs (pDCs) in cross-presenting antigen and activating CD8+ T cells, which is similar to mouse CD8α+ DCs as compared with CD11b+ DCs or pDCs, although with more moderate differences between human DC subsets. Yet, no specific marker was known to be shared between homologous DC subsets across species. We found that XC chemokine receptor 1 (XCR1) is specifically expressed and active in mouse CD8α+, human BDCA3+, and sheep CD26+ DCs and is conserved across species. The mRNA encoding the XCR1 ligand chemokine (C motif) ligand 1 (XCL1) is selectively expressed in natural killer (NK) and CD8+ T lymphocytes at steady-state and is enhanced upon activation. Moreover, the Xcl1 mRNA is selectively expressed at high levels in central memory compared with naive CD8+ T lymphocytes. Finally, XCR1−/− mice have decreased early CD8+ T cell responses to Listeria monocytogenes infection, which is associated with higher bacterial loads early in infection. Therefore, XCR1 constitutes the first conserved specific marker for cell subsets homologous to mouse CD8α+ DCs in higher vertebrates and promotes their ability to activate early CD8+ T cell defenses against an intracellular pathogenic bacteria. PMID:20479118

Potentiation by a novel alkaloid glycoside adjuvant of a protective cytotoxic T cell immune response specific for a preerythrocytic malaria vaccine candidate antigen.

PubMed

Heal, K G; Sheikh, N A; Hollingdale, M R; Morrow, W J; Taylor-Robinson, A W

2001-07-20

We have recently demonstrated that the novel glycoalkaloid tomatine, derived from leaves of the wild tomato Lycopersicon pimpinellifolium, can act as a powerful adjuvant for the elicitation of antigen-specific CD8+ T cell responses. Here, we have extended our previous investigation with the model antigen ovalbumin to an established malaria infection system in mice and evaluated the cellular immune response to a major preerythrocytic stage malaria vaccine candidate antigen when administered with tomatine. The defined MHC H-2kd class I-binding 9-mer peptide (amino acids 252-260) from Plasmodium berghei circumsporozoite (CS) protein was prepared with tomatine to form a molecular aggregate formulation and this used to immunise BALB/c (H-2kd) mice. Antigen-specific IFN-gamma secretion and cytotoxic T lymphocyte activity in vitro were both significantly enhanced compared to responses detected from similarly stimulated splenocytes from naive and tomatine-saline-immunised control mice. Moreover, when challenged with P. berghei sporozoites, mice immunised with the CS 9-mer-tomatine preparation had a significantly delayed onset of erythrocytic infection compared to controls. The data presented validate the use of tomatine to potentiate a cellular immune response to antigenic stimulus by testing in an important biologically relevant system. Specifically, the processing of the P. berghei CS 9-mer as an exogenous antigen and its presentation via MHC class I molecules to CD8+ T cells led to an immune response that is an in vitro correlate of protection against preerythrocytic malaria. This was confirmed by the protective capacity of the 9-mer-tomatine combination upon in vivo immunisation. These findings merit further work to optimise the use of tomatine as an adjuvant in malaria vaccine development.

Validation of RNA-based molecular clonotype analysis for virus-specific CD8+ T-cells in formaldehyde-fixed specimens isolated from peripheral blood

PubMed Central

van Bockel, David; Price, David A.; Asher, Tedi E.; Venturi, Vanessa; Suzuki, Kazuo; Warton, Kristina; Davenport, Miles P.; Cooper, David A.; Douek, Daniel C.; Kelleher, Anthony D.

2007-01-01

Recent advances in the field of molecular clonotype analysis have enabled detailed repertoire characterization of viably isolated antigen-specific T cell populations directly ex vivo. However, in the absence of a biologically contained FACS facility, peripheral blood mononuclear cell (PBMC) preparations derived from patients infected with agents such as HIV must be formaldehyde fixed to inactivate the pathogen; this procedure adversely affects nucleic acid template quality. Here, we developed and validated a method to amplify and sequence mRNA species derived from formaldehyde fixed PBMC specimens. Antigen-specific CD8+ cytotoxic T-lymphocyte populations were identified with standard fluorochrome-conjugated peptide-major histocompatibility complex class I tetramers refolded around synthetic peptides representing immunodominant epitopes from HIV p24 Gag (KRWII[M/L]GLNK/HLA B*2705) and CMV pp65 (NLVPMVATV/HLA A*0201 and TPRVTGGGAM/HLA B*0702), and acquired in separate laboratories with or without fixation. In the presence of proteinase K pre-treatment, the observed antigen-specific CD8+ T-cell repertoire determined by molecular clonotype analysis was statistically no different whether derived from fixed or unfixed PBMC. However, oligo-dT recovery methods were not suitable for use with fixed tissue as significant skewing of clonotypic representation was observed. Thus, we have developed a reliable RNA-based method for molecular clonotype analysis that is compatible with formaldehyde fixation and therefore suitable for use with primary human samples isolated by FACS outside the context of a biological safety level 3 containment facility. PMID:17716684

Characterization of extrathymic CD8αβ T cells in the liver and intestine in TAP-1 deficient mice

PubMed Central

Tsukada, Chika; Miyaji, Chikako; Kawamura, Hiroki; Miyakawa, Ryoko; Yokoyama, Hisashi; Ishimoto, Yuiko; Miyazawa, Shinobu; Watanabe, Hisami; Abo, Toru

2003-01-01

TAP-1 deficient (−/−) mice cannot transport MHC class I antigens onto the cell surface, which results in failure of the generation of CD8+ T cells in the thymus. In a series of recent studies, it has been proposed that extrathymic T cells are generated in the liver and at other extrathymic sites (e.g. the intestine). It was therefore investigated whether CD8+ extrathymic T cells require an interaction with MHC class I antigens for their differentiation in TAP-1(−/−) mice. Although CD8+ thymically derived T cells were confirmed to be absent in the spleen as well as in the thymus, CD8αβ+ T cells were abundant in the livers and intestines of TAP-1(−/−) mice. These CD8+ T cells expanded in the liver as a function of age and were mainly confined to a NK1·1−CD3int population which is known to be truly of extrathymic origin. Hepatic lymphocytes, which contained CD8+ T cells and which were isolated from TAP-1(−/−) mice (H-2b), responded to neither mutated MHC class I antigens (bm1) nor allogeneic MHC class I antigens (H-2d) in in vitro mixed lymphocyte cultures. However, the results from repeated in vivo stimulations with alloantigens (H-2d) were interesting. Allogeneic cytotoxicity was induced in liver lymphocytes in TAP-1(−/−) mice, although the magnitude of cytotoxicity was lower than that of liver lymphocytes in immunized B6 mice. All allogeneic cytotoxicity disappeared with the elimination of CD8+ cells in TAP-1(−/−) mice. These results suggest that the generation and function of CD8+ extrathymic T cells are independent of the existence of the MHC class I antigens of the mouse but have a limited allorecognition ability. PMID:12807479

Macrophages play an essential role in antigen-specific immune suppression mediated by T CD8⁺ cell-derived exosomes.

PubMed

Nazimek, Katarzyna; Ptak, Wlodzimierz; Nowak, Bernadeta; Ptak, Maria; Askenase, Philip W; Bryniarski, Krzysztof

2015-09-01

Murine contact sensitivity (CS) reaction could be antigen-specifically regulated by T CD8(+) suppressor (Ts) lymphocytes releasing microRNA-150 in antibody light-chain-coated exosomes that were formerly suggested to suppress CS through action on macrophages (Mφ). The present studies investigated the role of Mφ in Ts cell-exosome-mediated antigen-specific suppression as well as modulation of Mφ antigen-presenting function in humoral and cellular immunity by suppressive exosomes. Mice depleted of Mφ by clodronate liposomes could not be tolerized and did not produce suppressive exosomes. Moreover, isolated T effector lymphocytes transferring CS were suppressed by exosomes only in the presence of Mφ, demonstrating the substantial role of Mφ in the generation and action of Ts cell regulatory exosomes. Further, significant decrease of number of splenic B cells producing trinitrophenyl (TNP) -specific antibodies with the alteration of the ratio of serum titres of IgM to IgG was observed in recipients of exosome-treated, antigen-pulsed Mφ and the significant suppression of CS was demonstrated in recipients of exosome-treated, TNP-conjugated Mφ. Additionally, exosome-pulsed, TNP-conjugated Mφ mediated suppression of CS in mice pre-treated with a low-dose of cyclophosphamide, suggesting de novo induction of T regulatory (Treg) lymphocytes. Treg cell involvement in the effector phase of the studied suppression mechanism was proved by unsuccessful tolerization of DEREG mice depleted of Treg lymphocytes. Furthermore, the inhibition of proliferation of CS effector cells cultured with exosome-treated Mφ in a transmembrane manner was observed. Our results demonstrated the essential role of Mφ in antigen-specific immune suppression mediated by Ts cell-derived exosomes and realized by induction of Treg lymphocytes and inhibition of T effector cell proliferation. © 2015 John Wiley & Sons Ltd.

Efficient induction of CD25- iTreg by co-immunization requires strongly antigenic epitopes for T cells.

PubMed

Geng, Shuang; Yu, Yang; Kang, Youmin; Pavlakis, George; Jin, Huali; Li, Jinyao; Hu, Yanxin; Hu, Weibin; Wang, Shuang; Wang, Bin

2011-05-05

We previously showed that co-immunization with a protein antigen and a DNA vaccine coding for the same antigen induces CD40 low IL-10 high tolerogenic DCs, which in turn stimulates the expansion of antigen-specific CD4+CD25-Foxp3+ regulatory T cells (CD25- iTreg). However, it was unclear how to choose the antigen sequence to maximize tolerogenic antigen presentation and, consequently, CD25- iTreg induction. In the present study, we demonstrated the requirement of highly antigenic epitopes for CD25- iTreg induction. Firstly, we showed that the induction of CD25- iTreg by tolerogenic DC can be blocked by anti-MHC-II antibody. Next, both the number and the suppressive activity of CD25- iTreg correlated positively with the overt antigenicity of an epitope to activate T cells. Finally, in a mouse model of dermatitis, highly antigenic epitopes derived from a flea allergen not only induced more CD25- iTreg, but also more effectively prevented allergenic reaction to the allergen than did weakly antigenic epitopes. Our data thus indicate that efficient induction of CD25- iTreg requires highly antigenic peptide epitopes. This finding suggests that highly antigenic epitopes should be used for efficient induction of CD25- iTreg for clinical applications such as flea allergic dermatitis.

Parasite Fate and Involvement of Infected Cells in the Induction of CD4+ and CD8+ T Cell Responses to Toxoplasma gondii

PubMed Central

Dupont, Christopher D.; Christian, David A.; Selleck, Elizabeth M.; Pepper, Marion; Leney-Greene, Michael; Harms Pritchard, Gretchen; Koshy, Anita A.; Wagage, Sagie; Reuter, Morgan A.; Sibley, L. David; Betts, Michael R.; Hunter, Christopher A.

2014-01-01

During infection with the intracellular parasite Toxoplasma gondii, the presentation of parasite-derived antigens to CD4+ and CD8+ T cells is essential for long-term resistance to this pathogen. Fundamental questions remain regarding the roles of phagocytosis and active invasion in the events that lead to the processing and presentation of parasite antigens. To understand the most proximal events in this process, an attenuated non-replicating strain of T. gondii (the cpsII strain) was combined with a cytometry-based approach to distinguish active invasion from phagocytic uptake. In vivo studies revealed that T. gondii disproportionately infected dendritic cells and macrophages, and that infected dendritic cells and macrophages displayed an activated phenotype characterized by enhanced levels of CD86 compared to cells that had phagocytosed the parasite, thus suggesting a role for these cells in priming naïve T cells. Indeed, dendritic cells were required for optimal CD4+ and CD8+ T cell responses, and the phagocytosis of heat-killed or invasion-blocked parasites was not sufficient to induce T cell responses. Rather, the selective transfer of cpsII-infected dendritic cells or macrophages (but not those that had phagocytosed the parasite) to naïve mice potently induced CD4+ and CD8+ T cell responses, and conferred protection against challenge with virulent T. gondii. Collectively, these results point toward a critical role for actively infected host cells in initiating T. gondii-specific CD4+ and CD8+ T cell responses. PMID:24722202

Peptides containing antigenic and cationic domains have enhanced, multivalent immunogenicity when bound to DNA vaccines.

PubMed

Riedl, Petra; Reimann, Jörg; Schirmbeck, Reinhold

2004-02-01

We explored strategies to codeliver DNA- and peptide-based vaccines in a way that enhances the immunogenicity of both components of the combination vaccine for T cells. Specific CD8(+) T cell responses to an antigenic peptide are primed when the peptide is fused to a cationic peptide domain that is bound to plasmid DNA or oligonucleotides (ODN; with or without CpG motifs). Plasmid DNA mixed with antigenic/cationic peptides or histones forms large complexes with different biological properties depending on the molar ratios of peptide/protein and polynucleotide. Complexes containing high (but not low) molar ratios of cationic peptide to DNA facilitate transfection (DNA uptake and expression of the plasmid-encoded product) of cells. In contrast, complexes containing low (but not high) molar ratios of cationic peptide to DNA prime potent multispecific T cell responses after a single intramuscular injection of the complexes. The general validity of this observation was confirmed mixing different antigenic/cationic peptides with different DNA vaccines. In these vaccine formulations, multispecific CD8(+) T cell responses specific for epitopes of the peptide- as well as the DNA-based vaccine were efficiently coprimed, together with humoral antibody responses to conformational determinants of large viral antigens encoded by the DNA vaccine. The data indicate that mixtures of DNA vaccines with antigenic, cationic peptides are immunogenic vaccine formulations particularly suited for the induction of multispecific T cell responses.

Immunopathology of autoantibody-associated encephalitides: clues for pathogenesis.

PubMed

Bien, Christian G; Vincent, Angela; Barnett, Michael H; Becker, Albert J; Blümcke, Ingmar; Graus, Francesc; Jellinger, Kurt A; Reuss, David E; Ribalta, Teresa; Schlegel, Jürgen; Sutton, Ian; Lassmann, Hans; Bauer, Jan

2012-05-01

Classical paraneoplastic encephalitis syndromes with 'onconeural' antibodies directed to intracellular antigens, and the recently described paraneoplastic or non-paraneoplastic encephalitides and antibodies against both neural surface antigens (voltage-gated potassium channel-complexes, N-methyl-d-aspartate receptors) and intracellular antigens (glutamic acid decarboxylase-65), constitute an increasingly recognized group of immune-mediated brain diseases. Evidence for specific immune mechanisms, however, is scarce. Here, we report qualitative and quantitative immunopathology in brain tissue (biopsy or autopsy material) of 17 cases with encephalitis and antibodies to either intracellular (Hu, Ma2, glutamic acid decarboxylase) or surface antigenic targets (voltage-gated potassium channel-complex or N-methyl-d-aspartate receptors). We hypothesized that the encephalitides with antibodies against intracellular antigens (intracellular antigen-onconeural and intracellular antigen-glutamic acid decarboxylase groups) would show neurodegeneration mediated by T cell cytotoxicity and the encephalitides with antibodies against surface antigens would be antibody-mediated and would show less T cell involvement. We found a higher CD8/CD3 ratio and more frequent appositions of granzyme-B(+) cytotoxic T cells to neurons, with associated neuronal loss, in the intracellular antigen-onconeural group (anti-Hu and anti-Ma2 cases) compared to the patients with surface antigens (anti-N-methyl-d-aspartate receptors and anti-voltage-gated potassium channel complex cases). One of the glutamic acid decarboxylase antibody encephalitis cases (intracellular antigen-glutamic acid decarboxylase group) showed multiple appositions of GrB-positive T cells to neurons. Generally, however, the glutamic acid decarboxylase antibody cases showed less intense inflammation and also had relatively low CD8/CD3 ratios compared with the intracellular antigen-onconeural cases. Conversely, we found complement C9neo deposition on neurons associated with acute neuronal cell death in the surface antigen group only, specifically in the voltage-gated potassium channel-complex antibody patients. N-methyl-d-aspartate receptors-antibody cases showed no evidence of antibody and complement-mediated tissue injury and were distinguished from all other encephalitides by the absence of clear neuronal pathology and a low density of inflammatory cells. Although tissue samples varied in location and in the stage of disease, our findings strongly support a central role for T cell-mediated neuronal cytotoxicity in encephalitides with antibodies against intracellular antigens. In voltage-gated potassium channel-complex encephalitis, a subset of the surface antigen antibody encephalitides, an antibody- and complement-mediated immune response appears to be responsible for neuronal loss and cerebral atrophy; the apparent absence of these mechanisms in N-methyl-d-aspartate receptors antibody encephalitis is intriguing and requires further study.

Activated STAT5 promotes long-lived cytotoxic CD8+ T cells that induce regression of autochthonous melanoma.

PubMed

Grange, Magali; Buferne, Michel; Verdeil, Grégory; Leserman, Lee; Schmitt-Verhulst, Anne-Marie; Auphan-Anezin, Nathalie

2012-01-01

Immunotherapy based on adoptive transfer of tumor antigen-specific CD8(+) T cell (TC) is generally limited by poor in vivo expansion and tumor infiltration. In this study, we report that activated STAT5 transcription factors (STAT5CA) confer high efficiency on CD8(+) effector T cells (eTC) for host colonization after adoptive transfer. Engineered expression of STAT5CA in antigen-experienced TCs with poor replicative potential was also sufficient to convert them into long-lived antigen-responsive eTCs. In transplanted mastocytoma- or melanoma-bearing hosts, STAT5CA greatly enhanced the ability of eTCs to accumulate in tumors, become activated by tumor antigens, and to express the cytolytic factor granzyme B. Taken together, these properties contributed to an increase in tumor regression by STAT5CA-transduced, as compared with untransduced, TCs including when the latter control cells were combined with infusion of interleukin (IL)-2/anti-IL-2 complexes. In tumors arising in the autochthonous TiRP transgenic model of melanoma associated with systemic chronic inflammation, endogenous CD8(+) TCs were nonfunctional. In this setting, adoptive transfer of STAT5CA-transduced TCs produced superior antitumor effects compared with nontransduced TCs. Our findings imply that STAT5CA expression can render TCs resistant to the immunosuppressive environment of melanoma tumors, enhancing their ability to home to tumors and to maintain high granzyme B expression, as well as their capacity to stimulate granzyme B expression in endogenous TCs. ©2011 AACR.

MyD88-dependent expansion of an immature GR-1+CD11b+ population induces T cell suppression and Th2 polarization in sepsis

PubMed Central

Delano, Matthew J.; Scumpia, Philip O.; Weinstein, Jason S.; Coco, Dominique; Nagaraj, Srinivas; Kelly-Scumpia, Kindra M.; O'Malley, Kerri A.; Wynn, James L.; Antonenko, Svetlana; Al-Quran, Samer Z.; Swan, Ryan; Chung, Chun-Shiang; Atkinson, Mark A.; Ramphal, Reuben; Gabrilovich, Dmitry I.; Reeves, Wesley H.; Ayala, Alfred; Phillips, Joseph; LaFace, Drake; Heyworth, Paul G.; Clare-Salzler, Michael; Moldawer, Lyle L.

2007-01-01

Polymicrobial sepsis alters the adaptive immune response and induces T cell suppression and Th2 immune polarization. We identify a GR-1+CD11b+ population whose numbers dramatically increase and remain elevated in the spleen, lymph nodes, and bone marrow during polymicrobial sepsis. Phenotypically, these cells are heterogeneous, immature, predominantly myeloid progenitors that express interleukin 10 and several other cytokines and chemokines. Splenic GR-1+ cells effectively suppress antigen-specific CD8+ T cell interferon (IFN) γ production but only modestly suppress antigen-specific and nonspecific CD4+ T cell proliferation. GR-1+ cell depletion in vivo prevents both the sepsis-induced augmentation of Th2 cell–dependent and depression of Th1 cell–dependent antibody production. Signaling through MyD88, but not Toll-like receptor 4, TIR domain–containing adaptor-inducing IFN-β, or the IFN-α/β receptor, is required for complete GR-1+CD11b+ expansion. GR-1+CD11b+ cells contribute to sepsis-induced T cell suppression and preferential Th2 polarization. PMID:17548519

CD73-deficient mice have increased antitumor immunity and are resistant to experimental metastasis.

PubMed

Stagg, John; Divisekera, Upulie; Duret, Helene; Sparwasser, Tim; Teng, Michele W L; Darcy, Phillip K; Smyth, Mark J

2011-04-15

CD73 is a cell-surface enzyme that suppresses immune responses by producing extracellular adenosine. In this study, we employed CD73 gene-targeted mice to investigate the role of host-derived CD73 on antitumor immunity and tumor cell metastasis. We found that CD73 ablation significantly suppressed the growth of ovalbumin-expressing MC38 colon cancer, EG7 lymphoma, AT-3 mammary tumors, and B16F10 melanoma. The protective effect of CD73 deficiency on primary tumors was dependent on CD8(+) T cells and associated with an increased frequency of antigen-specific CD8(+) T cells in peripheral blood and tumors and increased antigen-specific IFN-γ production. Replicate studies in bone marrow chimeras established that both hematopoietic and nonhematopoietic expression of CD73 was important to promote tumor immune escape. Using adoptive reconstitution of T regulatory cell (Treg)-depleted DEREG (depletion of regulatory T cells) mice, we demonstrated that part of the protumorigenic effect of Tregs was dependent on their expression of CD73. CD73-deficient mice were also protected against pulmonary metastasis of B16F10 melanoma cells after intravenous injection. Unexpectedly, we found that the prometastatic effect of host-derived CD73 was dependent on CD73 expression on nonhematopoietic cells. CD73 expression on nonhematopoietic cells, most likely endothelial cells, was critical for promoting lung metastasis in a manner independent from immunosuppressive effects. Notably, in vivo blockade of CD73 with a selective inhibitor or anti-CD73 monoclonal antibody significantly reduced tumor growth and metastasis of CD73-negative tumors. Taken together, our findings indicate that CD73 may be targeted at multiple levels to induce anticancer effects including at the level of tumor cells, Tregs, and nonhematopoietic cells. ©2011 AACR.

Reducing persistent polyomavirus infection increases functionality of virus-specific memory CD8 T cells.

PubMed

Qin, Qingsong; Lauver, Matthew; Maru, Saumya; Lin, Eugene; Lukacher, Aron E

2017-02-01

Mouse polyomavirus (MuPyV) causes a smoldering persistent infection in immunocompetent mice. To lower MuPyV infection in acutely and persistently infected mice, and study the impact of a temporal reduction in viral loads on the memory CD8 T cell response, we created a recombinant MuPyV in which a loxP sequence was inserted into the A2 strain genome upstream of the early promoter and another loxP sequence was inserted in cis into the intron shared by all three T antigens. Using mice transgenic for tamoxifen-inducible Cre recombinase, we demonstrated that reduction in MuPyV load during persistent infection was associated with differentiation of virus-specific CD8 T cells having a superior recall response. Evidence presented here supports the concept that reduction in viral load during persistent infection can promote differentiation of protective virus-specific memory CD8 T cells in patients at risk for diseases caused by human polyomaviruses. Copyright © 2016 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takahashi, Takeshi, E-mail: takeshi-takahashi@ciea.or.jp; Katano, Ikumi; Ito, Ryoji

Highlights: • β-Lactoglobulin (BLG) specific TCR genes were introduced to human HSC by retrovirus. • Human HSC with BLG-specific TCR were transplanted into NOG-HLA-DR4 I-A{sup −/−} mice. • BLG-specific TCR induced positive selection of thymocytes. • BLG-specific TCR positive CD4{sup +} T cells mediated immune responses in humanized mice. - Abstract: The development of severe immunodeficient mouse strains containing various human genes, including cytokines or HLA, has enabled the reconstitution of functional human immune systems after transplantation of human hematopoietic stem cells (HSC). Accumulating evidence has suggested that HLA-restricted antigen-specific human T-cell responses can be generated in these humanized mice.more » To directly monitor immune responses of human CD4{sup +} T cells, we introduced β-lactoglobulin (BLG)-specific T cell receptor (TCR) genes derived from CD4{sup +} T-cell clones of cow-milk allergy patients into HSCs, and subsequently transplanted them into NOG-HLA-DR4 transgenic/I-Aβ deficient mice (NOG-DR4/I-A{sup o}). In the thymus, thymocytes with BLG-specific TCR preferentially differentiated into CD4{sup +}CD8{sup −} single-positive cells. Adoptive transfer of mature CD4{sup +} T cells expressing the TCR into recipient NOG-DR4/I-A{sup o} mice demonstrated that human CD4{sup +} T cells proliferated in response to antigenic stimulation and produced IFN-γ in vivo, suggesting that functional T-cell reactions (especially Th1-skewed responses) were induced in humanized mice.« less

The effect of IL-18 on IL-12-induced CD30 expression and IL-4 and IFN-γ production by allergen and PPD specific T cells

PubMed Central

Tarkowski, M; Chrul, S; Bodalski, J

2002-01-01

CD30 is expressed on activated T cells that, as has been suggested, preferentially produce IFN-γ. Interleukin 12 increases antigen-induced CD30 expression on T cells and IFN-γ production. Synthesis of IFN-γ can be augmented further by IL-18. The aim of our study was to investigate whether IL-18 affects the IL-12 induced CD30 expression and cytokine production by allergen or PPD specific T cells. Mononuclear cells of healthy or atopic volunteers were stimulated with PPD or allergen, respectively, to obtain specific T cell lines. T cells were restimulated with appropriate antigen and antigen-presenting cells in the presence of IL-12, IL-18 or a combination of these cytokines. After 3 days, expression of CD30 was investigated on CD4 and CD8 T cells and IFN-γ and IL-4 cytokine production was estimated in the culture supernatants. Flow cytometric analyses showed no effect of IL-18 on CD30 expression during IL-12 co-stimulation. At the same time after the optimal stimulation for CD30 expression, the levels of IFN-γ were high in PPD-stimulated cell lines but have not been up-regulated by IL-18. IFN-γ levels were much lower in allergen-stimulated T cells and although they were up-regulated by IL-12 there was no additional or synergistic effect from IL-18. IL-18, however, increased production of IL-4 in allergen-stimulated cell lines. Our studies provide new information about IL-18 activity on human cells and question its exclusive role in Th1 mediated responses. PMID:11882036

Naturally occurring tolerance acquisition to foods in previously allergic children is characterized by antigen specificity and associated with increased subsets of regulatory T cells.

PubMed

Qamar, N; Fishbein, A B; Erickson, K A; Cai, M; Szychlinski, C; Bryce, P J; Schleimer, R P; Fuleihan, R L; Singh, A M

2015-11-01

Food allergy affects approximately 6-8% of children, and increasing in prevalence. Some children naturally outgrow their food allergy without intervention, but the mechanisms by which this occurs remain poorly understood. We sought to investigate the role of regulatory T cells in the development of naturally acquired tolerance. Fifty-eight children (1-18 years) with either egg or peanut allergy, recent acquisition of natural tolerance to egg or peanut, or no food allergy were studied. Peripheral blood mononuclear cells (PBMC) from these groups were stimulated with relevant antigen for 48 h and flow cytometry performed to characterize both surface (CD3, CD4, CD25, CD14, CD19, and CD127) and intracellular markers (IL-10, Foxp3, and IL-5). Resting PBMC from naturally tolerant patients had significantly increased CD3+CD4+CD25+CD127loFoxp3+ cells, when compared to allergic or control patients (mean 6.36 vs. 2.37 vs. 2.62%, respectively, P < 0.05). Upon stimulation with relevant antigen, naturally tolerant patients also had increased IL-10-expressing CD25+CD127lo cells (6.33 vs. 1.65 vs. 0.7, P < 0.01), Foxp3+ cells (mean 12.6 vs. 5.42 vs. 3%, P < 0.01), and CD4+ cells (mean 4.48 vs. 1.59 vs. 0.87%, P < 0.01); the increase was not observed in PBMCs from allergic or control patients. Additionally, this upregulation was only seen with relevant antigen stimulation and not upon stimulation with unrelated antigen. The increased CD3+CD4+CD25+CD127lo cells at baseline and upon stimulation and increased induction of IL-10-producing cells of several types, including Tr1 cells, from naturally tolerant patients suggests an important role for regulatory T cell subsets in the acquisition of natural tolerance. © 2015 John Wiley & Sons Ltd.

Naturally Occurring Tolerance Acquisition to Foods in Previously Allergic Children is Characterized by Antigen Specificity and Associated with Increased Subsets of Regulatory T cells

PubMed Central

Qamar, Nashmia; Fishbein, Anna B.; Erickson, Kristin A.; Cai, Miao; Szychlinski, Christine; Bryce, Paul J.; Schleimer, Robert P.; Fuleihan, Ramsay L.; Singh, Anne Marie

2015-01-01

Background Food allergy affects approximately 6–8% of children, and increasing in prevalence. Some children naturally outgrow their food allergy without intervention but the mechanisms by which this occurs remain poorly understood. We sought to investigate the role of regulatory T cells in the development of naturally acquired tolerance. Methods Fifty-eight children (1 to 18 years) with either egg or peanut allergy, recent acquisition of natural tolerance to egg or peanut, or no food allergy were studied. Peripheral blood mononuclear cells (PBMC) from these groups were stimulated with relevant antigen for 48 hours and flow cytometry performed to characterize both surface (CD3, CD4, CD25, CD14, CD19, CD127) and intracellular markers (IL-10, Foxp3, and IL-5). Results Resting PBMC from naturally tolerant patients had significantly increased CD3+CD4+CD25+CD127loFoxp3+ cells, when compared to allergic or control patients [mean 6.36 vs 2.37 vs 2.62%, respectively, p<0.05]. Upon stimulation with relevant antigen, naturally tolerant patients also had increased IL-10-expressing CD25+CD127lo cells [6.33 vs 1.65 vs 0.7, p<0.01], Foxp3+ cells [mean 12.6 vs 5.42 vs 3%, p<0.01] and CD4+ cells [mean 4.48 vs 1.59 vs 0.87%, p<0.01]; the increase was not observed in PBMCs from allergic or control patients. Additionally, this upregulation was only seen with relevant antigen stimulation and not upon stimulation with unrelated antigen. Conclusion The increased CD3+CD4+CD25+CD127lo cells at baseline and upon stimulation and increased induction of IL-10-producing cells of several types, including Tr1 cells, from naturally tolerant patients suggests an important role for regulatory T cell subsets in the acquisition of natural tolerance. PMID:25989379

Virological outcome after structured interruption of antiretroviral therapy for human immunodeficiency virus infection is associated with the functional profile of virus-specific CD8+ T cells.

PubMed

Daucher, Marybeth; Price, David A; Brenchley, Jason M; Lamoreaux, Laurie; Metcalf, Julia A; Rehm, Catherine; Nies-Kraske, Elizabeth; Urban, Elizabeth; Yoder, Christian; Rock, Diane; Gumkowski, Julie; Betts, Michael R; Dybul, Mark R; Douek, Daniel C

2008-04-01

A clear understanding of the antiviral effects of CD8(+) T cells in the context of chronic human immunodeficiency virus (HIV) infection is critical for the development of prophylactic vaccines and therapeutics designed to support T-cell-mediated immunity. However, defining the potential correlates of effective CD8(+) T-cell immunity has proven difficult; notably, comprehensive analyses have demonstrated that the size and shape of the CD8(+) T-cell response are not necessarily indicative of efficacy determined by measures of plasma viral load. Here, we conducted a detailed quantitative and qualitative analysis of CD8(+) T-cell responses to autologous virus in a cohort of six HIV-infected individuals with a history of structured interruption of antiretroviral therapy (ART) (SIT). The magnitude and breadth of the HIV-specific response did not, by themselves, explain the changes observed in plasma virus levels after the cessation of ART. Furthermore, mutational escape from targeted epitopes could not account for the differential virological outcomes in this cohort. However, the functionality of HIV-specific CD8(+) T-cell populations upon antigen encounter, determined by the simultaneous and independent measurement of five CD8(+) T-cell functions (degranulation and gamma interferon, macrophage inflammatory protein 1beta, tumor necrosis factor alpha, and interleukin-2 levels) reflected the emergent level of plasma virus, with multiple functions being elicited in those individuals with lower levels of viremia after SIT. These data show that the quality of the HIV-specific CD8(+) T-cell response, rather than the quantity, is associated with the dynamics of viral replication in the absence of ART and suggest that the effects of SIT can be assessed by measuring the functional profile of HIV-specific CD8(+) T cells.

[Some aspects of immune response in toxoplasmosis].

PubMed

Dziubek, Z; Zarnowska, H; Basiak, W; Górski, A; Kajfasz, P

2001-01-01

The study was performed to determine cell-mediated and humoral responses related to the clinical course of disease in adults with different forms of toxoplasmosis. The cellular response was estimated by blastogenic transformation of T-lymphocytes induced by OKT3 mitogenic stimulus, as well as by a specific soluble toxoplasmic antigen. The phenotype analysis of T-lymphocytes was performed using monoclonal antibody on FACS flow cytometer. The humoral response was examined by determining the levels of toxoplasma-specific IgA, IgM and IgG antibodies and total serum immunoglobulins. Significant disruption of the cell-mediated response was not observed (T-lymphocyte proliferation test). In the group of patients with lymphadenopathy, an increased proportion of CD8+ lymphocytes was only noted in the first month of the disease, although enlargement of the cervical lymph nodes was observed for up to a year. The individuals in question were found to have IgA antibodies against the p30 antigen of T. gondii, mainly in the period of disease up to 7 months. The proportion of CD4+ lymphocytes was sub-normal in all patients of this group who were studied in the first two months of the disease, and in most of those studied later. Three out of 14 patients with the ocular form of toxoplasmosis only had recent foci of inflammation in the retina, and were also found to have specific IgM. These patients had depressed CD4+ levels, and showed no reaction from CD8+ lymphocytes. Sub-normal CD4+ population could have been the cause of the weak stimulation of CD8+ cells and an expression of this is the long course of disease. Sub-normal CD4+ cells may be also results the suppressing effect of the parasite.

Ethnic and socio-cultural specificities in Tunisia have no impact on the prevalence of anti-Saccharomyces cerevisiae antibodies in Crohn's disease patients, their relatives or associated clinical factors.

PubMed

Hadrich, Ines; Vandewalle, Peggy; Cheikhrouhou, Fatma; Makni, Fattouma; Krichen, Mohamed Salah; Sendid, Boualem; Standaert-Vitse, Annie; Ayadi, Ali; Poulain, Daniel

2007-06-01

In Western Europe and the USA, the presence of anti-Saccharomyces cerevisiae antibodies (ASCAs) in Crohn's disease (CD) patients and their healthy relatives suggests that ASCAs may be influenced by genetic and/or environmental factors. To assess the prevalence of ASCAs in Tunisian patients with CD or ulcerative colitis (UC), and unaffected family members, in relation to clinical phenotype. Patients and methods. Seventy-seven patients (39 CD, 38 UC), 66 healthy relatives of CD patients, 16 relatives of UC patients and 70 healthy controls were studied. ASCAs were quantified with a new isotype-specific ELISA test involving an antigenic extract from S. cerevisiae strain W303 and by the original test which detects total immunoglobulins against S. cerevisiae Su1 mannan. The specificity of the two tests was identical (91%). The isotype-specific ASCA W303 test was more sensitive than the ASCA Su1 test for immunoglobulin detection, but some CD patients were positive only with this latter test. A high percentage of patients with CD (72%) and their unaffected family members (35%) were ASCA-positive in contrast to UC patients (16%) and their relatives (0%) and controls (8.6%). ASCAs were shown to be independent of rural or urban living, disease activity, but were associated with ileal location. The antigen of S. cerevisiae strain W303 discriminated patients depending on age at onset or location of the disease. This study confirms the antigenic heterogeneity of S. cerevisiae strains in their ability to detect ASCA. It suggests that ASCAs are markers of immunoregulatory disturbance in CD, independently of ethnic/cultural differences between Europe, the USA and North Africa.

The Fas/CD95 Receptor Regulates the Death of Autoreactive B Cells and the Selection of Antigen-Specific B Cells

PubMed Central

Koncz, Gabor; Hueber, Anne-Odile

2012-01-01

Cell death receptors have crucial roles in the regulation of immune responses. Here we review recent in vivo data confirming that the Fas death receptor (TNFSR6) on B cells is important for the regulation of autoimmunity since the impairment of only Fas function on B cells results in uncontrolled autoantibody production and autoimmunity. Fas plays a role in the elimination of the non-specific and autoreactive B cells in germinal center, while during the selection of antigen-specific B cells different escape signals ensure the resistance to Fas-mediated apoptosis. Antigen-specific survival such as BCR or MHCII signal or coreceptors (CD19) cooperating with BCR inhibits the formation of death inducing signaling complex. Antigen-specific survival can be reinforced by antigen-independent signals of IL-4 or CD40 overproducing the anti-apoptotic members of the Bcl-2 family proteins. PMID:22848207

N-ras couples antigen receptor signaling to Eomesodermin and to functional CD8+ T cell memory but not to effector differentiation

PubMed Central

Iborra, Salvador; Ramos, Manuel; Arana, David M.; Lázaro, Silvia; Aguilar, Francisco; Santos, Eugenio; López, Daniel

2013-01-01

Signals from the TCR that specifically contribute to effector versus memory CD8+ T cell differentiation are poorly understood. Using mice and adoptively transferred T lymphocytes lacking the small GTPase N-ras, we found that N-ras–deficient CD8+ T cells differentiate efficiently into antiviral primary effectors but have a severe defect in generating protective memory cells. This defect was rescued, although only partly, by rapamycin-mediated inhibition of mammalian target of rapamycin (mTOR) in vivo. The memory defect correlated with a marked impairment in vitro and in vivo of the antigen-mediated early induction of T-box transcription factor Eomesodermin (Eomes), whereas T-bet was unaffected. Besides N-ras, early Eomes induction in vitro required phosphoinositide 3-kinase (PI3K)–AKT but not extracellular signal-regulated kinase (ERK) activation, and it was largely insensitive to rapamycin. Consistent with N-ras coupling Eomes to T cell memory, retrovirally enforced expression of Eomes in N-ras–deficient CD8+ T cells effectively rescued their memory differentiation. Thus, our study identifies a critical role for N-ras as a TCR-proximal regulator of Eomes for early determination of the CD8+ T cell memory fate. PMID:23776078

Detection of ABCB5 tumour antigen-specific CD8+ T cells in melanoma patients and implications for immunotherapy.

PubMed

Borchers, S; Maβlo, C; Müller, C A; Tahedl, A; Volkind, J; Nowak, Y; Umansky, V; Esterlechner, J; Frank, M H; Ganss, C; Kluth, M A; Utikal, J

2018-01-01

ATP binding cassette subfamily B member 5 (ABCB5) has been identified as a tumour-initiating cell marker and is expressed in various malignancies, including melanoma. Moreover, treatment with anti-ABCB5 monoclonal antibodies has been shown to inhibit tumour growth in xenotransplantation models. Therefore, ABCB5 represents a potential target for cancer immunotherapy. However, cellular immune responses against ABCB5 in humans have not been described so far. Here, we investigated whether ABCB5-reactive T cells are present in human melanoma patients and tested the applicability of ABCB5-derived peptides for experimental induction of human T cell responses. Peripheral blood mononuclear cells (PBMNC) isolated from blood samples of melanoma patients (n = 40) were stimulated with ABCB5 peptides, followed by intracellular cytokine staining (ICS) for interferon (IFN)-γ and tumour necrosis factor (TNF)-α. To evaluate immunogenicity of ABCB5 peptides in naive healthy donors, CD8 T cells were co-cultured with ABCB5 antigen-loaded autologous dendritic cells (DC). ABCB5 reactivity in expanded T cells was assessed similarly by ICS. ABCB5-reactive CD8 + T cells were detected ex vivo in 19 of 29 patients, melanoma antigen recognised by T cells (MART-1)-reactive CD8 + T cells in six of 21 patients. In this small, heterogeneous cohort, reactivity against ABCB5 was significantly higher than against MART-1. It occurred significantly more often and independently of clinical characteristics. Reactivity against ABCB5 could be induced in 14 of 16 healthy donors in vitro by repeated stimulation with peptide-loaded autologous DC. As ABCB5-reactive CD8 T cells can be found in the peripheral blood of melanoma patients and an ABCB5-specific response can be induced in vitro in naive donors, ABCB5 could be a new target for immunotherapies in melanoma. © 2017 British Society for Immunology.

BK virus-specific T cells for use in cellular therapy show specificity to multiple antigens and polyfunctional cytokine responses.

PubMed

Blyth, Emily; Clancy, Leighton; Simms, Renee; Gaundar, Shivashni; O'Connell, Philip; Micklethwaite, Kenneth; Gottlieb, David J

2011-11-27

BK virus (BKV) infection causes hemorrhagic cystitis posthemopoietic stem-cell transplant and graft loss in renal transplant recipients. Reactivation occurs in up to 60% of patients in both groups. Treatment-related cellular immunosuppression is a major contributor to the development of BKV-related disease, but the targets of the immune response are not well characterized. Immunotherapy by adoptive transfer of cellular effectors has been shown to be effective in controlling and preventing some virus-related diseases in transplant recipients, particularly Epstein-Barr virus and cytomegalovirus. Infusion of BKV-specific T cells may potentially reconstitute functional BKV immunity and reduce clinical complications of BKV infection. BKV-specific T cells for clinical use in adoptive immunotherapy were generated using monocyte-derived dendritic cells pulsed with overlapping peptide mixes spanning the five BKV proteins VP1, VP2, VP3, large T antigen, and small T antigen. Phenotypic and functional characteristics of the cells were investigated as well as their antigen specificity. Expanded CD4(+) and CD8(+) cells responded to restimulation with BKV peptides principally from VP1, large T, or small T antigens; produced multiple cytokines; and showed cytotoxic activity against antigen-coated targets. Possible clinical uses for BKV-specific T cells generated using this method include immune reconstitution posthemopoietic stem-cell transplantation or prophylaxis and treatment of immune deficiency in renal transplant recipients, fulfilling the need for effective therapy for BKV-related hemorrhagic cystitis and renal dysfunction.

Probing the Effector and Suppressive Functions of Human T Cell Subsets Using Antigen-Specific Engineered T Cell Receptors

PubMed Central

Imberg, Keren; Mercer, Frances; Zhong, Shi; Krogsgaard, Michelle; Unutmaz, Derya

2013-01-01

Activation of T cells through the engagement of the T cell receptors (TCRs) with specific peptide-MHC complexes on antigen presenting cells (APCs) is the major determinant for their proliferation, differentiation and display of effector functions. To assess the role of quantity and quality of peptide-MHC presentation in eliciting T cell activation and suppression functions, we genetically engineered human T cells with two TCRs that recognize HLA-A*0201-restricted peptides derived from either HIV or melanoma antigens. The engineered-TCRs are highly functional in both CD8+ and CD4+ T cells as assessed by the upregulation of activation markers, induction of cytokine secretion and cytotoxicity. We further demonstrated that engineered-TCRs can also be expressed on naïve human T cells, which are stimulated through APCs presenting specific peptides to induce T cell proliferation and acquire effector functions. Furthermore, regulatory T cells (Tregs) ectopically expressing the engineered-TCRs are activated in an antigen-specific fashion and suppress T cell proliferation. In this system, the inhibitory activity of peptide-stimulated Tregs require the presence of dendritic cells (DCs) in the culture, either as presenters or as bystander cells, pointing to a critical role for DCs in suppression by Tregs. In conclusion, the engineered-TCR system reported here advances our ability to understand the differentiation pathways of naïve T cells into antigen-specific effector cells and the role of antigen-specific signaling in Treg-mediated immune suppression. PMID:23437112

CD40 inhibits replication of hepatitis C virus in primary human hepatocytes by c-Jun N terminal kinase activation independent from the interferon pathway.

PubMed

Rau, Sibylle J; Hildt, Eberhard; Himmelsbach, Kiyoshi; Thimme, Robert; Wakita, Takaji; Blum, Hubert E; Fischer, Richard

2013-01-01

CD40, a member of the tumor necrosis factor receptor family, and its ligand, CD40L (CD154), are important regulators of the antiviral immune response. CD40L is up-regulated on lymphocytes and CD40 on hepatocytes during infection with hepatitis C virus (HCV); we investigated the role of CD40 signaling during HCV replication in hepatocytes. Viral replication was studied in primary human hepatocytes (PHH) and Huh7.5 cells using the infectious HCV Japanese fulminate hepatitis 1 isolate (JFH1) culture system, and in coculture with HCV antigen-specific CD8+ T cells. CD40L rapidly and transiently inhibits expression of the HCV nonstructural proteins NS3 and NS5A as well as HCV structural proteins core and E2 in Huh7.5 cells. Similarly, CD40L prevented replication of HCV in PHH, in synergy with interferon (IFN)-alpha. In Huh7.5 cells with replicating HCV, CD40L prevented production of infectious viral particles. When HCV antigen-specific CD8+ T cells were cocultured with HLA-A2-expressing Huh7 cells that had replicating virus, the T cells became activated, up-regulated CD40L, and inhibited HCV replication. Inhibition of CD40L partially prevented the antiviral activity of the CD8+ T cells. The antiviral effect of CD40L required activation of c-Jun N terminal kinases (JNK)1/2, but not induction of apoptosis or the JAK/STAT pathway that is necessary for the antiviral effects of IFNs. CD40 inhibits HCV replication by a novel, innate immune mechanism. This pathway might mediate viral clearance, and disruptions might be involved in the pathogenesis of HCV infection. Copyright © 2012 American Association for the Study of Liver Diseases.

Prophylactic Herpes Simplex Virus 2 (HSV-2) Vaccines Adjuvanted with Stable Emulsion and Toll-Like Receptor 9 Agonist Induce a Robust HSV-2-Specific Cell-Mediated Immune Response, Protect against Symptomatic Disease, and Reduce the Latent Viral Reservoir

PubMed Central

Marshall, Jason D.; Dorwart, Michael R.; Heeke, Darren S.; Rao, Eileen; Tummala, Padmaja; Yu, Li; Cohen, Gary H.; Eisenberg, Roselyn J.

2017-01-01

ABSTRACT Several prophylactic vaccines targeting herpes simplex virus 2 (HSV-2) have failed in the clinic to demonstrate sustained depression of viral shedding or protection from recurrences. Although these vaccines have generated high titers of neutralizing antibodies (NAbs), their induction of robust CD8 T cells has largely been unreported, even though evidence for the importance of HSV-2 antigen-specific CD8 T cells is mounting in animal models and in translational studies involving subjects with active HSV-2-specific immune responses. We developed a subunit vaccine composed of the NAb targets gD and gB and the novel T cell antigen and tegument protein UL40, and we compared this vaccine to a whole-inactivated-virus vaccine (formaldehyde-inactivated HSV-2 [FI-HSV-2]). We evaluated different formulations in combination with several Th1-inducing Toll-like receptor (TLR) agonists in vivo. In mice, the TLR9 agonist cytosine-phosphate-guanine (CpG) oligodeoxynucleotide formulated in a squalene-based oil-in-water emulsion promoted most robust, functional HSV-2 antigen-specific CD8 T cell responses and high titers of neutralizing antibodies, demonstrating its superiority to vaccines adjuvanted by monophosphoryl lipid A (MPL)-alum. We further established that FI-HSV-2 alone or in combination with adjuvants as well as adjuvanted subunit vaccines were successful in the induction of NAbs and T cell responses in guinea pigs. These immunological responses were coincident with a suppression of vaginal HSV-2 shedding, low lesion scores, and a reduction in latent HSV-2 DNA in dorsal root ganglia to undetectable levels. These data support the further preclinical and clinical development of prophylactic HSV-2 vaccines that contain appropriate antigen and adjuvant components responsible for programming elevated CD8 T cell responses. IMPORTANCE Millions of people worldwide are infected with herpes simplex virus 2 (HSV-2), and to date, an efficacious prophylactic vaccine has not met the rigors of clinical trials. Attempts to develop a vaccine have focused primarily on glycoproteins necessary for HSV-2 entry as target antigens and to which the dominant neutralizing antibody response is directed during natural infection. Individuals with asymptomatic infection have exhibited T cell responses against specific HSV-2 antigens not observed in symptomatic individuals. We describe for the first time the immunogenicity profile in animal models of UL40, a novel HSV-2 T cell antigen that has been correlated with asymptomatic HSV-2 disease. Additionally, vaccine candidates adjuvanted by a robust formulation of the CpG oligonucleotide delivered in emulsion were superior to unadjuvanted or MPL-alum-adjuvanted formulations at eliciting a robust cell-mediated immune response and blocking the establishment of a latent viral reservoir in the guinea pig challenge model of HSV-2 infection. PMID:28228587

Prophylactic Herpes Simplex Virus 2 (HSV-2) Vaccines Adjuvanted with Stable Emulsion and Toll-Like Receptor 9 Agonist Induce a Robust HSV-2-Specific Cell-Mediated Immune Response, Protect against Symptomatic Disease, and Reduce the Latent Viral Reservoir.

PubMed

Hensel, Michael T; Marshall, Jason D; Dorwart, Michael R; Heeke, Darren S; Rao, Eileen; Tummala, Padmaja; Yu, Li; Cohen, Gary H; Eisenberg, Roselyn J; Sloan, Derek D

2017-05-01

Several prophylactic vaccines targeting herpes simplex virus 2 (HSV-2) have failed in the clinic to demonstrate sustained depression of viral shedding or protection from recurrences. Although these vaccines have generated high titers of neutralizing antibodies (NAbs), their induction of robust CD8 T cells has largely been unreported, even though evidence for the importance of HSV-2 antigen-specific CD8 T cells is mounting in animal models and in translational studies involving subjects with active HSV-2-specific immune responses. We developed a subunit vaccine composed of the NAb targets gD and gB and the novel T cell antigen and tegument protein UL40, and we compared this vaccine to a whole-inactivated-virus vaccine (formaldehyde-inactivated HSV-2 [FI-HSV-2]). We evaluated different formulations in combination with several Th1-inducing Toll-like receptor (TLR) agonists in vivo In mice, the TLR9 agonist cytosine-phosphate-guanine (CpG) oligodeoxynucleotide formulated in a squalene-based oil-in-water emulsion promoted most robust, functional HSV-2 antigen-specific CD8 T cell responses and high titers of neutralizing antibodies, demonstrating its superiority to vaccines adjuvanted by monophosphoryl lipid A (MPL)-alum. We further established that FI-HSV-2 alone or in combination with adjuvants as well as adjuvanted subunit vaccines were successful in the induction of NAbs and T cell responses in guinea pigs. These immunological responses were coincident with a suppression of vaginal HSV-2 shedding, low lesion scores, and a reduction in latent HSV-2 DNA in dorsal root ganglia to undetectable levels. These data support the further preclinical and clinical development of prophylactic HSV-2 vaccines that contain appropriate antigen and adjuvant components responsible for programming elevated CD8 T cell responses. IMPORTANCE Millions of people worldwide are infected with herpes simplex virus 2 (HSV-2), and to date, an efficacious prophylactic vaccine has not met the rigors of clinical trials. Attempts to develop a vaccine have focused primarily on glycoproteins necessary for HSV-2 entry as target antigens and to which the dominant neutralizing antibody response is directed during natural infection. Individuals with asymptomatic infection have exhibited T cell responses against specific HSV-2 antigens not observed in symptomatic individuals. We describe for the first time the immunogenicity profile in animal models of UL40, a novel HSV-2 T cell antigen that has been correlated with asymptomatic HSV-2 disease. Additionally, vaccine candidates adjuvanted by a robust formulation of the CpG oligonucleotide delivered in emulsion were superior to unadjuvanted or MPL-alum-adjuvanted formulations at eliciting a robust cell-mediated immune response and blocking the establishment of a latent viral reservoir in the guinea pig challenge model of HSV-2 infection. Copyright © 2017 American Society for Microbiology.

B cells and TCR avidity determine distinct functions of CD4+ T cells in retroviral infection1

PubMed Central

Ploquin, Mickaël J-Y; Eksmond, Urszula; Kassiotis, George

2011-01-01

The T-cell-dependent B-cell response relies on cognate interaction between B cells and CD4+ Th cells. However, the consequences of this interaction for CD4+ T cells are not entirely known. B cells generally promote CD4+ T-cell responses to pathogens, albeit to a variable degree. In contrast, CD4+ T-cell responses to self or tumor antigens are often suppressed by B cells. Here we demonstrated that interaction with B cells dramatically inhibited the function of virus-specific CD4+ T cells in retroviral infection. We have used Friend virus (FV) infection of mice as a model for retroviral infection, in which the behavior of virus-specific CD4+ T cells was monitored according to their TCR avidity. We report that avidity for antigen and interaction with B cells determine distinct aspects of the primary CD4+ T-cell response to FV infection. Virus-specific CD4+ T cells followed exclusive Th1 and T follicular helper (Tfh) differentiation. High avidity for antigen facilitated expansion during priming and enhanced the capacity for IFN-γ and IL-21 production. In contrast, Tfh differentiation was not affected by avidity for antigen. By reducing or preventing B-cell interaction we found that B cells promoted Tfh differentiation, induced programmed death 1 (PD-1) expression and inhibited IFN-γ production by virus-specific CD4+ T cells. Ultimately, B cells protected hosts from CD4+ T-cell-mediated immune pathology, at the detriment of CD4+ T-cell-mediated protective immunity. Our results suggest that B-cell presentation of vaccine antigens could be manipulated to direct the appropriate CD4+ T-cell response. PMID:21841129

Purification of a polyclonal antibody against CD147 for ELISA using antigen-immunoaffinity chromatography

PubMed Central

Liu, Shuangshuang; Li, Shasha; Zhang, Yang; Wang, Ye; Zhu, Yumeng; Wang, Bin; Chen, Zhi-Nan

2017-01-01

The immunoglobulin superfamily member CD147 is a widely expressed glycoprotein that occurs in both a membrane-spanning and soluble form. Sandwich ELISA is a powerful tool for analyzing soluble antigens. The aim of the present study was to obtain a highly specific polyclonal antibody against human CD147 that can be used for sandwich ELISA analysis. Expression of recombinant CD147 by a eukaryotic expression system was used to immunize rabbits to obtain antiserum. A highly specific polyclonal antibody that was able to detect soluble CD147 in sandwich ELISA was obtained by antigen-immunoaffinity chromatography purification. The purity of rabbit anti-CD147 polyclonal antibodies was ~99%, and ELISA analysis was able to determine the titer of the rabbit anti-CD147 polyclonal antibodies at 1:512,000. The lowest concentration of the standard CD147 antigen that the sandwich ELISA was able to detect was 31.25 pg/ml. The sandwich ELISA system was composed of anti-hepatoma HAb18 monoclonal antibodies and purified rabbit anti-CD147 polyclonal antibodies. The present study demonstrated that antigen-immunoaffinity chromatography may be a good technique for the purification of polyclonal antibodies, which may be used to detect antigen in sandwich ELISAs. PMID:28487989

2B4-SAP signaling is required for the priming of naive CD8+ T cells by antigen-expressing B cells and B lymphoma cells

PubMed Central

2017-01-01

ABSTRACT Mutations in SH2D1A gene that encodes SAP (SLAM-associated protein) result in X-linked lymphoproliferative disease (XLP), a rare primary immunodeficiency disease defined by exquisite sensitivity to the B-lymphotropic Epstein–Barr virus (EBV) and B cell lymphomas. However, the precise mechanism of how the loss of SAP function contributes to extreme vulnerability to EBV and the development of B cell lymphomas remains unclear. Here, we investigate the hypothesis that SAP is critical for CD8+ T cell immune surveillance of antigen (Ag)-expressing B cells or B lymphoma cells under conditions of defined T cell receptor (TCR) signaling. Sh2d1a−/− CD8+ T cells exhibited greatly diminished proliferation relative to wild type when Ag-presenting-B cells or -B lymphoma cells served as the primary Ag-presenting cell (APC). By contrast, Sh2d1a−/− CD8+ T cells responded equivalently to wild-type CD8+ T cells when B cell-depleted splenocytes, melanoma cells or breast carcinoma cells performed Ag presentation. Through application of signaling lymphocyte activation molecule (SLAM) family receptor blocking antibodies or SLAM family receptor-deficient CD8+ T cells and APCs, we found that CD48 engagement on the B cell surface by 2B4 is crucial for initiating SAP-dependent signaling required for the Ag-driven CD8+ T cell proliferation and differentiation. Altogether, a pivotal role for SAP in promoting the expansion and differentiation of B cell-primed viral-specific naive CD8+ T cells may explain the selective immune deficiency of XLP patients to EBV and B cell lymphomas. PMID:28344876

2B4-SAP signaling is required for the priming of naive CD8+ T cells by antigen-expressing B cells and B lymphoma cells.

PubMed

Huang, Yu-Hsuan; Tsai, Kevin; Tan, Sara Y; Kang, Sohyeong; Ford, Mandy L; Harder, Kenneth W; Priatel, John J

2017-01-01

Mutations in SH2D1A gene that encodes SAP (SLAM-associated protein) result in X-linked lymphoproliferative disease (XLP), a rare primary immunodeficiency disease defined by exquisite sensitivity to the B-lymphotropic Epstein-Barr virus (EBV) and B cell lymphomas. However, the precise mechanism of how the loss of SAP function contributes to extreme vulnerability to EBV and the development of B cell lymphomas remains unclear. Here, we investigate the hypothesis that SAP is critical for CD8 + T cell immune surveillance of antigen (Ag)-expressing B cells or B lymphoma cells under conditions of defined T cell receptor (TCR) signaling. Sh2d1a - / - CD8 + T cells exhibited greatly diminished proliferation relative to wild type when Ag-presenting-B cells or -B lymphoma cells served as the primary Ag-presenting cell (APC). By contrast, Sh2d1a - / - CD8 + T cells responded equivalently to wild-type CD8 + T cells when B cell-depleted splenocytes, melanoma cells or breast carcinoma cells performed Ag presentation. Through application of signaling lymphocyte activation molecule (SLAM) family receptor blocking antibodies or SLAM family receptor-deficient CD8 + T cells and APCs, we found that CD48 engagement on the B cell surface by 2B4 is crucial for initiating SAP-dependent signaling required for the Ag-driven CD8 + T cell proliferation and differentiation. Altogether, a pivotal role for SAP in promoting the expansion and differentiation of B cell-primed viral-specific naive CD8 + T cells may explain the selective immune deficiency of XLP patients to EBV and B cell lymphomas.

Lymphocyte and neuronal antigens in neuropsychiatric lupus: presence of an elutable, immunoprecipitable lymphocyte/neuronal 52 kd reactivity.

PubMed Central

Denburg, J A; Behmann, S A

1994-01-01

OBJECTIVE--To examine specific lymphocyte or neuronal antigens immuno-precipitated by systemic lupus erythematosus (SLE) sera. METHOD--SLE sera were screened for the presence of antibodies binding to surface antigens of CD4(+) HUT-78 or SK-N-SH and IMR-6 neuroblastoma cells using Western blotting or radioimmunoprecipitation. RESULTS--IgG eluates from both lymphocytes and neuroblastoma cells recognised a 52 kd band in HUT 78 cell lysates. Eight sera studied further using radioimmunoprecipitation also demonstrated binding to a 52 kd antigen (4/8 on HUT-78, 8/8 on SK-N-SH cells), partially depleted by absorption with viable HUT-78. CONCLUSION--A 52 kd antigen recognised by SLE sera on lymphocytes and neuronal cells may play a role in the pathogenesis of neuropsychiatric-SLE. Images PMID:8017983

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation.

PubMed

Imai, Jun; Otani, Mayu; Sakai, Takahiro; Hatta, Shinichi

2017-08-21

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of naïve CD8 + T cells and memory CD8 + T cells for infectious and tumor immunity but also in the inactivation of self-acting naïve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

Cancer vaccine enhanced, non-tumor-reactive CD8(+) T cells exhibit a distinct molecular program associated with "division arrest anergy".

PubMed

Beyer, Marc; Karbach, Julia; Mallmann, Michael R; Zander, Thomas; Eggle, Daniela; Classen, Sabine; Debey-Pascher, Svenja; Famulok, Michael; Jäger, Elke; Schultze, Joachim L

2009-05-15

Immune-mediated tumor rejection relies on fully functional T-cell responses and neutralization of an adverse tumor microenvironment. In clinical trials, we detected peptide-specific but non-tumor-reactive and therefore not fully functional CD8(+) T cells post-vaccination against tumor antigens. Understanding the molecular mechanisms behind nontumor reactivity will be a prerequisite to overcome this CD8(+) T-cell deviation. We report that these non-tumor-reactive CD8(+) T cells are characterized by a molecular program associated with hallmarks of "division arrest anergy." Non-tumor-reactive CD8(+) T cells are characterized by coexpression of CD7, CD25, and CD69 as well as elevated levels of lck(p505) and p27(kip1). In vivo quantification revealed high prevalence of non-tumor-reactive CD8(+) T cells with increased levels during cancer vaccination. Furthermore, their presence was associated with a trend toward shorter survival. Dynamics and frequencies of non-target-reactive CD8(+) T cells need to be further addressed in context of therapeutic vaccine development in cancer, chronic infections, and autoimmune diseases.

Adenosine regulates CD8 T-cell priming by inhibition of membrane-proximal T-cell receptor signalling

PubMed Central

Linnemann, Carsten; Schildberg, Frank A; Schurich, Anna; Diehl, Linda; Hegenbarth, Silke I; Endl, Elmar; Lacher, Svenja; Müller, Christa E; Frey, Jürgen; Simeoni, Luca; Schraven, Burkhart; Stabenow, Dirk; Knolle, Percy A

2009-01-01

Adenosine is a well-described anti-inflammatory modulator of immune responses within peripheral tissues. Extracellular adenosine accumulates in inflamed and damaged tissues and inhibits the effector functions of various immune cell populations, including CD8 T cells. However, it remains unclear whether extracellular adenosine also regulates the initial activation of naïve CD8 T cells by professional and semi-professional antigen-presenting cells, which determines their differentiation into effector or tolerant CD8 T cells, respectively. We show that adenosine inhibited the initial activation of murine naïve CD8 T cells after αCD3/CD28-mediated stimulation. Adenosine caused inhibition of activation, cytokine production, metabolic activity, proliferation and ultimately effector differentiation of naïve CD8 T cells. Remarkably, adenosine interfered efficiently with CD8 T-cell priming by professional antigen-presenting cells (dendritic cells) and semi-professional antigen-presenting cells (liver sinusoidal endothelial cells). Further analysis of the underlying mechanisms demonstrated that adenosine prevented rapid tyrosine phosphorylation of the key kinase ZAP-70 as well as Akt and ERK1/2 in naïve αCD3/CD28-stimulated CD8 cells. Consequently, αCD3/CD28-induced calcium-influx into CD8 cells was reduced by exposure to adenosine. Our results support the notion that extracellular adenosine controls membrane-proximal T-cell receptor signalling and thereby also differentiation of naïve CD8 T cells. These data raise the possibility that extracellular adenosine has a physiological role in the regulation of CD8 T-cell priming and differentiation in peripheral organs. PMID:19740334

Adenosine regulates CD8 T-cell priming by inhibition of membrane-proximal T-cell receptor signalling.

PubMed

Linnemann, Carsten; Schildberg, Frank A; Schurich, Anna; Diehl, Linda; Hegenbarth, Silke I; Endl, Elmar; Lacher, Svenja; Müller, Christa E; Frey, Jürgen; Simeoni, Luca; Schraven, Burkhart; Stabenow, Dirk; Knolle, Percy A

2009-09-01

Adenosine is a well-described anti-inflammatory modulator of immune responses within peripheral tissues. Extracellular adenosine accumulates in inflamed and damaged tissues and inhibits the effector functions of various immune cell populations, including CD8 T cells. However, it remains unclear whether extracellular adenosine also regulates the initial activation of naïve CD8 T cells by professional and semi-professional antigen-presenting cells, which determines their differentiation into effector or tolerant CD8 T cells, respectively. We show that adenosine inhibited the initial activation of murine naïve CD8 T cells after alphaCD3/CD28-mediated stimulation. Adenosine caused inhibition of activation, cytokine production, metabolic activity, proliferation and ultimately effector differentiation of naïve CD8 T cells. Remarkably, adenosine interfered efficiently with CD8 T-cell priming by professional antigen-presenting cells (dendritic cells) and semi-professional antigen-presenting cells (liver sinusoidal endothelial cells). Further analysis of the underlying mechanisms demonstrated that adenosine prevented rapid tyrosine phosphorylation of the key kinase ZAP-70 as well as Akt and ERK1/2 in naïve alphaCD3/CD28-stimulated CD8 cells. Consequently, alphaCD3/CD28-induced calcium-influx into CD8 cells was reduced by exposure to adenosine. Our results support the notion that extracellular adenosine controls membrane-proximal T-cell receptor signalling and thereby also differentiation of naïve CD8 T cells. These data raise the possibility that extracellular adenosine has a physiological role in the regulation of CD8 T-cell priming and differentiation in peripheral organs.

T inflammatory memory CD8 T cells participate to antiviral response and generate secondary memory cells with an advantage in XCL1 production.

PubMed

Jubin, Virginie; Ventre, Erwan; Leverrier, Yann; Djebali, Sophia; Mayol, Katia; Tomkowiak, Martine; Mafille, Julien; Teixeira, Marie; Teoh, Denise Y-L; Lina, Bruno; Walzer, Thierry; Arpin, Christophe; Marvel, Jacqueline

2012-06-01

Besides the classically described subsets of memory CD8 T cells generated under infectious conditions, are T inflammatory memory cells generated under sterile priming conditions, such as sensitization to allergens. Although not fully differentiated as pathogen-induced memory cells, they display memory properties that distinguish them from naive CD8 T cells. Given these memory cells are generated in an antigen-specific context that is devoid of pathogen-derived danger signals and CD4 T cell help, we herein questioned whether they maintained their activation and differentiation potential, could be recruited in an immune response directed against a pathogen expressing their cognate antigen and further differentiate in fully competent secondary memory cells. We show that T inflammatory memory cells can indeed take part to the immune response triggered by a viral infection, differentiate into secondary effectors and further generate typical central memory CD8 T cells and effector memory CD8 T cells. Furthermore, the secondary memory cells they generate display a functional advantage over primary memory cells in their capacity to produce TNF-α and the XCL1 chemokine. These results suggest that cross-reactive stimulations and differentiation of cells directed against allergens or self into fully competent pathogen-induced memory cells might have incidences in inflammatory immuno-pathologies.

Surgical Stress Abrogates Pre-Existing Protective T Cell Mediated Anti-Tumor Immunity Leading to Postoperative Cancer Recurrence

PubMed Central

Lansdell, Casey; Alkayyal, Almohanad A.; Baxter, Katherine E.; Angka, Leonard; Zhang, Jiqing; Tanese de Souza, Christiano; Stephenson, Kyle B.; Parato, Kelley; Bramson, Jonathan L.; Bell, John C.; Lichty, Brian D.; Auer, Rebecca C.

2016-01-01

Anti-tumor CD8+ T cells are a key determinant for overall survival in patients following surgical resection for solid malignancies. Using a mouse model of cancer vaccination (adenovirus expressing melanoma tumor-associated antigen (TAA)—dopachrome tautomerase (AdDCT) and resection resulting in major surgical stress (abdominal nephrectomy), we demonstrate that surgical stress results in a reduction in the number of CD8+ T cell that produce cytokines (IFNγ, TNFα, Granzyme B) in response to TAA. This effect is secondary to both reduced proliferation and impaired T cell function following antigen binding. In a prophylactic model, surgical stress completely abrogates tumor protection conferred by vaccination in the immediate postoperative period. In a clinically relevant surgical resection model, vaccinated mice undergoing a positive margin resection with surgical stress had decreased survival compared to mice with positive margin resection alone. Preoperative immunotherapy with IFNα significantly extends survival in surgically stressed mice. Importantly, myeloid derived suppressor cell (MDSC) population numbers and functional impairment of TAA-specific CD8+ T cell were altered in surgically stressed mice. Our observations suggest that cancer progression may result from surgery-induced suppression of tumor-specific CD8+ T cells. Preoperative immunotherapies aimed at targeting the prometastatic effects of cancer surgery will reduce recurrence and improve survival in cancer surgery patients. PMID:27196057

Intratumoral Infection with Murine Cytomegalovirus Synergizes with PD-L1 Blockade to Clear Melanoma Lesions and Induce Long-term Immunity

PubMed Central

Erkes, Dan A; Xu, Guangwu; Daskalakis, Constantine; Zurbach, Katherine A; Wilski, Nicole A; Moghbeli, Toktam; Hill, Ann B; Snyder, Christopher M

2016-01-01

Cytomegalovirus is an attractive cancer vaccine platform because it induces strong, functional CD8+ T-cell responses that accumulate over time and migrate into most tissues. To explore this, we used murine cytomegalovirus expressing a modified gp100 melanoma antigen. Therapeutic vaccination by the intraperitoneal and intradermal routes induced tumor infiltrating gp100-specific CD8+ T-cells, but provided minimal benefit for subcutaneous lesions. In contrast, intratumoral infection of established tumor nodules greatly inhibited tumor growth and improved overall survival in a CD8+ T-cell-dependent manner, even in mice previously infected with murine cytomegalovirus. Although murine cytomegalovirus could infect and kill B16F0s in vitro, infection was restricted to tumor-associated macrophages in vivo. Surprisingly, the presence of a tumor antigen in the virus only slightly increased the efficacy of intratumoral infection and tumor-specific CD8+ T-cells in the tumor remained dysfunctional. Importantly, combining intratumoral murine cytomegalovirus infection with anti-PD-L1 therapy was synergistic, resulting in tumor clearance from over half of the mice and subsequent protection against tumor challenge. Thus, while a murine cytomegalovirus-based vaccine was poorly effective against established subcutaneous tumors, direct infection of tumor nodules unexpectedly delayed tumor growth and synergized with immune checkpoint blockade to promote tumor clearance and long-term protection. PMID:27434584

Functional impairment of Tax-specific but not cytomegalovirus-specific CD8+ T lymphocytes in a minor population of asymptomatic human T-cell leukemia virus type 1-carriers

PubMed Central

2011-01-01

Background Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a small percentage of infected individuals. ATL is often associated with general immune suppression and an impaired HTLV-1-specific T-cell response, an important host defense system. We previously found that a small fraction of asymptomatic HTLV-1-carriers (AC) already showed impaired T-cell responses against the major target antigen, Tax. However, it is unclear whether the impaired HTLV-1 Tax-specific T-cell response in these individuals is an HTLV-1-specific phenomenon, or merely reflects general immune suppression. In this study, in order to characterize the impaired HTLV-1-specific T-cell response, we investigated the function of Tax-specific CD8+ T-cells in various clinical status of HTLV-1 infection. Results By using tetramers consisting of HLA-A*0201, -A*2402, or -A*1101, and corresponding Tax epitope peptides, we detected Tax-specific CD8+ T-cells in the peripheral blood from 87.0% of ACs (n = 20/23) and 100% of HAM/TSP patients (n = 18/18) tested. We also detected Tax-specific CD8+ T-cells in 38.1% of chronic type ATL (cATL) patients (n = 8/21), although its frequencies in peripheral blood CD8+ T cells were significantly lower than those of ACs or HAM/TSP patients. Tax-specific CD8+ T-cells detected in HAM/TSP patients proliferated well in culture and produced IFN-γ when stimulated with Tax peptides. However, such functions were severely impaired in the Tax-specific CD8+ T-cells detected in cATL patients. In ACs, the responses of Tax-specific CD8+ T-cells were retained in most cases. However, we found one AC sample whose Tax-specific CD8+ T-cells hardly produced IFN-γ, and failed to proliferate and express activation (CD69) and degranulation (CD107a) markers in response to Tax peptide. Importantly, the same AC sample contained cytomegalovirus (CMV) pp65-specific CD8+ T-cells that possessed functions upon CMV pp65 peptide stimulation. We further examined additional samples of two smoldering type ATL patients and found that they also showed dysfunctions of Tax-specific but not CMV-specific CD8+ T-cells. Conclusions These findings indicated that Tax-specific CD8+ T-cells were scarce and dysfunctional not only in ATL patients but also in a limited AC population, and that the dysfunction was selective for HTLV-1-specifc CD8+ T-cells in early stages. PMID:22151736

Induction of influenza-specific local CD8 T-cells in the respiratory tract after aerosol delivery of vaccine antigen or virus in the Babraham inbred pig.

PubMed

Tungatt, Katie; Dolton, Garry; Morgan, Sophie B; Attaf, Meriem; Fuller, Anna; Whalley, Thomas; Hemmink, Johanneke D; Porter, Emily; Szomolay, Barbara; Montoya, Maria; Hammond, John A; Miles, John J; Cole, David K; Townsend, Alain; Bailey, Mick; Rizkallah, Pierre J; Charleston, Bryan; Tchilian, Elma; Sewell, Andrew K

2018-05-01

There is increasing evidence that induction of local immune responses is a key component of effective vaccines. For respiratory pathogens, for example tuberculosis and influenza, aerosol delivery is being actively explored as a method to administer vaccine antigens. Current animal models used to study respiratory pathogens suffer from anatomical disparity with humans. The pig is a natural and important host of influenza viruses and is physiologically more comparable to humans than other animal models in terms of size, respiratory tract biology and volume. It may also be an important vector in the birds to human infection cycle. A major drawback of the current pig model is the inability to analyze antigen-specific CD8+ T-cell responses, which are critical to respiratory immunity. Here we address this knowledge gap using an established in-bred pig model with a high degree of genetic identity between individuals, including the MHC (Swine Leukocyte Antigen (SLA)) locus. We developed a toolset that included long-term in vitro pig T-cell culture and cloning and identification of novel immunodominant influenza-derived T-cell epitopes. We also generated structures of the two SLA class I molecules found in these animals presenting the immunodominant epitopes. These structures allowed definition of the primary anchor points for epitopes in the SLA binding groove and established SLA binding motifs that were used to successfully predict other influenza-derived peptide sequences capable of stimulating T-cells. Peptide-SLA tetramers were constructed and used to track influenza-specific T-cells ex vivo in blood, the lungs and draining lymph nodes. Aerosol immunization with attenuated single cycle influenza viruses (S-FLU) induced large numbers of CD8+ T-cells specific for conserved NP peptides in the respiratory tract. Collectively, these data substantially increase the utility of pigs as an effective model for studying protective local cellular immunity against respiratory pathogens.

Induction of influenza-specific local CD8 T-cells in the respiratory tract after aerosol delivery of vaccine antigen or virus in the Babraham inbred pig

PubMed Central

Morgan, Sophie B.; Attaf, Meriem; Szomolay, Barbara; Miles, John J.; Townsend, Alain; Bailey, Mick; Charleston, Bryan; Tchilian, Elma

2018-01-01

There is increasing evidence that induction of local immune responses is a key component of effective vaccines. For respiratory pathogens, for example tuberculosis and influenza, aerosol delivery is being actively explored as a method to administer vaccine antigens. Current animal models used to study respiratory pathogens suffer from anatomical disparity with humans. The pig is a natural and important host of influenza viruses and is physiologically more comparable to humans than other animal models in terms of size, respiratory tract biology and volume. It may also be an important vector in the birds to human infection cycle. A major drawback of the current pig model is the inability to analyze antigen-specific CD8+ T-cell responses, which are critical to respiratory immunity. Here we address this knowledge gap using an established in-bred pig model with a high degree of genetic identity between individuals, including the MHC (Swine Leukocyte Antigen (SLA)) locus. We developed a toolset that included long-term in vitro pig T-cell culture and cloning and identification of novel immunodominant influenza-derived T-cell epitopes. We also generated structures of the two SLA class I molecules found in these animals presenting the immunodominant epitopes. These structures allowed definition of the primary anchor points for epitopes in the SLA binding groove and established SLA binding motifs that were used to successfully predict other influenza-derived peptide sequences capable of stimulating T-cells. Peptide-SLA tetramers were constructed and used to track influenza-specific T-cells ex vivo in blood, the lungs and draining lymph nodes. Aerosol immunization with attenuated single cycle influenza viruses (S-FLU) induced large numbers of CD8+ T-cells specific for conserved NP peptides in the respiratory tract. Collectively, these data substantially increase the utility of pigs as an effective model for studying protective local cellular immunity against respiratory pathogens. PMID:29772011

Engineered artificial antigen presenting cells facilitate direct and efficient expansion of tumor infiltrating lymphocytes

PubMed Central

2011-01-01

Background Development of a standardized platform for the rapid expansion of tumor-infiltrating lymphocytes (TILs) with anti-tumor function from patients with limited TIL numbers or tumor tissues challenges their clinical application. Methods To facilitate adoptive immunotherapy, we applied genetically-engineered K562 cell-based artificial antigen presenting cells (aAPCs) for the direct and rapid expansion of TILs isolated from primary cancer specimens. Results TILs outgrown in IL-2 undergo rapid, CD28-independent expansion in response to aAPC stimulation that requires provision of exogenous IL-2 cytokine support. aAPCs induce numerical expansion of TILs that is statistically similar to an established rapid expansion method at a 100-fold lower feeder cell to TIL ratio, and greater than those achievable using anti-CD3/CD28 activation beads or extended IL-2 culture. aAPC-expanded TILs undergo numerical expansion of tumor antigen-specific cells, remain amenable to secondary aAPC-based expansion, and have low CD4/CD8 ratios and FOXP3+ CD4+ cell frequencies. TILs can also be expanded directly from fresh enzyme-digested tumor specimens when pulsed with aAPCs. These "young" TILs are tumor-reactive, positively skewed in CD8+ lymphocyte composition, CD28 and CD27 expression, and contain fewer FOXP3+ T cells compared to parallel IL-2 cultures. Conclusion Genetically-enhanced aAPCs represent a standardized, "off-the-shelf" platform for the direct ex vivo expansion of TILs of suitable number, phenotype and function for use in adoptive immunotherapy. PMID:21827675

Evaluation of profile and functionality of memory T cells in pulmonary tuberculosis.

PubMed

Tonaco, Marcela M; Moreira, Jôsimar D; Nunes, Fernanda F C; Loures, Cristina M G; Souza, Larissa R; Martins, Janaina M; Silva, Henrique R; Porto, Arthur Henrique R; Toledo, Vicente Paulo C P; Miranda, Silvana S; Guimarães, Tânia Mara P D

2017-12-01

The cells T CD4+ T and CD8+ can be subdivided into phenotypes naïve, T of central memory, T of effector memory and effector, according to the expression of surface molecules CD45RO and CD27. The T lymphocytes are cells of long life with capacity of rapid expansion and function, after a new antigenic exposure. In tuberculosis, it was found that specific memory T cells are present, however, gaps remain about the role of such cells in the disease immunology. In this study, the phenotypic profile was analyzed and characterized the functionality of CD4+ T lymphocytes and CD8+ T cells of memory and effector, in response to specific stimuli in vitro, in patients with active pulmonary TB, compared to individuals with latent infection with Mycobacterium tuberculosis the ones treated with pulmonary TB. It was observed that the group of patients with active pulmonary tuberculosis was the one which presented the highest proportion of cells T CD4+ of central memory IFN-ɣ+ e TNF-α+, suggesting that in TB, these T of central memory cells would have a profile of protective response, being an important target of study for the development of more effective vaccines; this group also developed lower proportion of CD8+ T effector lymphocytes than the others, a probable cause of specific and less effective response against the bacillus in these individuals; the ones treated for pulmonary tuberculosis were those who developed higher proportion of T CD4+ of memory central IL-17+ cells, indicating that the stimulation of long duration, with high antigenic load, followed by elimination of the pathogen, contribute to more significant generation of such cells; individuals with latent infection by M. tuberculosis and treated for pulmonary tuberculosis, showed greater response of CD8+ T effector lymphocytes IFN-ɣ+ than the controls, suggesting that these cells, as well as CD4+ T lymphocytes, have crucial role of protection against M. tuberculosis. These findings have contributed to a better understanding of the immunologic changes in M. tuberculosis infection and the development of new strategies for diagnosis and prevention of tuberculosis. Copyright © 2017. Published by Elsevier B.V.

Different roles of CD4, CD8 and γδ T-lymphocytes in naive and vaccinated chickens during Salmonella Enteritidis infection.

PubMed

Sekelova, Zuzana; Polansky, Ondrej; Stepanova, Hana; Fedr, Radek; Faldynova, Marcela; Rychlik, Ivan; Vlasatikova, Lenka

2017-07-01

Lymphocytes represent the key antigen-specific leukocyte subpopulation. Despite their importance in mounting an immune response, an unbiased description of proteins expressed by chicken lymphocytes has not been presented. In this study, we therefore intravenously infected chickens with Salmonella Enteritidis, sorted CD4, CD8 and γδ T-lymphocytes from the spleen by flow cytometry and determined the proteome of each population by LC-MS/MS. CD4 T-lymphocyte characteristic proteins included ubiquitin SUMO-like domain and BAR domain containing proteins. CD8 T-lymphocyte specific proteins were characterized by purine ribonucleoside triphosphate binding and were involved in cell differentiation, cell activation and regulation of programmed cell death. γδ T-lymphocyte specific proteins exhibited enrichment of small GTPase of Rab type and GTP binding. Following infection, inducible proteins in CD4 lymphocytes included ribosomal proteins and downregulated proteins localized to the lysosome. CD8 T-lymphocytes induced MCM complex proteins, proteins required for DNA replication and machinery for protein processing in the endoplasmic reticulum. Proteins inducible in γδ T-lymphocytes belonged to immune system response, oxidative phosphorylation and the spliceosome. In this study, we predicted the likely events in lymphocyte response to systemic bacterial infection and identified proteins which can be used as markers specific for each lymphocyte subpopulation. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

ESAT-6–dependent cytosolic pattern recognition drives noncognate tuberculosis control in vivo

PubMed Central

Zedler, Ulrike; Stäber, Manuela; Perdomo, Carolina; Dorhoi, Anca

2016-01-01

IFN-γ is a critical mediator of host defense against Mycobacterium tuberculosis (Mtb) infection. Antigen-specific CD4+ T cells have long been regarded as the main producer of IFN-γ in tuberculosis (TB), and CD4+ T cell immunity is the main target of current TB vaccine candidates. However, given the recent failures of such a TB vaccine candidate in clinical trials, strategies to harness CD4-independent mechanisms of protection should be included in future vaccine design. Here, we have reported that noncognate IFN-γ production by Mtb antigen–independent memory CD8+ T cells and NK cells is protective during Mtb infection and evaluated the mechanistic regulation of IFN-γ production by these cells in vivo. Transfer of arenavirus- or protein-specific CD8+ T cells or NK cells reduced the mortality and morbidity rates of mice highly susceptible to TB in an IFN-γ–dependent manner. Secretion of IFN-γ by these cell populations required IL-18, sensing of mycobacterial viability, Mtb protein 6-kDa early secretory antigenic target–mediated (ESAT-6–mediated) cytosolic contact, and activation of NLR family pyrin domain–containing protein 3 (NLRP3) inflammasomes in CD11c+ cell subsets. Neutralization of IL-18 abrogated protection in susceptible recipient mice that had received noncognate cells. Moreover, improved Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine–induced protection was lost in the absence of ESAT-6–dependent cytosolic contact. Our findings provide a comprehensive mechanistic framework for antigen-independent IFN-γ secretion in response to Mtb with critical implications for future intervention strategies against TB. PMID:27111234

Autoimmune vitiligo does not require the ongoing priming of naïve CD8 T cells for disease progression or associated protection against melanoma1

PubMed Central

Byrne, Katelyn T.; Zhang, Peisheng; Steinberg, Shannon M.; Turk, Mary Jo

2014-01-01

Vitiligo is a CD8 T cell-mediated autoimmune disease that has been shown to promote the longevity of memory T cell responses to melanoma. However mechanisms whereby melanocyte/melanoma antigen-specific T cell responses are perpetuated in the context of vitiligo are not well understood. The present studies investigate the possible phenomenon of naïve T cell priming in hosts with melanoma-initiated, self-perpetuating, autoimmune vitiligo. Using naïve pmel (gp10025-33-specific) transgenic CD8 T cells, we demonstrate that autoimmune melanocyte destruction induces naive T cell proliferation in skin-draining lymph nodes, in an antigen-dependent fashion. These pmel T cells upregulate expression of CD44, P-selectin ligand, and granzyme B. However, they do not downregulate CD62L, nor do they acquire the ability to produce IFN-γ, indicating a lack of functional priming. Accordingly, adult thymectomized mice exhibit no reduction in the severity or kinetics of depigmentation or long-lived protection against melanoma, indicating that the continual priming of naïve T cells is not required for vitiligo or its associated anti-tumor immunity. Despite this, depletion of CD4 T cells during the course of vitiligo rescues the priming of naïve pmel T cells that are capable of producing IFN-γ and persisting as memory, suggesting an ongoing and dominant mechanism of suppression by regulatory T cells. This work reveals the complex regulation of self-reactive CD8 T cells in vitiligo, and demonstrates the overall poorly immunogenic nature of this autoimmune disease setting. PMID:24403535

Supraphysiologic control over HIV-1 replication mediated by CD8 T cells expressing a re-engineered CD4-based chimeric antigen receptor

PubMed Central

Richardson, Max W.; Ellebrecht, Christoph T.; Glover, Joshua A.; Secreto, Anthony J.; Kulikovskaya, Irina; Yi, Yanjie; Wang, Jianbin; Dufendach, Keith A.; Holmes, Michael C.; Collman, Ronald G.

2017-01-01

HIV is adept at avoiding naturally generated T cell responses; therefore, there is a need to develop HIV-specific T cells with greater potency for use in HIV cure strategies. Starting with a CD4-based chimeric antigen receptor (CAR) that was previously used without toxicity in clinical trials, we optimized the vector backbone, promoter, HIV targeting moiety, and transmembrane and signaling domains to determine which components augmented the ability of T cells to control HIV replication. This re-engineered CAR was at least 50-fold more potent in vitro at controlling HIV replication than the original CD4 CAR, or a TCR-based approach, and substantially better than broadly neutralizing antibody-based CARs. A humanized mouse model of HIV infection demonstrated that T cells expressing optimized CARs were superior at expanding in response to antigen, protecting CD4 T cells from infection, and reducing viral loads compared to T cells expressing the original, clinical trial CAR. Moreover, in a humanized mouse model of HIV treatment, CD4 CAR T cells containing the 4-1BB costimulatory domain controlled HIV spread after ART removal better than analogous CAR T cells containing the CD28 costimulatory domain. Together, these data indicate that potent HIV-specific T cells can be generated using improved CAR design and that CAR T cells could be important components of an HIV cure strategy. PMID:29023549

Primed tumor-reactive multifunctional CD62L+ human CD8+T-cells for immunotherapy

PubMed Central

Wölfl, Matthias; Merker, Katharina; Morbach, Henner; Van Gool, Stefaan W.; Eyrich, Matthias; Greenberg, Philip D.; Schlegel, Paul G.

2011-01-01

T-cell mediated immunotherapy against malignancies has been shown to be effective for certain types of cancer. However ex vivo expansion of tumor-reactive T-cells has been hindered by the low precursor frequency of such cells, often requiring multiple rounds of stimulation, resulting in full differentiation, loss of homing receptors and potential exhaustion of the expanded T-cells. Here we show that when using highly purified naïve CD8+ T-cells, a single stimulation with peptide pulsed, IFNγ/LPS-matured dendritic cells in combination with the sequential use of IL-21, IL-7 and IL-15 is sufficient for extensive expansion of antigen-specific T-cells. Short-term expanded T-cells were tumor-reactive, multifunctional and retained a central memory-like phenotype (CD62L+, CCR7+, CD28+). The procedure is highly reproducible and robust as demonstrated for different healthy donors and for cancer patients. Such short-term tumor-antigen-primed, multifunctional T-cells may therefore serve as a platform to target different malignancies accessible to immunotherapy. PMID:20972785

A novel therapeutic hepatitis B vaccine induces cellular and humoral immune responses and breaks tolerance in hepatitis B virus (HBV) transgenic mice.

PubMed

Buchmann, Pascale; Dembek, Claudia; Kuklick, Larissa; Jäger, Clemens; Tedjokusumo, Raindy; von Freyend, Miriam John; Drebber, Uta; Janowicz, Zbigniew; Melber, Karl; Protzer, Ulrike

2013-02-06

Therapeutic vaccines are currently being developed for chronic hepatitis B and C. As an alternative to long-term antiviral treatment or to support only partially effective therapy, they should activate the patient's immune system effectively to fight and finally control the virus. A paradigm of therapeutic vaccination is the potent induction of T-cell responses against key viral antigens - besides activation of a humoral immune response. We have evaluated the potential of a novel vaccine formulation comprising particulate hepatitis B surface (HBsAg) and core antigen (HBcAg), and the saponin-based ISCOMATRIX™ adjuvant for its ability to stimulate T and B cell responses in C57BL/6 mice and its ability to break tolerance in syngeneic HBV transgenic (HBVtg) mice. In C57BL/6 mice, the vaccine induced multifunctional HBsAg- and HBcAg-specific CD8+ T cells detected by staining for IFNγ, TNFα and IL-2, as well as high antibody titers against both antigens. Vaccination of HBVtg animals induced potent HBsAg- and HBcAg-specific CD8+ T-cell responses in spleens and HBcAg-specific CD8+ T-cell responses in livers as well as anti-HBs seroconversion two weeks post injection. Vaccination further reduced HBcAg expression in livers of HBVtg mice without causing liver damage. In summary, this study demonstrates therapeutic efficacy of a novel vaccine formulation in a mouse model of immunotolerant, chronic HBV infection. Copyright © 2013 Elsevier Ltd. All rights reserved.

Improved Innate and Adaptive Immunostimulation by Genetically Modified HIV-1 Protein Expressing NYVAC Vectors

PubMed Central

Quakkelaar, Esther D.; Redeker, Anke; Haddad, Elias K.; Harari, Alexandre; McCaughey, Stella Mayo; Duhen, Thomas; Filali-Mouhim, Abdelali; Goulet, Jean-Philippe; Loof, Nikki M.; Ossendorp, Ferry; Perdiguero, Beatriz; Heinen, Paul; Gomez, Carmen E.; Kibler, Karen V.; Koelle, David M.; Sékaly, Rafick P.; Sallusto, Federica; Lanzavecchia, Antonio; Pantaleo, Giuseppe; Esteban, Mariano; Tartaglia, Jim; Jacobs, Bertram L.; Melief, Cornelis J. M.

2011-01-01

Attenuated poxviruses are safe and capable of expressing foreign antigens. Poxviruses are applied in veterinary vaccination and explored as candidate vaccines for humans. However, poxviruses express multiple genes encoding proteins that interfere with components of the innate and adaptive immune response. This manuscript describes two strategies aimed to improve the immunogenicity of the highly attenuated, host-range restricted poxvirus NYVAC: deletion of the viral gene encoding type-I interferon-binding protein and development of attenuated replication-competent NYVAC. We evaluated these newly generated NYVAC mutants, encoding HIV-1 env, gag, pol and nef, for their ability to stimulate HIV-specific CD8 T-cell responses in vitro from blood mononuclear cells of HIV-infected subjects. The new vectors were evaluated and compared to the parental NYVAC vector in dendritic cells (DCs), RNA expression arrays, HIV gag expression and cross-presentation assays in vitro. Deletion of type-I interferon-binding protein enhanced expression of interferon and interferon-induced genes in DCs, and increased maturation of infected DCs. Restoration of replication competence induced activation of pathways involving antigen processing and presentation. Also, replication-competent NYVAC showed increased Gag expression in infected cells, permitting enhanced cross-presentation to HIV-specific CD8 T cells and proliferation of HIV-specific memory CD8 T-cells in vitro. The recombinant NYVAC combining both modifications induced interferon-induced genes and genes involved in antigen processing and presentation, as well as increased Gag expression. This combined replication-competent NYVAC is a promising candidate for the next generation of HIV vaccines. PMID:21347234

The role of cytokines in T-cell memory in health and disease.

PubMed

Raeber, Miro E; Zurbuchen, Yves; Impellizzieri, Daniela; Boyman, Onur

2018-05-01

Upon stimulation with their cognate antigen, naive T cells undergo proliferation and differentiation into effector cells, followed by apoptosis or survival as precursors of long-lived memory cells. These phases of a T-cell response and the ensuing maintenance of memory T cells are shaped by cytokines, most notably interleukin-2 (IL-2), IL-7, and IL-15 that share the common γ chain (γ c ) cytokine receptor. Steady-state production of IL-7 and IL-15 is necessary for background proliferation and homeostatic survival of CD4 + and CD8 + memory T cells. During immune responses, augmented levels of IL-2, IL-15, IL-21, IL-12, IL-18, and type-I interferons determine the memory potential of antigen-specific effector CD8 + cells, while increased IL-2 and IL-15 cause bystander proliferation of heterologous CD4 + and CD8 + memory T cells. Limiting availability of γ c cytokines, reduction in regulatory T cells or IL-10, and persistence of inflammation or cognate antigen can result in memory T cells, which fail to become cytokine-dependent long-lived cells. Conversely, increased IL-7 and IL-15 can expand memory T cells, including pathogenic tissue-resident memory T cells, as seen in lymphopenia and certain chronic-inflammatory disorders and malignancies. These abovementioned factors impact immunotherapy and vaccines directed at memory T cells in cancer and chronic infection. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Role of novel type I interferon epsilon in viral infection and mucosal immunity

PubMed Central

Xi, Yang; Day, Stephanie L; Jackson, Ronald J; Ranasinghe, Charani

2012-01-01

Intranasal infection with vaccinia virus co-expressing interferon epsilon (VV-HIV-IFN-ɛ) was used to evaluate the role of IFN-ɛ in mucosal immunity. VV-HIV- IFN-ɛ infection induced a rapid VV clearance in lung that correlated with (i) an elevated lung VV-specific CD8+CD107a+IFN-γ+ population expressing activation markers CD69/CD103, (ii) enhanced lymphocyte recruitment to lung alveoli with reduced inflammation, and (iii) an heightened functional/cytotoxic CD8+CD4+ T-cell subset (CD3hiCCR7hiCD62Llo) in lung lymph nodes. These responses were different to that observed with intranasal VV-HA-IFN-α4 or VV-HA-IFN-β infections. When IFN-ɛ was used in an intranasal/intramuscular heterologous HIV prime-boost immunization, elevated HIV-specific effector, but not memory CD8+T cells responses, were observed in spleen, genito-rectal nodes, and Peyer's patch. Homing marker α4β7 and CCR9 analysis indicated that unlike other type I IFNs, IFN-ɛ could promote migration of antigen-specific CD8+T cells to the gut. Our results indicate that IFN-ɛ has a unique role in the mucosae and most likely can be used to control local lung and/or gut infections (i.e., microbicide) such as tuberculosis, HIV-1, or sexually transmitted diseases. PMID:22617838

Use of a novel antigen expressing system to study the Salmonella enterica serovar Typhi protein recognition by T cells

PubMed Central

Lou, David; Steiner, Stephanie; Rezwanul, Tasmia; Guo, Qin; Picking, William D.; Nene, Vishvanath; Sztein, Marcelo B.

2017-01-01

Salmonella enterica serovar Typhi (S. Typhi), the causative agent of the typhoid fever, is a pathogen of great public health importance. Typhoid vaccines have the potential to be cost-effective measures towards combating this disease, yet the antigens triggering host protective immune responses are largely unknown. Given the key role of cellular-mediated immunity in S. Typhi protection, it is crucial to identify S. Typhi proteins involved in T-cell responses. Here, cells from individuals immunized with Ty21a typhoid vaccine were collected before and after immunization and used as effectors. We also used an innovative antigen expressing system based on the infection of B-cells with recombinant Escherichia coli (E. coli) expressing one of four S. Typhi gene products (i.e., SifA, OmpC, FliC, GroEL) as targets. Using flow cytometry, we found that the pattern of response to specific S. Typhi proteins was variable. Some individuals responded to all four proteins while others responded to only one or two proteins. We next evaluated whether T-cells responding to recombinant E. coli also possess the ability to respond to purified proteins. We observed that CD4+ cell responses, but not CD8+ cell responses, to recombinant E. coli were significantly associated with the responses to purified proteins. Thus, our results demonstrate the feasibility of using an E. coli expressing system to uncover the antigen specificity of T-cells and highlight its applicability to vaccine studies. These results also emphasize the importance of selecting the stimuli appropriately when evaluating CD4+ and CD8+ cell responses. PMID:28873442

T cell receptor alpha variable 12-2 bias in the immunodominant response to Yellow fever virus.

PubMed

Bovay, Amandine; Zoete, Vincent; Dolton, Garry; Bulek, Anna M; Cole, David K; Rizkallah, Pierre J; Fuller, Anna; Beck, Konrad; Michielin, Olivier; Speiser, Daniel E; Sewell, Andrew K; Fuertes Marraco, Silvia A

2018-02-01

The repertoire of human αβ T-cell receptors (TCRs) is generated via somatic recombination of germline gene segments. Despite this enormous variation, certain epitopes can be immunodominant, associated with high frequencies of antigen-specific T cells and/or exhibit bias toward a TCR gene segment. Here, we studied the TCR repertoire of the HLA-A*0201-restricted epitope LLWNGPMAV (hereafter, A2/LLW) from Yellow Fever virus, which generates an immunodominant CD8 + T cell response to the highly effective YF-17D vaccine. We discover that these A2/LLW-specific CD8 + T cells are highly biased for the TCR α chain TRAV12-2. This bias is already present in A2/LLW-specific naïve T cells before vaccination with YF-17D. Using CD8 + T cell clones, we show that TRAV12-2 does not confer a functional advantage on a per cell basis. Molecular modeling indicated that the germline-encoded complementarity determining region (CDR) 1α loop of TRAV12-2 critically contributes to A2/LLW binding, in contrast to the conventional dominant dependence on somatically rearranged CDR3 loops. This germline component of antigen recognition may explain the unusually high precursor frequency, prevalence and immunodominance of T-cell responses specific for the A2/LLW epitope. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Plasmodium parasite as an effective hepatocellular carcinoma antigen glypican-3 delivery vector.

PubMed

Liu, Quan; Yang, Yijun; Tan, Xuefang; Tao, Zhu; Adah, Dickson; Yu, Songlin; Lu, Junnan; Zhao, Siting; Qin, Limei; Qin, Li; Chen, Xiaoping

2017-04-11

We have previously demonstrated that malaria parasite infection has an anti-tumor effect in a mouse model. This research aimed to investigate the possibility of using Plasmodium parasite as a novel vaccine vector for hepatocellular carcinoma (HCC) immunotherapy. We constructed a Plasmodium yoelii 17XNL strain (P.y) expressing murine glypican-3 (GPC3) protein (P.y-GPC3), and examined its therapeutic potency in a murine Hepa1-6-induced hepatoma model that highly expressed GPC3 protein. The prerequisites for invoking a CD8+ T cell response were assessed after P.y-based immunization, which included obviously increased concentrations of T helper cell type 1 (Th1)-associated cytokines, such as IL-2, IFN-γ and TNF-α, in serum and preferential expansion of the CD8α+ dendritic cell (DC) subset with higher expression of CD80 and CD86 molecules. Compared with uninfected and wild-type P.y-infected mice, a significant GPC3-specific cytotoxic T lymphocyte (CTL) response was detected in P.y-GPC3 vaccinated mice. Furthermore, P.y-GPC3-based vaccination dramatically inhibited Hepa1-6-induced tumor growth in the implanted HCC and prolonged the survival of tumor-bearing mice. We concluded that a Plasmodium-based vector is highly efficient in inducing tumor antigen-specific T cell-mediated immunity and protection against tumor cells. More broadly, this strategy supported our hypothesis that Plasmodium parasites, as novel therapeutic antigen vectors, may be applicable to tumor immunotherapy for patients with HCC.

Cryopreservation-related loss of antigen-specific IFNγ producing CD4+ T-cells can skew immunogenicity data in vaccine trials: Lessons from a malaria vaccine trial substudy.

PubMed

Ford, Tom; Wenden, Claire; Mbekeani, Alison; Dally, Len; Cox, Josephine H; Morin, Merribeth; Winstone, Nicola; Hill, Adrian V S; Gilmour, Jill; Ewer, Katie J

2017-04-04

Ex vivo functional immunoassays such as ELISpot and intracellular cytokine staining (ICS) by flow cytometry are crucial tools in vaccine development both in the identification of novel immunogenic targets and in the immunological assessment of samples from clinical trials. Cryopreservation and subsequent thawing of PBMCs via validated processes has become a mainstay of clinical trials due to processing restrictions inherent in the disparate location and capacity of trial centres, and also in the need to standardize biological assays at central testing facilities. Logistical and financial requirement to batch process samples from multiple study timepoints are also key. We used ELISpot and ICS assays to assess antigen-specific immunogenicity in blood samples taken from subjects enrolled in a phase II malaria heterologous prime-boost vaccine trial and showed that the freeze thaw process can result in a 3-5-fold reduction of malaria antigen-specific IFNγ-producing CD3 + CD4 + effector populations from PBMC samples taken post vaccination. We have also demonstrated that peptide responsive CD8 + T cells are relatively unaffected, as well as CD4 + T cell populations that do not produce IFNγ. These findings contribute to a growing body of data that could be consolidated and synthesised as guidelines for clinical trials with the aim of increasing the efficiency of vaccine development pipelines. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Exposure to sequestered self-antigens in vivo is not sufficient for the induction of autoimmune diabetes

PubMed Central

Chan, Olivia; Hall, Håkan; Elford, Alisha R.; Yen, Patty; Calzascia, Thomas; Spencer, David M.; Ohashi, Pamela S.

2017-01-01

Although the role of T cells in autoimmunity has been explored for many years, the mechanisms leading to the initial priming of an autoimmune T cell response remain enigmatic. The ‘hit and run’ model suggests that self-antigens released upon cell death can provide the initial signal for a self-sustaining autoimmune response. Using a novel transgenic mouse model where we could induce the release of self-antigens via caspase-dependent apoptosis. We tracked the fate of CD8+ T cells specific for the self-antigen. Our studies demonstrated that antigens released from apoptotic cells were cross-presented by CD11c+ cells in the draining lymph node. This cross-presentation led to proliferation of self-antigen specific T cells, followed by a transient ability to produce IFN-γ, but did not lead to the development of autoimmune diabetes. Using this model we examined the consequences on T cell immunity when apoptosis was combined with dendritic cell maturation signals, an autoimmune susceptible genetic background, and the deletion of Tregs. The results of our study demonstrate that autoimmune diabetes cannot be initiated by the presentation of antigens released from apoptotic cells in vivo even in the presence of factors known to promote autoimmunity. PMID:28257518

Transforming growth factor-beta inhibits human antigen-specific CD4+ T cell proliferation without modulating the cytokine response.

PubMed

Tiemessen, Machteld M; Kunzmann, Steffen; Schmidt-Weber, Carsten B; Garssen, Johan; Bruijnzeel-Koomen, Carla A F M; Knol, Edward F; van Hoffen, Els

2003-12-01

Transforming growth factor (TGF)-beta has been demonstrated to play a key role in the regulation of the immune response, mainly by its suppressive function towards cells of the immune system. In humans, the effect of TGF-beta on antigen-specific established memory T cells has not been investigated yet. In this study antigen-specific CD4(+) T cell clones (TCC) were used to determine the effect of TGF-beta on antigen-specific proliferation, the activation status of the T cells and their cytokine production. This study demonstrates that TGF-beta is an adequate suppressor of antigen-specific T cell proliferation, by reducing the cell-cycle rate rather than induction of apoptosis. Addition of TGF-beta resulted in increased CD69 expression and decreased CD25 expression on T cells, indicating that TGF-beta is able to modulate the activation status of in vivo differentiated T cells. On the contrary, the antigen-specific cytokine production was not affected by TGF-beta. Although TGF-beta was suppressive towards the majority of the T cells, insensitivity of a few TCC towards TGF-beta was also observed. This could not be correlated to differential expression of TGF-beta signaling molecules such as Smad3, Smad7, SARA (Smad anchor for receptor activation) and Hgs (hepatocyte growth factor-regulated tyrosine kinase substrate). In summary, TGF-beta has a pronounced inhibitory effect on antigen-specific T cell proliferation without modulating their cytokine production.

Self-Amplifying mRNA Vaccines Expressing Multiple Conserved Influenza Antigens Confer Protection against Homologous and Heterosubtypic Viral Challenge

PubMed Central

Magini, Diletta; Giovani, Cinzia; Mangiavacchi, Simona; Maccari, Silvia; Cecchi, Raffaella; Ulmer, Jeffrey B.; De Gregorio, Ennio; Geall, Andrew J.; Brazzoli, Michela; Bertholet, Sylvie

2016-01-01

Current hemagglutinin (HA)-based seasonal influenza vaccines induce vaccine strain-specific neutralizing antibodies that usually fail to provide protection against mismatched circulating viruses. Inclusion in the vaccine of highly conserved internal proteins such as the nucleoprotein (NP) and the matrix protein 1 (M1) was shown previously to increase vaccine efficacy by eliciting cross-reactive T-cells. However, appropriate delivery systems are required for efficient priming of T-cell responses. In this study, we demonstrated that administration of novel self-amplifying mRNA (SAM®) vectors expressing influenza NP (SAM(NP)), M1 (SAM(M1)), and NP and M1 (SAM(M1-NP)) delivered with lipid nanoparticles (LNP) induced robust polyfunctional CD4 T helper 1 cells, while NP-containing SAM also induced cytotoxic CD8 T cells. Robust expansions of central memory (TCM) and effector memory (TEM) CD4 and CD8 T cells were also measured. An enhanced recruitment of NP-specific cytotoxic CD8 T cells was observed in the lungs of SAM(NP)-immunized mice after influenza infection that paralleled with reduced lung viral titers and pathology, and increased survival after homologous and heterosubtypic influenza challenge. Finally, we demonstrated for the first time that the co-administration of RNA (SAM(M1-NP)) and protein (monovalent inactivated influenza vaccine (MIIV)) was feasible, induced simultaneously NP-, M1- and HA-specific T cells and HA-specific neutralizing antibodies, and enhanced MIIV efficacy against a heterologous challenge. In conclusion, systemic administration of SAM vectors expressing conserved internal influenza antigens induced protective immune responses in mice, supporting the SAM® platform as another promising strategy for the development of broad-spectrum universal influenza vaccines. PMID:27525409

Induction and function of virus-specific CD4+ T cell responses

PubMed Central

Whitmire, Jason K.

2010-01-01

CD4+ T cells -- often referred to as T-helper cells -- play a central role in immune defense and pathogenesis. Virus infections and vaccines stimulate and expand populations of antigen-specific CD4+ T cells in mice and in man. These virus-specific CD4+ T cells are extremely important in antiviral protection: deficiencies in CD4+ T cells are associated with virus reactivation, generalized susceptibility to opportunistic infections, and poor vaccine efficacy. As described below, CD4+ T cells influence effector and memory CD8+ T cell responses, humoral immunity, and the antimicrobial activity of macrophages and are involved in recruiting cells to sites of infection. This review summarizes a few key points about the dynamics of the CD4+ T cell response to virus infection, the positive role of pro-inflammatory cytokines in the differentiation of virus-specific CD4+ T cells, and new areas of investigation to improve vaccines against virus infection. PMID:21236461

Langerhans cells utilize CD1a and langerin to efficiently present nonpeptide antigens to T cells

PubMed Central

Hunger, Robert E.; Sieling, Peter A.; Ochoa, Maria Teresa; Sugaya, Makoto; Burdick, Anne E.; Rea, Thomas H.; Brennan, Patrick J.; Belisle, John T.; Blauvelt, Andrew; Porcelli, Steven A.; Modlin, Robert L.

2004-01-01

Langerhans cells (LCs) constitute a subset of DCs that initiate immune responses in skin. Using leprosy as a model, we investigated whether expression of CD1a and langerin, an LC-specific C-type lectin, imparts a specific functional role to LCs. LC-like DCs and freshly isolated epidermal LCs presented nonpeptide antigens of Mycobacterium leprae to T cell clones derived from a leprosy patient in a CD1a-restricted and langerin-dependent manner. LC-like DCs were more efficient at CD1a-restricted antigen presentation than monocyte-derived DCs. LCs in leprosy lesions coexpress CD1a and langerin, placing LCs in position to efficiently present a subset of antigens to T cells as part of the host response to human infectious disease. PMID:14991068

Quantitative impact of thymic selection on Foxp3+ and Foxp3- subsets of self-peptide/MHC class II-specific CD4+ T cells.

PubMed

Moon, James J; Dash, Pradyot; Oguin, Thomas H; McClaren, Jennifer L; Chu, H Hamlet; Thomas, Paul G; Jenkins, Marc K

2011-08-30

It is currently thought that T cells with specificity for self-peptide/MHC (pMHC) ligands are deleted during thymic development, thereby preventing autoimmunity. In the case of CD4(+) T cells, what is unclear is the extent to which self-peptide/MHC class II (pMHCII)-specific T cells are deleted or become Foxp3(+) regulatory T cells. We addressed this issue by characterizing a natural polyclonal pMHCII-specific CD4(+) T-cell population in mice that either lacked or expressed the relevant antigen in a ubiquitous pattern. Mice expressing the antigen contained one-third the number of pMHCII-specific T cells as mice lacking the antigen, and the remaining cells exhibited low TCR avidity. In mice lacking the antigen, the pMHCII-specific T-cell population was dominated by phenotypically naive Foxp3(-) cells, but also contained a subset of Foxp3(+) regulatory cells. Both Foxp3(-) and Foxp3(+) pMHCII-specific T-cell numbers were reduced in mice expressing the antigen, but the Foxp3(+) subset was more resistant to changes in number and TCR repertoire. Therefore, thymic selection of self-pMHCII-specific CD4(+) T cells results in incomplete deletion within the normal polyclonal repertoire, especially among regulatory T cells.

Antigen-specific and non-specific CD4{sup +} T cell recruitment and proliferation during influenza infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chapman, Timothy J.; Castrucci, Maria R.; Padrick, Ryan C.

To track epitope-specific CD4{sup +} T cells at a single-cell level during influenza infection, the MHC class II-restricted OVA{sub 323-339} epitope was engineered into the neuraminidase stalk of influenza/A/WSN, creating a surrogate viral antigen. The recombinant virus, influenza A/WSN/OVA{sub II}, replicated well, was cleared normally, and stimulated both wild-type and DO11.10 or OT-II TCR transgenic OVA-specific CD4{sup +} T cells. OVA-specific CD4 T cells proliferated during infection only when the OVA epitope was present. However, previously primed (but not naive) transgenic CD4{sup +} T cells were recruited to the infected lung both in the presence and absence of the OVA{submore » 323-339} epitope. These data show that, when primed, CD4{sup +} T cells may traffic to the lung in the absence of antigen, but do not proliferate. These results also document a useful tool for the study of CD4 T cells in influenza infection.« less

Tumour cell lysate-loaded dendritic cell vaccine induces biochemical and memory immune response in castration-resistant prostate cancer patients

PubMed Central

Reyes, D; Salazar, L; Espinoza, E; Pereda, C; Castellón, E; Valdevenito, R; Huidobro, C; Inés Becker, M; Lladser, A; López, M N; Salazar-Onfray, F

2013-01-01

Background: Recently, we produced a tumour antigen-presenting cells (TAPCells) vaccine using a melanoma cell lysate, called TRIMEL, as an antigen source and an activation factor. Tumour antigen-presenting cells induced immunological responses and increased melanoma patient survival. Herein, we investigated the effect of TAPCells loaded with prostate cancer cell lysates (PCCL) as an antigen source, and TRIMEL as a dendritic cell (DC) activation factor; which were co-injected with the Concholepas concholepas haemocyanin (CCH) as an adjuvant on castration-resistant prostate cancer (CRPC) patients. Methods: The lysate mix capacity, for inducing T-cell activation, was analysed by flow cytometry and Elispot. Delayed-type hypersensitivity (DTH) reaction against PCCL, frequency of CD8+ memory T cells (Tm) in blood and prostate-specific antigen (PSA) levels in serum were measured in treated patients. Results: The lysate mix induced functional mature DCs that were capable of activating PCCL-specific T cells. No relevant adverse reactions were observed. Six out of 14 patients showed a significant decrease in levels of PSA. DTH+ patients showed a prolonged PSA doubling-time after treatment. Expansion of functional central and effector CD8+ Tm were detected. Conclusion: Treatment of CRPC patients with lysate-loaded TAPCells and CCH as an adjuvant is safe: generating biochemical and memory immune responses. However, the limited number of cases requires confirmation in a phase II clinical trial. PMID:23989944

Tumour cell lysate-loaded dendritic cell vaccine induces biochemical and memory immune response in castration-resistant prostate cancer patients.

PubMed

Reyes, D; Salazar, L; Espinoza, E; Pereda, C; Castellón, E; Valdevenito, R; Huidobro, C; Inés Becker, M; Lladser, A; López, M N; Salazar-Onfray, F

2013-09-17

Recently, we produced a tumour antigen-presenting cells (TAPCells) vaccine using a melanoma cell lysate, called TRIMEL, as an antigen source and an activation factor. Tumour antigen-presenting cells induced immunological responses and increased melanoma patient survival. Herein, we investigated the effect of TAPCells loaded with prostate cancer cell lysates (PCCL) as an antigen source, and TRIMEL as a dendritic cell (DC) activation factor; which were co-injected with the Concholepas concholepas haemocyanin (CCH) as an adjuvant on castration-resistant prostate cancer (CRPC) patients. The lysate mix capacity, for inducing T-cell activation, was analysed by flow cytometry and Elispot. Delayed-type hypersensitivity (DTH) reaction against PCCL, frequency of CD8(+) memory T cells (Tm) in blood and prostate-specific antigen (PSA) levels in serum were measured in treated patients. The lysate mix induced functional mature DCs that were capable of activating PCCL-specific T cells. No relevant adverse reactions were observed. Six out of 14 patients showed a significant decrease in levels of PSA. DTH(+) patients showed a prolonged PSA doubling-time after treatment. Expansion of functional central and effector CD8(+) Tm were detected. Treatment of CRPC patients with lysate-loaded TAPCells and CCH as an adjuvant is safe: generating biochemical and memory immune responses. However, the limited number of cases requires confirmation in a phase II clinical trial.

Pathogen boosted adoptive cell transfer immunotherapy to treat solid tumors.

PubMed

Xin, Gang; Schauder, David M; Jing, Weiqing; Jiang, Aimin; Joshi, Nikhil S; Johnson, Bryon; Cui, Weiguo

2017-01-24

Because of insufficient migration and antitumor function of transferred T cells, especially inside the immunosuppressive tumor microenvironment (TME), the efficacy of adoptive cell transfer (ACT) is much curtailed in treating solid tumors. To overcome these challenges, we sought to reenergize ACT (ReACT) with a pathogen-based cancer vaccine. To bridge ACT with a pathogen, we genetically engineered tumor-specific CD8 T cells in vitro with a second T-cell receptor (TCR) that recognizes a bacterial antigen. We then transferred these dual-specific T cells in combination with intratumoral bacteria injection to treat solid tumors in mice. The dual-specific CD8 T cells expanded vigorously, migrated to tumor sites, and robustly eradicated primary tumors. The mice cured from ReACT also developed immunological memory against tumor rechallenge. Mechanistically, we have found that this combined approach reverts the immunosuppressive TME and recruits CD8 T cells with an increased number and killing ability to the tumors.

Efficient Capsid Antigen Presentation From Adeno-Associated Virus Empty Virions In Vivo.

PubMed

Pei, Xiaolei; Earley, Lauriel Freya; He, Yi; Chen, Xiaojing; Hall, Nikita Elexa; Samulski, Richard Jude; Li, Chengwen

2018-01-01

Adeno-associated virus (AAV) vectors have been successfully applied in clinical trials for hemophilic patients. Although promising, the clinical results suggest that the capsid-specific CD8+T cell response has a negative effect on therapeutic success. In an in vitro analysis using an engineered AAV virus carrying immune-dominant SIINFEKL peptide in the capsid backbone, we have previously demonstrated that capsid antigen presentation from full (genome containing) AAV capsids requires endosome escape and is proteasome dependent and that no capsid antigen presentation is induced from empty virions. In the present study, we examined capsid antigen presentation from administration of empty virions in animal models. In wild-type mice, similar to AAV full particles, capsid antigen presentation from AAV empty virion infection was dose dependent, and the kinetics studies showed that antigen presentation was detected from 2 to 40 days after AAV empty virion administration. In the transporter associated with antigen processing 1 deficient (TAP-/-) mice, capsid antigen presentation was inhibited from both AAV full and empty virions, but higher inhibition was achieved from AAV full particle administration than that from empty virions. This indicates that the pathway of capsid antigen presentation from AAV transduction is dependent on proteasome-mediated degradation of AAV capsids (mainly for full particles) and that the endosomal pathway may also play a role in antigen presentation from empty particles but not full virions. The capsid antigen presentation efficiency from AAV preparations was positively correlated with the amount of empty virions contaminated with full particles. Collectively, the results indicate that contamination of AAV empty virions induces efficient antigen presentation in vivo and the mechanism of capsid antigen presentation from empty virions involves both endosomal and proteasomal pathways. The elucidation of capsid antigen presentation from AAV empty virions may allow us to rationally design effective strategies to prevent elimination of AAV transduced target cells by capsid specific CD8+ T cells.

Efficient Capsid Antigen Presentation From Adeno-Associated Virus Empty Virions In Vivo

PubMed Central

Pei, Xiaolei; Earley, Lauriel Freya; He, Yi; Chen, Xiaojing; Hall, Nikita Elexa; Samulski, Richard Jude; Li, Chengwen

2018-01-01

Adeno-associated virus (AAV) vectors have been successfully applied in clinical trials for hemophilic patients. Although promising, the clinical results suggest that the capsid-specific CD8+T cell response has a negative effect on therapeutic success. In an in vitro analysis using an engineered AAV virus carrying immune-dominant SIINFEKL peptide in the capsid backbone, we have previously demonstrated that capsid antigen presentation from full (genome containing) AAV capsids requires endosome escape and is proteasome dependent and that no capsid antigen presentation is induced from empty virions. In the present study, we examined capsid antigen presentation from administration of empty virions in animal models. In wild-type mice, similar to AAV full particles, capsid antigen presentation from AAV empty virion infection was dose dependent, and the kinetics studies showed that antigen presentation was detected from 2 to 40 days after AAV empty virion administration. In the transporter associated with antigen processing 1 deficient (TAP−/−) mice, capsid antigen presentation was inhibited from both AAV full and empty virions, but higher inhibition was achieved from AAV full particle administration than that from empty virions. This indicates that the pathway of capsid antigen presentation from AAV transduction is dependent on proteasome-mediated degradation of AAV capsids (mainly for full particles) and that the endosomal pathway may also play a role in antigen presentation from empty particles but not full virions. The capsid antigen presentation efficiency from AAV preparations was positively correlated with the amount of empty virions contaminated with full particles. Collectively, the results indicate that contamination of AAV empty virions induces efficient antigen presentation in vivo and the mechanism of capsid antigen presentation from empty virions involves both endosomal and proteasomal pathways. The elucidation of capsid antigen presentation from AAV empty virions may allow us to rationally design effective strategies to prevent elimination of AAV transduced target cells by capsid specific CD8+ T cells. PMID:29725339

Characterization of Human CD8 T Cell Responses in Dengue Virus-Infected Patients from India

PubMed Central

Chandele, Anmol; Sewatanon, Jaturong; Gunisetty, Sivaram; Singla, Mohit; Onlamoon, Nattawat; Akondy, Rama S.; Kissick, Haydn Thomas; Nayak, Kaustuv; Reddy, Elluri Seetharami; Kalam, Haroon; Kumar, Dhiraj; Verma, Anil; Panda, HareKrushna; Wang, Siyu; Angkasekwinai, Nasikarn; Pattanapanyasat, Kovit; Chokephaibulkit, Kulkanya; Lodha, Rakesh; Kabra, Sushil; Ahmed, Rafi

2016-01-01

ABSTRACT Epidemiological studies suggest that India has the largest number of dengue virus infection cases worldwide. However, there is minimal information about the immunological responses in these patients. CD8 T cells are important in dengue, because they have been implicated in both protection and immunopathology. Here, we provide a detailed analysis of HLA-DR+ CD38+ and HLA-DR− CD38+ effector CD8 T cell subsets in dengue patients from India and Thailand. Both CD8 T cell subsets expanded and expressed markers indicative of antigen-driven proliferation, tissue homing, and cytotoxic effector functions, with the HLA-DR+ CD38+ subset being the most striking in these effector qualities. The breadth of the dengue-specific CD8 T cell response was diverse, with NS3-specific cells being the most dominant. Interestingly, only a small fraction of these activated effector CD8 T cells produced gamma interferon (IFN-γ) when stimulated with dengue virus peptide pools. Transcriptomics revealed downregulation of key molecules involved in T cell receptor (TCR) signaling. Consistent with this, the majority of these CD8 T cells remained IFN-γ unresponsive even after TCR-dependent polyclonal stimulation (anti-CD3 plus anti-CD28) but produced IFN-γ by TCR-independent polyclonal stimulation (phorbol 12-myristate 13-acetate [PMA] plus ionomycin). Thus, the vast majority of these proliferating, highly differentiated effector CD8 T cells probably acquire TCR refractoriness at the time the patient is experiencing febrile illness that leads to IFN-γ unresponsiveness. Our studies open novel avenues for understanding the mechanisms that fine-tune the balance between CD8 T cell-mediated protective versus pathological effects in dengue. IMPORTANCE Dengue is becoming a global public health concern. Although CD8 T cells have been implicated both in protection and in the cytokine-mediated immunopathology of dengue, how the balance is maintained between these opposing functions remains unknown. We comprehensively characterized CD8 T cell subsets in dengue patients from India and Thailand and show that these cells expand massively and express phenotypes indicative of overwhelming antigenic stimulus and tissue homing/cytotoxic-effector functions but that a vast majority of them fail to produce IFN-γ in vitro. Interestingly, the cells were fully capable of producing the cytokine when stimulated in a T cell receptor (TCR)-independent manner but failed to do so in TCR-dependent stimulation. These results, together with transcriptomics, revealed that the vast majority of these CD8 T cells from dengue patients become cytokine unresponsive due to TCR signaling insufficiencies. These observations open novel avenues for understanding the mechanisms that fine-tune the balance between CD8-mediated protective versus pathological effects. PMID:27707928

Cetuximab-activated natural killer (NK) and dendritic cells (DC) collaborate to trigger tumor antigen-specific T cell immunity in head and neck cancer patients

PubMed Central

Srivastava, Raghvendra M.; Lee, Steve C.; Filho, Pedro A. Andrade; Lord, Christopher A.; Jie, Hyun-bae; Davidson, H. Carter; López-Albaitero, Andrés; Gibson, Sandra P.; Gooding, William E.; Ferrone, Soldano; Ferris, Robert L.

2013-01-01

Purpose Tumor antigen (TA)-specific monoclonal antibodies (mAb) block oncogenic signaling and induce Fcγ receptor (FcγR)-mediated cytotoxicity. However, the role of CD8+ cytotoxic T lymphocyte (CTL) and FcγR in initiating innate and adaptive immune responses in mAb-treated human cancer patients is still emerging. Experimental Design FcγRIIIa codon 158 polymorphism was correlated with survival in 107 cetuximab-treated head and neck cancer (HNC) patients. Flow cytometry was performed to quantify EGFR-specific T cells in cetuximab-treated HNC patients. The effect of cetuximab on NK cell, dendritic cell (DC), and T cell activation was measured using IFN-γ release assays and flow cytometry. Results FcγR IIIa polymorphism did not predict clinical outcome in cetuximab-treated HNC patients, however elevated circulating EGFR -specific CD8+ 853-861 T cells were found in cetuximab-treated HNC patients (p<0.005). Cetuximab promoted EGFR-specific cellular immunity through the interaction of EGFR+ tumor cells and FcγRIIIa on NK cells, but not on the polymorphism per se. Cetuximab-activated NK cells induced IFN-γ dependent expression of DC maturation markers, antigen presentation machinery (APM) components such as TAP-1/2, and Th1 chemokines through NKG2D/MICA binding. Cetuximab initiated adaptive immune responses via NK-cell induced DC maturation, which enhanced cross-presentation to CTL specific for EGFR as well as another TA, MAGE-3. Conclusion Cetuximab-activated NK cells promote DC maturation and CD8+ T cell priming, leading to TA spreading and Th1 cytokine release through ‘NK-DC cross-talk.’ FcγRIIIa polymorphism did not predict clinical response to cetuximab, but was necessary for NK-DC interaction and mAb induced cross-presentation. EGFR-specific T cells in cetuximab treated HNC patients may contribute to clinical response. PMID:23444227

Immunity in the spleen and blood of mice immunized with irradiated Toxoplasma gondii tachyzoites.

PubMed

Zorgi, Nahiara Esteves; Galisteo, Andrés Jimenez; Sato, Maria Notomi; do Nascimento, Nanci; de Andrade, Heitor Franco

2016-08-01

Toxoplasma gondii infection induces a strong and long-lasting immune response that is able to prevent most reinfections but allows tissue cysts. Irradiated, sterilized T. gondii tachyzoites are an interesting vaccine, and they induce immunity that is similar to infection, but without cysts. In this study, we evaluated the cellular immune response in the blood and spleen of mice immunized with this preparation by mouth (v.o.) or intraperitoneally (i.p.) and analyzed the protection after challenge with viable parasites. BALB/c mice were immunized with three i.p. or v.o. doses of irradiated T. gondii tachyzoites. Oral challenge with ten cysts of the ME-49 or VEG strain at 90 days after the last dose resulted in high levels of protection with low parasite burden in the immunized animals. There were higher levels of specific IgG, IgA and IgM antibodies in the serum, and the i.p. immunized mice had higher levels of the high-affinity IgG and IgM antibodies than the orally immunized mice, which had more high-affinity IgA antibodies. B cells (CD19(+)), plasma cells (CD138(+)) and the CD4(+) and CD8(+) T cell populations were increased in both the blood and spleen. Cells from the spleen of the i.p. immunized mice also showed antigen-induced production of interleukin-10 (IL-10), interferon gamma (IFN-γ) and interleukin 4 (IL-4). The CD4(+) T cells, B cells and likely CD8(+) T cells from the spleens of the i.p. immunized mice proliferated with a specific antigen. The protection was correlated with the spleen and blood CD8(+) T cell, high-affinity IgG and IgM and antigen-induced IL-10 and IL-4 production. Immunization with irradiated T. gondii tachyzoites induces an immune response that is mediated by B cells and CD4(+) and CD8(+) T cells, with increased humoral and cellular immune responses that are necessary for host protection after infection. The vaccine is similar to natural infection, but free of tissue cysts; this immunity restrains infection at challenge and can be an attractive and efficient model for vaccine development in toxoplasmosis.

Dysfunctional BLK in common variable immunodeficiency perturbs B-cell proliferation and ability to elicit antigen-specific CD4+ T-cell help.

PubMed

Compeer, Ewoud B; Janssen, Willemijn; van Royen-Kerkhof, Annet; van Gijn, Marielle; van Montfrans, Joris M; Boes, Marianne

2015-05-10

Common Variable Immunodeficiency (CVID) is the most prevalent primary antibody deficiency, and characterized by defective generation of high-affinity antibodies. Patients have therefore increased risk to recurrent infections of the respiratory and intestinal tract. Development of high-affinity antigen-specific antibodies involves two key actions of B-cell receptors (BCR): transmembrane signaling through BCR-complexes to induce B-cell differentiation and proliferation, and BCR-mediated antigen internalization for class-II MHC-mediated presentation to acquire antigen-specific CD4(+) T-cell help.We identified a variant (L3P) in the B-lymphoid tyrosine kinase (BLK) gene of 2 related CVID-patients, which was absent in healthy relatives. BLK belongs to the Src-kinases family and involved in BCR-signaling. Here, we sought to clarify BLK function in healthy human B-cells and its association to CVID.BLK expression was comparable in patient and healthy B-cells. Functional analysis of L3P-BLK showed reduced BCR crosslinking-induced Syk phosphorylation and proliferation, in both primary B-cells and B-LCLs. B-cells expressing L3P-BLK showed accelerated destruction of BCR-internalized antigen and reduced ability to elicit CD40L-expression on antigen-specific CD4(+) T-cells.In conclusion, we found a novel BLK gene variant in CVID-patients that causes suppressed B-cell proliferation and reduced ability of B-cells to elicit antigen-specific CD4(+) T-cell responses. Both these mechanisms may contribute to hypogammaglobulinemia in CVID-patients.

Antigen Availability Shapes T Cell Differentiation and Function during Tuberculosis.

PubMed

Moguche, Albanus O; Musvosvi, Munyaradzi; Penn-Nicholson, Adam; Plumlee, Courtney R; Mearns, Helen; Geldenhuys, Hennie; Smit, Erica; Abrahams, Deborah; Rozot, Virginie; Dintwe, One; Hoff, Søren T; Kromann, Ingrid; Ruhwald, Morten; Bang, Peter; Larson, Ryan P; Shafiani, Shahin; Ma, Shuyi; Sherman, David R; Sette, Alessandro; Lindestam Arlehamn, Cecilia S; McKinney, Denise M; Maecker, Holden; Hanekom, Willem A; Hatherill, Mark; Andersen, Peter; Scriba, Thomas J; Urdahl, Kevin B

2017-06-14

CD4 T cells are critical for protective immunity against Mycobacterium tuberculosis (Mtb), the cause of tuberculosis (TB). Yet to date, TB vaccine candidates that boost antigen-specific CD4 T cells have conferred little or no protection. Here we examined CD4 T cell responses to two leading TB vaccine antigens, ESAT-6 and Ag85B, in Mtb-infected mice and in vaccinated humans with and without underlying Mtb infection. In both species, Mtb infection drove ESAT-6-specific T cells to be more differentiated than Ag85B-specific T cells. The ability of each T cell population to control Mtb in the lungs of mice was restricted for opposite reasons: Ag85B-specific T cells were limited by reduced antigen expression during persistent infection, whereas ESAT-6-specific T cells became functionally exhausted due to chronic antigenic stimulation. Our findings suggest that different vaccination strategies will be required to optimize protection mediated by T cells recognizing antigens expressed at distinct stages of Mtb infection. Copyright © 2017 Elsevier Inc. All rights reserved.

On-chip activation and subsequent detection of individual antigen-specific T cells

PubMed Central

Song, Qing; Han, Qing; Bradshaw, Elizabeth M.; Kent, Sally C.; Raddassi, Khadir; Nilsson, Björn; Nepom, Gerald T.; Hafler, David A.; Love, J. Christopher

2010-01-01

The frequencies of antigen-specific CD4+ T cells in samples of human tissue has been difficult to determine accurately ex vivo, particularly for autoimmune diseases such as multiple sclerosis or Type 1 diabetes. Conventional approaches involve the expansion of primary T cells in vitro to increase the numbers of cells, and a subsequent assessment of the frequencies of antigen-specific T cells in the expanded population by limiting dilution or by using fluorescently labeled tetramers of peptide-loaded major histocompatibility complex (MHC) receptors. Here we describe an alternative approach that uses arrays of subnanoliter wells coated with recombinant peptide-loaded MHC Class II monomers to isolate and stimulate individual CD4+ T cells in an antigen-specific manner. In these experiments, activation was monitored using microengraving to capture two cytokines (IFNγ and IL-17) released from single cells. This new method should enable direct enumeration of antigen-specific CD4+ T cells ex vivo from clinical samples. PMID:20000848

Antigen-Specific CD8+ T Cells and Protective Immunity to Tuberculosis

PubMed Central

2017-01-01

The continuing HIV/AIDS epidemic and the spread of multi-drug resistant Mycobacterium tuberculosis has led to the perpetuation of the worldwide tuberculosis epidemic. While M. bovis BCG is widely used as a vaccine, it lacks efficacy in preventing pulmonary tuberculosis in adults [1]. To combat this ongoing scourge, vaccine development for tuberculosis is a global priority. Most infected individuals develop long-lived protective immunity, which controls and contains M. tuberculosis in a T cell-dependent manner. An effective T cells response determines whether the infection resolves or develops into clinically evident disease. Consequently, there is great interest in determining which T cells subsets mediate anti-mycobacterial immunity, delineating their effector functions, and evaluating whether vaccination can elicit these T cells subsets and induce protective immunity. CD4+ T cells are critical for resistance to M. tuberculosis in both humans and rodent models. CD4+ T cells are required to control the initial infection as well as to prevent recrudescence in both humans and mice [2]. While it is generally accepted that class II MHC-restricted CD4+ T cells are essential for immunity to tuberculosis, M. tuberculosis infection elicits CD8+ T cells responses in both people and in experimental animals. CD8+ T cells are also recruited to the lung during M. tuberculosis infection and are found in the granulomas of infected people. Thus, how CD8+ T cells contribute to overall immunity to tuberculosis and whether antigens recognized by CD8+ T cells would enhance the efficacy of vaccine strategies continue to be important questions. PMID:23468108

Systemic Tolerance Mediated by Melanoma Brain Tumors is Reversible by Radiotherapy and Vaccination

PubMed Central

Jackson, Christopher M.; Kochel, Christina M.; Nirschl, Christopher J.; Durham, Nicholas M.; Ruzevick, Jacob; Alme, Angela; Francica, Brian J.; Elias, Jimmy; Daniels, Andrew; Dubensky, Thomas W.; Lauer, Peter; Brockstedt, Dirk G.; Baxi, Emily G.; Calabresi, Peter A.; Taube, Janis M.; Pardo, Carlos A.; Brem, Henry; Pardoll, Drew M.; Lim, Michael; Drake, Charles G.

2016-01-01

Purpose Immune responses to antigens originating in the CNS are generally attenuated, since collateral damage can have devastating consequences. The significance of this finding for the efficacy of tumor-targeted immunotherapies is largely unknown. Experimental Design The B16 murine melanoma model was used to compare cytotoxic responses against established tumors in the CNS and in the periphery. Cytokine analysis of tissues from brain tumor-bearing mice detected elevated TGF-β secretion from microglia and in the serum and TGF-β signaling blockade reversed tolerance of tumor antigen-directed CD8 T cells. Additionally, a treatment regimen using focal radiation therapy and recombinant Listeria monocytogenes was evaluated for immunologic activity and efficacy in this model. Results CNS melanomas were more tolerogenic than equivalently progressed tumors outside the CNS as antigen-specific CD8 T cells were deleted and exhibited impaired cytotoxicity. Tumor-bearing mice had elevated serum levels of TGF-β; however, blocking TGF-β signaling with a small molecule inhibitor or a monoclonal antibody did not improve survival. Conversely, tumor antigen-specific vaccination in combination with focal radiation therapy reversed tolerance and improved survival. This treatment regimen was associated with increased polyfunctionality of CD8 T cells, elevated T effector to T regulatory cell ratios and decreased TGF-β secretion from microglia. Conclusions These data suggest that CNS tumors may impair systemic antitumor immunity and consequently accelerate cancer progression locally as well as outside the CNS while antitumor immunity may be restored by combining vaccination with radiation therapy. These findings are hypothesis-generating and warrant further study in more contemporary melanoma models as well as human trials. PMID:26490306

Immunologic hierarchy, class II MHC promiscuity, and epitope spreading of a melanoma helper peptide vaccine.

PubMed

Hu, Yinin; Petroni, Gina R; Olson, Walter C; Czarkowski, Andrea; Smolkin, Mark E; Grosh, William W; Chianese-Bullock, Kimberly A; Slingluff, Craig L

2014-08-01

Immunization with a combination melanoma helper peptide (6MHP) vaccine has been shown to induce CD4(+) T cell responses, which are associated with patient survival. In the present study, we define the relative immunogenicity and HLA allele promiscuity of individual helper peptides and identify helper peptide-mediated augmentation of specific CD8(+) T cell responses. Thirty-seven participants with stage IIIB-IV melanoma were vaccinated with 6MHP in incomplete Freund's adjuvant. The 6MHP vaccine is comprised of 6 peptides representing melanocytic differentiation proteins gp100, tyrosinase, Melan-A/MART-1, and cancer testis antigens from the MAGE family. CD4(+) and CD8(+) T cell responses were assessed in peripheral blood and in sentinel immunized nodes (SIN) by thymidine uptake after exposure to helper peptides and by direct interferon-γ ELIspot assay against 14 MHC class I-restricted peptides. Vaccine-induced CD4(+) T cell responses to individual epitopes were detected in the SIN of 63 % (22/35) and in the peripheral blood of 38 % (14/37) of participants for an overall response rate of 65 % (24/37). The most frequently immunogenic peptides were MAGE-A3281-295 (49 %) and tyrosinase386-406 (32 %). Responses were not limited to HLA restrictions originally described. Vaccine-associated CD8(+) T cell responses against class I-restricted peptides were observed in 45 % (5/11) of evaluable participants. The 6MHP vaccine induces both CD4(+) and CD8(+) T cell responses against melanoma antigens. CD4(+) T cell responses were detected beyond reported HLA-DR restrictions. Induction of CD8(+) T cell responses suggests epitope spreading and systemic activity mediated at the tumor site.

Therapeutic cancer vaccines

PubMed Central

Melief, Cornelis J.M.; van Hall, Thorbald; Arens, Ramon; Ossendorp, Ferry; van der Burg, Sjoerd H.

2015-01-01

The clinical benefit of therapeutic cancer vaccines has been established. Whereas regression of lesions was shown for premalignant lesions caused by HPV, clinical benefit in cancer patients was mostly noted as prolonged survival. Suboptimal vaccine design and an immunosuppressive cancer microenvironment are the root causes of the lack of cancer eradication. Effective cancer vaccines deliver concentrated antigen to both HLA class I and II molecules of DCs, promoting both CD4 and CD8 T cell responses. Optimal vaccine platforms include DNA and RNA vaccines and synthetic long peptides. Antigens of choice include mutant sequences, selected cancer testis antigens, and viral antigens. Drugs or physical treatments can mitigate the immunosuppressive cancer microenvironment and include chemotherapeutics, radiation, indoleamine 2,3-dioxygenase (IDO) inhibitors, inhibitors of T cell checkpoints, agonists of selected TNF receptor family members, and inhibitors of undesirable cytokines. The specificity of therapeutic vaccination combined with such immunomodulation offers an attractive avenue for the development of future cancer therapies. PMID:26214521

Dendritic Cells Enhance Polyfunctionality of Adoptively Transferred T Cells That Target Cytomegalovirus in Glioblastoma.

PubMed

Reap, Elizabeth A; Suryadevara, Carter M; Batich, Kristen A; Sanchez-Perez, Luis; Archer, Gary E; Schmittling, Robert J; Norberg, Pamela K; Herndon, James E; Healy, Patrick; Congdon, Kendra L; Gedeon, Patrick C; Campbell, Olivia C; Swartz, Adam M; Riccione, Katherine A; Yi, John S; Hossain-Ibrahim, Mohammed K; Saraswathula, Anirudh; Nair, Smita K; Dunn-Pirio, Anastasie M; Broome, Taylor M; Weinhold, Kent J; Desjardins, Annick; Vlahovic, Gordana; McLendon, Roger E; Friedman, Allan H; Friedman, Henry S; Bigner, Darell D; Fecci, Peter E; Mitchell, Duane A; Sampson, John H

2018-01-01

Median survival for glioblastoma (GBM) remains <15 months. Human cytomegalovirus (CMV) antigens have been identified in GBM but not normal brain, providing an unparalleled opportunity to subvert CMV antigens as tumor-specific immunotherapy targets. A recent trial in recurrent GBM patients demonstrated the potential clinical benefit of adoptive T-cell therapy (ATCT) of CMV phosphoprotein 65 (pp65)-specific T cells. However, ex vivo analyses from this study found no change in the capacity of CMV pp65-specific T cells to gain multiple effector functions or polyfunctionality, which has been associated with superior antitumor efficacy. Previous studies have shown that dendritic cells (DC) could further enhance tumor-specific CD8 + T-cell polyfunctionality in vivo when administered as a vaccine. Therefore, we hypothesized that vaccination with CMV pp65 RNA-loaded DCs would enhance the frequency of polyfunctional CMV pp65-specific CD8 + T cells after ATCT. Here, we report prospective results of a pilot trial in which 22 patients with newly diagnosed GBM were initially enrolled, of which 17 patients were randomized to receive CMV pp65-specific T cells with CMV-DC vaccination (CMV-ATCT-DC) or saline (CMV-ATCT-saline). Patients who received CMV-ATCT-DC vaccination experienced a significant increase in the overall frequencies of IFNγ + , TNFα + , and CCL3 + polyfunctional, CMV-specific CD8 + T cells. These increases in polyfunctional CMV-specific CD8 + T cells correlated ( R = 0.7371, P = 0.0369) with overall survival, although we cannot conclude this was causally related. Our data implicate polyfunctional T-cell responses as a potential biomarker for effective antitumor immunotherapy and support a formal assessment of this combination approach in a larger randomized study. Significance: A randomized pilot trial in patients with GBM implicates polyfunctional T-cell responses as a biomarker for effective antitumor immunotherapy. Cancer Res; 78(1); 256-64. ©2017 AACR . ©2017 American Association for Cancer Research.

Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA–peptide tetramers

PubMed Central

Cobbold, Mark; Khan, Naeem; Pourgheysari, Batoul; Tauro, Sudhir; McDonald, Dorothy; Osman, Husam; Assenmacher, Mario; Billingham, Lucinda; Steward, Colin; Crawley, Charles; Olavarria, Eduardo; Goldman, John; Chakraverty, Ronjon; Mahendra, Premini; Craddock, Charles; Moss, Paul A.H.

2005-01-01

Stem cell transplantation is used widely in the management of a range of diseases of the hemopoietic system. Patients are immunosuppressed profoundly in the early posttransplant period, and reactivation of cytomegalovirus (CMV) remains a significant cause of morbidity and mortality. Adoptive transfer of donor-derived CMV-specific CD8+ T cell clones has been shown to reduce the rate of viral reactivation; however, the complexity of this approach severely limits its clinical application. We have purified CMV-specific CD8+ T cells from the blood of stem cell transplant donors using staining with HLA–peptide tetramers followed by selection with magnetic beads. CMV-specific CD8+ cells were infused directly into nine patients within 4 h of selection. Median cell dosage was 8.6 × 103/kg with a purity of 98% of all T cells. CMV-specific CD8+ T cells became detectable in all patients within 10 d of infusion, and TCR clonotype analysis showed persistence of infused cells in two patients studied. CMV viremia was reduced in every case and eight patients cleared the infection, including one patient who had a prolonged history of CMV infection that was refractory to antiviral therapy. This novel approach to adoptive transfer has considerable potential for antigen-specific T cell therapy. PMID:16061727

The effect of cyclosporin A, FK506 and rapamycin on the murine contact sensitivity reaction

PubMed Central

Salerno, A; Bonanno, C T; Caccamo, N; Cigna, D; Dominici, R; Ferro, C; Sireci, G; Dieli, F

1998-01-01

We have evaluated the effects of three potent immunosuppressive agents, cyclosporin A (CsA), FK506 and rapamycin, on the murine contact sensitivity (CS) reaction to the hapten trinitrochlorobenzene. Development of CS reaction requires participation of three distinct T cell subsets: αβ+, CD4+ T lymphocytes, which are the classical effector cell of the CS reaction, γδ+ T lymphocytes, and αβ+, double-negative (CD4− CD8−) T lymphocytes that express the B220 molecule and produce IL-4. We found that all three drugs inhibit the development of the CS reaction, but they affect different target cells. In fact, rapamycin and FK-506 block both αβ+, CD4+ and γδ+ T lymphocytes, while CsA inhibits only the αβ+, CD4+ T lymphocyte. None of the three drugs exerted any inhibitory activity on the αβ+, double-negative (CD4− CD8−) T lymphocytes. Hapten-immune lymph node cells from mice treated in vivo with CsA or FK506 failed to proliferate and to produce IL-2 when re-exposed to the specific antigen in vitro. In contrast, immune lymph node cells from mice that had been treated in vivo with rapamycin gave optimal antigen-specific proliferation and IL-2 production in vitro. The implications of these observations are discussed in relation to the use of these immunosuppressive agents for prevention of allograft rejection. PMID:9566798

Two Theileria parva CD8 T Cell Antigen Genes Are More Variable in Buffalo than Cattle Parasites, but Differ in Pattern of Sequence Diversity

PubMed Central

Pelle, Roger; Graham, Simon P.; Njahira, Moses N.; Osaso, Julius; Saya, Rosemary M.; Odongo, David O.; Toye, Philip G.; Spooner, Paul R.; Musoke, Anthony J.; Mwangi, Duncan M.; Taracha, Evans L. N.; Morrison, W. Ivan; Weir, William; Silva, Joana C.; Bishop, Richard P.

2011-01-01

Background Theileria parva causes an acute fatal disease in cattle, but infections are asymptomatic in the African buffalo (Syncerus caffer). Cattle can be immunized against the parasite by infection and treatment, but immunity is partially strain specific. Available data indicate that CD8+ T lymphocyte responses mediate protection and, recently, several parasite antigens recognised by CD8+ T cells have been identified. This study set out to determine the nature and extent of polymorphism in two of these antigens, Tp1 and Tp2, which contain defined CD8+ T-cell epitopes, and to analyse the sequences for evidence of selection. Methodology/Principal Findings Partial sequencing of the Tp1 gene and the full-length Tp2 gene from 82 T. parva isolates revealed extensive polymorphism in both antigens, including the epitope-containing regions. Single nucleotide polymorphisms were detected at 51 positions (∼12%) in Tp1 and in 320 positions (∼61%) in Tp2. Together with two short indels in Tp1, these resulted in 30 and 42 protein variants of Tp1 and Tp2, respectively. Although evidence of positive selection was found for multiple amino acid residues, there was no preferential involvement of T cell epitope residues. Overall, the extent of diversity was much greater in T. parva isolates originating from buffalo than in isolates known to be transmissible among cattle. Conclusions/Significance The results indicate that T. parva parasites maintained in cattle represent a subset of the overall T. parva population, which has become adapted for tick transmission between cattle. The absence of obvious enrichment for positively selected amino acid residues within defined epitopes indicates either that diversity is not predominantly driven by selection exerted by host T cells, or that such selection is not detectable by the methods employed due to unidentified epitopes elsewhere in the antigens. Further functional studies are required to address this latter point. PMID:21559495

Two Theileria parva CD8 T cell antigen genes are more variable in buffalo than cattle parasites, but differ in pattern of sequence diversity.

PubMed

Pelle, Roger; Graham, Simon P; Njahira, Moses N; Osaso, Julius; Saya, Rosemary M; Odongo, David O; Toye, Philip G; Spooner, Paul R; Musoke, Anthony J; Mwangi, Duncan M; Taracha, Evans L N; Morrison, W Ivan; Weir, William; Silva, Joana C; Bishop, Richard P

2011-04-29

Theileria parva causes an acute fatal disease in cattle, but infections are asymptomatic in the African buffalo (Syncerus caffer). Cattle can be immunized against the parasite by infection and treatment, but immunity is partially strain specific. Available data indicate that CD8(+) T lymphocyte responses mediate protection and, recently, several parasite antigens recognised by CD8(+) T cells have been identified. This study set out to determine the nature and extent of polymorphism in two of these antigens, Tp1 and Tp2, which contain defined CD8(+) T-cell epitopes, and to analyse the sequences for evidence of selection. Partial sequencing of the Tp1 gene and the full-length Tp2 gene from 82 T. parva isolates revealed extensive polymorphism in both antigens, including the epitope-containing regions. Single nucleotide polymorphisms were detected at 51 positions (∼12%) in Tp1 and in 320 positions (∼61%) in Tp2. Together with two short indels in Tp1, these resulted in 30 and 42 protein variants of Tp1 and Tp2, respectively. Although evidence of positive selection was found for multiple amino acid residues, there was no preferential involvement of T cell epitope residues. Overall, the extent of diversity was much greater in T. parva isolates originating from buffalo than in isolates known to be transmissible among cattle. The results indicate that T. parva parasites maintained in cattle represent a subset of the overall T. parva population, which has become adapted for tick transmission between cattle. The absence of obvious enrichment for positively selected amino acid residues within defined epitopes indicates either that diversity is not predominantly driven by selection exerted by host T cells, or that such selection is not detectable by the methods employed due to unidentified epitopes elsewhere in the antigens. Further functional studies are required to address this latter point.

Circulating rotavirus-specific T cells have a poor functional profile.

PubMed

Parra, Miguel; Herrera, Daniel; Jácome, María Fernanda; Mesa, Martha C; Rodríguez, Luz-Stella; Guzmán, Carolina; Angel, Juana; Franco, Manuel A

2014-11-01

Frequencies of circulating T cells producing IFN-γ, TNF-α, and IL-2, and percentages of T cells proliferating after stimulation with rotavirus (RV), tetanus toxoid, and influenza were evaluated in PBMC derived from healthy adults and children. In addition, the potential anergic state of RV-specific T cells was analyzed by stimulation of PBMC with RV antigen in the presence of three anergy inhibitors (rIL-2, rIL-12, or DGKα-i). The quality and magnitude of RV-T cell responses were significantly lower than those of tetanus toxoid and influenza antigens. RV-CD4 T cell response was enriched in monofunctional IFN-γ(+) cells, while influenza-CD4 and tetanus toxoid-CD4 T cell responses were enriched in multifunctional T cells. Moreover, rIL-2--unlike rIL-12 or DGKα-i--increased the frequencies of RV-CD4 TNF-α(+), CD4 IFN-γ(+), and CD8 IFN-γ(+) cells. Thus, circulating RV-T cells seem to have a relatively poor functional profile that may be partially reversed in vitro by the addition of rIL-2. Copyright © 2014 Elsevier Inc. All rights reserved.

T-cells from HLA-B*57:01+ human subjects are activated with abacavir through two independent pathways and induce cell death by multiple mechanisms.

PubMed

Bell, Catherine C; Faulkner, Lee; Martinsson, Klara; Farrell, John; Alfirevic, Ana; Tugwood, Jonathan; Pirmohamed, Munir; Naisbitt, Dean J; Park, B Kevin

2013-05-20

Susceptibility to abacavir hypersensitivity has been attributed to possession of the specific human leukocyte antigen allele HLA-B*57:01. HLA-B*57:01-restricted activation of CD8+ T-cells provides a link between the genetic association and the iatrogenic disease. The objectives of this study were to characterize the functionality of drug-responsive CD8+ T-cell clones generated from HLA-B*57:01+ drug-naive subjects and to explore the relationship between abacavir accumulation in antigen presenting cells and the T-cell response. Seventy-four CD8+ clones expressing different Vβ receptors were shown to proliferate and kill target cells via different mechanisms when exposed to abacavir. Certain clones were activated with abacavir in the absence of antigen presenting cells. Analysis of the remaining clones revealed two pathways of drug-dependent T-cell activation. Overnight incubation of antigen presenting cells with abacavir, followed by repeated washing to remove soluble drug, activated approximately 50% of the clones, and the response was blocked by glutaraldehyde fixation. In contrast, a 1 h antigen presenting cell pulse did not activate any of the clones. Accumulation of abacavir in antigen presenting cells was rapid (less than 1 h), and the intracellular concentrations were maintained for 16 h. However, intracellular abacavir was not detectable by mass spectrometry after pulsing. These data suggest that T-cells can be activated by abacavir through a direct interaction with surface and intracellular major histocompatibility complex (MHC) molecules. With the former, abacavir seemingly participates in the MHC T-cell receptor binding interaction. In contrast, the latter pathway likely involves MHC binding peptides displayed as a consequence of abacavir exposure, but not abacavir itself.

Antigen targeting to antigen-presenting cells enhances presentation to class II-restricted T lymphocytes.

PubMed Central

Scardino, A; Paroli, M; De Petrillo, G; Michel, M L; Barnaba, V

1994-01-01

Receptor-mediated uptake increases by several orders of magnitude the efficiency of APC to internalize Ag, and is stringently required for the Ag-presenting function of T lymphocytes due to their inability to take up Ag non-specifically. We have previously reported that hepatitis B envelope antigen (HBenvAg) can be internalized by T cells via transferrin receptor (TfR). To evaluate if Ag targeting to receptors expressed on APC could be an effective tool for promoting Ag uptake and presentation, we tested the capacity of activated T cells not expressing TfR to induce HBenvAg-specific T-cell responses when pulsed with a hybrid particle containing HBenvAg coupled to gp120 of human immunodeficiency virus (HIV), exploiting the ability of gp120 to bind to CD4 receptor. We found that CD4+/TfR- T cells pulsed either with the hybrid particle or peptide (S193-207) but not with S, L Ag, a recombinant form of HBenvAg, induced a specific proliferative response of a T-cell clone recognizing peptide (S193-207) of HBenvAg. The finding that the addition of anti-CD4 monoclonal antibody (mAb) before the pulsing of CD4+/TfR- T cells with the hybrid particle drastically blocked the specific T-cell response, together with the finding that CD8+/TfR- T cells were unable to serve as APC even if pulsed with this molecule, demonstrated that CD4 receptor was crucial for the HBenvAg internalization. On the other hand, HBenvAg presentation by CD4+/TfR+ T cells pulsed with the hybrid particle was inhibited only when both anti-CD4 and anti-TfR were added before the pulsing. These results suggest that Ag targeting to APC receptors may be usefully exploited to improve Ag-presentation efficiency in potential immunotherapeutic approaches. PMID:7907575

The Role of B Cells for in Vivo T Cell Responses to a Friend Virus-Induced Leukemia

NASA Astrophysics Data System (ADS)

Schultz, Kirk R.; Klarnet, Jay P.; Gieni, Randall S.; Hayglass, Kent T.; Greenberg, Philip D.

1990-08-01

B cells can function as antigen-presenting cells and accessory cells for T cell responses. This study evaluated the role of B cells in the induction of protective T cell immunity to a Friend murine leukemia virus (F-MuLV)-induced leukemia (FBL). B cell-deficient mice exhibited significantly reduced tumor-specific CD4^+ helper and CD8^+ cytotoxic T cell responses after priming with FBL or a recombinant vaccinia virus containing F-MuLV antigens. Moreover, these mice had diminished T cell responses to the vaccinia viral antigens. Tumor-primed T cells transferred into B cell-deficient mice effectively eradicated disseminated FBL. Thus, B cells appear necessary for efficient priming but not expression of tumor and viral T cell immunity.

Expression, Polyubiquitination, and Therapeutic Potential of Recombinant E6E7 from HPV16 Antigens Fused to Ubiquitin.

PubMed

de Oliveira, Liliane M Fernandes; Morale, Mirian G; Chaves, Agtha A M; Demasi, Marilene; Ho, Paulo L

2017-01-01

Ubiquitin-proteasome system plays an essential role in the immune response due to its involvement in the antigen generation and presentation to CD8 + T cells. Hereby, ubiquitin fused to antigens has been explored as an immunotherapeutic strategy that requires the activation of cytotoxic T lymphocytes. Here we propose to apply this ubiquitin fusion approach to a recombinant vaccine against human papillomavirus 16-infected cells. E6E7 multi-epitope antigen was fused genetically at its N- or C-terminal end to ubiquitin and expressed in Escherichia coli as inclusion bodies. The antigens were solubilized using urea and purified by nickel affinity chromatography in denatured condition. Fusion of ubiquitin to E6E7 resulted in marked polyubiquitination in vitro mainly when fused to the E6E7 N-terminal. When tested in a therapeutic scenario, the fusion of ubiquitin to E6E7 reinforced the anti-tumor protection and increased the E6/E7-specific cellular immune responses. Present results encourage the investigation of the adjuvant potential of the ubiquitin fusion to recombinant vaccines requiring CD8 + T cells.

Costimulatory Function of Cd58/Cd2 Interaction in Adaptive Humoral Immunity in a Zebrafish Model.

PubMed

Shao, Tong; Shi, Wei; Zheng, Jia-Yu; Xu, Xiao-Xiao; Lin, Ai-Fu; Xiang, Li-Xin; Shao, Jian-Zhong

2018-01-01

CD58 and CD2 have long been known as a pair of reciprocal adhesion molecules involved in the immune modulations of CD8 + T and NK-mediated cellular immunity in humans and several other mammals. However, the functional roles of CD58 and CD2 in CD4 + T-mediated adaptive humoral immunity remain poorly defined. Moreover, the current functional observations of CD58 and CD2 were mainly acquired from in vitro assays, and in vivo investigation is greatly limited due to the absence of a Cd58 homology in murine models. In this study, we identified cd58 and cd2 homologs from the model species zebrafish ( Danio rerio ). These two molecules share conserved structural features to their mammalian counterparts. Functionally, cd58 and cd2 were significantly upregulated on antigen-presenting cells and Cd4 + T cells upon antigen stimulation. Blockade or knockdown of Cd58 and Cd2 dramatically impaired the activation of antigen-specific Cd4 + T and mIgM + B cells, followed by the inhibition of antibody production and host defense against bacterial infections. These results indicate that CD58/CD2 interaction was required for the full activation of CD4 + T-mediated adaptive humoral immunity. The interaction of Cd58 with Cd2 was confirmed by co-immunoprecipitation and functional competitive assays by introducing a soluble Cd2 protein. This study highlights a new costimulatory mechanism underlying the regulatory network of adaptive immunity and makes zebrafish an attractive model organism for the investigation of CD58/CD2-mediated immunology and disorders. It also provides a cross-species understanding of the evolutionary history of costimulatory signals from fish to mammals as a whole.

Different Vaccine Vectors Delivering the Same Antigen Elicit CD8plus T Cell Responses with Distinct Clonotype and Epitope Specificity

DOE Office of Scientific and Technical Information (OSTI.GOV)

M Honda; R Wang; W Kong

Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8{sup +} cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8{sup +} T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2D{sup d} better than the native sequences, and clones with distinct specificities were elicited by alternativemore » vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8{sup +} T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.« less

Different Vaccine Vectors Delivering the Same Antigen Elicit CD8+ T Cell Responses with Distinct Clonotype and Epitope Specificity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Honda, M.; Robinson, H.; Wang, R.

Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8{sup +} cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8{sup +} T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2D{sup d} better than the native sequences, and clones with distinct specificities were elicited by alternativemore » vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8{sup +} T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.« less

The Extended Family of CD1d-Restricted NKT Cells: Sifting through a Mixed Bag of TCRs, Antigens, and Functions

PubMed Central

Macho-Fernandez, Elodie; Brigl, Manfred

2015-01-01

Natural killer T (NKT) cells comprise a family of specialized T cells that recognize lipid antigens presented by CD1d. Based on their T cell receptor (TCR) usage and antigen specificities, CD1d-restricted NKT cells have been divided into two main subsets: type I NKT cells that use a canonical invariant TCR α-chain and recognize α-galactosylceramide (α-GalCer), and type II NKT cells that use a more diverse αβ TCR repertoire and do not recognize α-GalCer. In addition, α-GalCer-reactive NKT cells that use non-canonical αβ TCRs and CD1d-restricted T cells that use γδ or δ/αβ TCRs have recently been identified, revealing further diversity among CD1d-restricted T cells. Importantly, in addition to their distinct antigen specificities, functional differences are beginning to emerge between the different members of the CD1d-restricted T cell family. In this review, while using type I NKT cells as comparison, we will focus on type II NKT cells and the other non-invariant CD1d-restricted T cell subsets, and discuss our current understanding of the antigens they recognize, the formation of stimulatory CD1d/antigen complexes, the modes of TCR-mediated antigen recognition, and the mechanisms and consequences of their activation that underlie their function in antimicrobial responses, anti-tumor immunity, and autoimmunity. PMID:26284062

Perforin and gamma interferon expression are required for CD4+ and CD8+ T-cell-dependent protective immunity against a human parasite, Trypanosoma cruzi, elicited by heterologous plasmid DNA prime-recombinant adenovirus 5 boost vaccination.

PubMed

de Alencar, Bruna C G; Persechini, Pedro M; Haolla, Filipe A; de Oliveira, Gabriel; Silverio, Jaline C; Lannes-Vieira, Joseli; Machado, Alexandre V; Gazzinelli, Ricardo T; Bruna-Romero, Oscar; Rodrigues, Mauricio M

2009-10-01

A heterologous prime-boost strategy using plasmid DNA, followed by replication-defective recombinant adenovirus 5, is being proposed as a powerful way to elicit CD4(+) and CD8(+) T-cell-mediated protective immunity against intracellular pathogens. We confirmed this concept and furthered existing research by providing evidence that the heterologous prime-boost regimen using the gene encoding amastigote surface protein 2 elicited CD4(+) and CD8(+) T-cell-mediated protective immunity (reduction of acute parasitemia and prolonged survival) against experimental infection with Trypanosoma cruzi. Protective immunity correlated with the presence of in vivo antigen-specific cytotoxic activity prior to challenge. Based on this, our second goal was to determine the outcome of infection after heterologous prime-boost immunization of perforin-deficient mice. These mice were highly susceptible to infection. A detailed analysis of the cell-mediated immune responses in immunized perforin-deficient mice showed an impaired gamma interferon (IFN-gamma) secretion by immune spleen cells upon restimulation in vitro with soluble recombinant antigen. In spite of a normal numeric expansion, specific CD8(+) T cells presented several functional defects detected in vivo (cytotoxicity) and in vitro (simultaneous expression of CD107a/IFN-gamma or IFN-gamma/tumor necrosis factor alpha) paralleled by a decreased expression of CD44 and KLRG-1. Our final goal was to determine the importance of IFN-gamma in the presence of highly cytotoxic T cells. Vaccinated IFN-gamma-deficient mice developed highly cytotoxic cells but failed to develop any protective immunity. Our study thus demonstrated a role for perforin and IFN-gamma in a number of T-cell-mediated effector functions and in the antiparasitic immunity generated by a heterologous plasmid DNA prime-adenovirus boost vaccination strategy.

Perforin and Gamma Interferon Expression Are Required for CD4+ and CD8+ T-Cell-Dependent Protective Immunity against a Human Parasite, Trypanosoma cruzi, Elicited by Heterologous Plasmid DNA Prime-Recombinant Adenovirus 5 Boost Vaccination▿

PubMed Central

de Alencar, Bruna C. G.; Persechini, Pedro M.; Haolla, Filipe A.; de Oliveira, Gabriel; Silverio, Jaline C.; Lannes-Vieira, Joseli; Machado, Alexandre V.; Gazzinelli, Ricardo T.; Bruna-Romero, Oscar; Rodrigues, Mauricio M.

2009-01-01

A heterologous prime-boost strategy using plasmid DNA, followed by replication-defective recombinant adenovirus 5, is being proposed as a powerful way to elicit CD4+ and CD8+ T-cell-mediated protective immunity against intracellular pathogens. We confirmed this concept and furthered existing research by providing evidence that the heterologous prime-boost regimen using the gene encoding amastigote surface protein 2 elicited CD4+ and CD8+ T-cell-mediated protective immunity (reduction of acute parasitemia and prolonged survival) against experimental infection with Trypanosoma cruzi. Protective immunity correlated with the presence of in vivo antigen-specific cytotoxic activity prior to challenge. Based on this, our second goal was to determine the outcome of infection after heterologous prime-boost immunization of perforin-deficient mice. These mice were highly susceptible to infection. A detailed analysis of the cell-mediated immune responses in immunized perforin-deficient mice showed an impaired gamma interferon (IFN-γ) secretion by immune spleen cells upon restimulation in vitro with soluble recombinant antigen. In spite of a normal numeric expansion, specific CD8+ T cells presented several functional defects detected in vivo (cytotoxicity) and in vitro (simultaneous expression of CD107a/IFN-γ or IFN-γ/tumor necrosis factor alpha) paralleled by a decreased expression of CD44 and KLRG-1. Our final goal was to determine the importance of IFN-γ in the presence of highly cytotoxic T cells. Vaccinated IFN-γ-deficient mice developed highly cytotoxic cells but failed to develop any protective immunity. Our study thus demonstrated a role for perforin and IFN-γ in a number of T-cell-mediated effector functions and in the antiparasitic immunity generated by a heterologous plasmid DNA prime-adenovirus boost vaccination strategy. PMID:19651871

Targeting LAG-3 and PD-1 to Enhance T Cell Activation by Antigen-Presenting Cells

PubMed Central

Lichtenegger, Felix S.; Rothe, Maurine; Schnorfeil, Frauke M.; Deiser, Katrin; Krupka, Christina; Augsberger, Christian; Schlüter, Miriam; Neitz, Julia; Subklewe, Marion

2018-01-01

Immune checkpoint inhibition has been shown to successfully reactivate endogenous T cell responses directed against tumor-associated antigens, resulting in significantly prolonged overall survival in patients with various tumor entities. For malignancies with low endogenous immune responses, this approach has not shown a clear clinical benefit so far. Therapeutic vaccination, particularly dendritic cell (DC) vaccination, is a strategy to induce T cell responses. Interaction of DCs and T cells is dependent on receptor–ligand interactions of various immune checkpoints. In this study, we analyzed the influence of blocking antibodies targeting programmed cell death protein 1 (PD-1), HVEM, CD244, TIM-3, and lymphocyte activation gene 3 (LAG-3) on the proliferation and cytokine secretion of T cells after stimulation with autologous TLR-matured DCs. In this context, we found that LAG-3 blockade resulted in superior T cell activation compared to inhibition of other pathways, including PD-1/PD-L1. This result was consistent across different methods to measure T cell stimulation (proliferation, IFN-γ secretion), various stimulatory antigens (viral and bacterial peptide pool, specific viral antigen, specific tumor antigen), and seen for both CD4+ and CD8+ T cells. Only under conditions with a weak antigenic stimulus, particularly when combining antigen presentation by peripheral blood mononuclear cells with low concentrations of peptides, we observed the highest T cell stimulation with dual blockade of LAG-3 and PD-1 blockade. We conclude that priming of novel immune responses can be strongly enhanced by blockade of LAG-3 or dual blockade of LAG-3 and PD-1, depending on the strength of the antigenic stimulus. PMID:29535740

Targeting LAG-3 and PD-1 to Enhance T Cell Activation by Antigen-Presenting Cells.

PubMed

Lichtenegger, Felix S; Rothe, Maurine; Schnorfeil, Frauke M; Deiser, Katrin; Krupka, Christina; Augsberger, Christian; Schlüter, Miriam; Neitz, Julia; Subklewe, Marion

2018-01-01

Immune checkpoint inhibition has been shown to successfully reactivate endogenous T cell responses directed against tumor-associated antigens, resulting in significantly prolonged overall survival in patients with various tumor entities. For malignancies with low endogenous immune responses, this approach has not shown a clear clinical benefit so far. Therapeutic vaccination, particularly dendritic cell (DC) vaccination, is a strategy to induce T cell responses. Interaction of DCs and T cells is dependent on receptor-ligand interactions of various immune checkpoints. In this study, we analyzed the influence of blocking antibodies targeting programmed cell death protein 1 (PD-1), HVEM, CD244, TIM-3, and lymphocyte activation gene 3 (LAG-3) on the proliferation and cytokine secretion of T cells after stimulation with autologous TLR-matured DCs. In this context, we found that LAG-3 blockade resulted in superior T cell activation compared to inhibition of other pathways, including PD-1/PD-L1. This result was consistent across different methods to measure T cell stimulation (proliferation, IFN-γ secretion), various stimulatory antigens (viral and bacterial peptide pool, specific viral antigen, specific tumor antigen), and seen for both CD4 + and CD8 + T cells. Only under conditions with a weak antigenic stimulus, particularly when combining antigen presentation by peripheral blood mononuclear cells with low concentrations of peptides, we observed the highest T cell stimulation with dual blockade of LAG-3 and PD-1 blockade. We conclude that priming of novel immune responses can be strongly enhanced by blockade of LAG-3 or dual blockade of LAG-3 and PD-1, depending on the strength of the antigenic stimulus.

Bicistronic DNA vaccines simultaneously encoding HIV, HSV and HPV antigens promote CD8⁺ T cell responses and protective immunity.

PubMed

Santana, Vinicius C; Diniz, Mariana O; Cariri, Francisco A M O; Ventura, Armando M; Cunha-Neto, Edécio; Almeida, Rafael R; Campos, Marco A; Lima, Graciela K; Ferreira, Luís C S

2013-01-01

Millions of people worldwide are currently infected with human papillomavirus (HPV), herpes simplex virus (HSV) or human immunodeficiency virus (HIV). For this enormous contingent of people, the search for preventive and therapeutic immunological approaches represents a hope for the eradication of latent infection and/or virus-associated cancer. To date, attempts to develop vaccines against these viruses have been mainly based on a monovalent concept, in which one or more antigens of a virus are incorporated into a vaccine formulation. In the present report, we designed and tested an immunization strategy based on DNA vaccines that simultaneously encode antigens for HIV, HSV and HPV. With this purpose in mind, we tested two bicistronic DNA vaccines (pIRES I and pIRES II) that encode the HPV-16 oncoprotein E7 and the HIV protein p24 both genetically fused to the HSV-1 gD envelope protein. Mice i.m. immunized with the DNA vaccines mounted antigen-specific CD8⁺ T cell responses, including in vivo cytotoxic responses, against the three antigens. Under experimental conditions, the vaccines conferred protective immunity against challenges with a vaccinia virus expressing the HIV-derived protein Gag, an HSV-1 virus strain and implantation of tumor cells expressing the HPV-16 oncoproteins. Altogether, our results show that the concept of a trivalent HIV, HSV, and HPV vaccine capable to induce CD8⁺ T cell-dependent responses is feasible and may aid in the development of preventive and/or therapeutic approaches for the control of diseases associated with these viruses.

Abacavir-Reactive Memory T Cells Are Present in Drug Naïve Individuals

PubMed Central

Lucas, Andrew; Lucas, Michaela; Strhyn, Anette; Keane, Niamh M.; McKinnon, Elizabeth; Pavlos, Rebecca; Moran, Ellen M.; Meyer-Pannwitt, Viola; Gaudieri, Silvana; D’Orsogna, Lloyd; Kalams, Spyros; Ostrov, David A.; Buus, Søren; Peters, Bjoern; Mallal, Simon; Phillips, Elizabeth

2015-01-01

Background Fifty-five percent of individuals with HLA-B*57:01 exposed to the antiretroviral drug abacavir develop a hypersensitivity reaction (HSR) that has been attributed to naïve T-cell responses to neo-antigen generated by the drug. Immunologically confirmed abacavir HSR can manifest clinically in less than 48 hours following first exposure suggesting that, at least in some cases, abacavir HSR is due to re-stimulation of a pre-existing memory T-cell population rather than priming of a high frequency naïve T-cell population. Methods To determine whether a pre-existing abacavir reactive memory T-cell population contributes to early abacavir HSR symptoms, we studied the abacavir specific naïve or memory T-cell response using HLA-B*57:01 positive HSR patients or healthy controls using ELISpot assay, intra-cellular cytokine staining and tetramer labelling. Results Abacavir reactive CD8+ T-cell responses were detected in vitro in one hundred percent of abacavir unexposed HLA-B*57:01 positive healthy donors. Abacavir-specific CD8+ T cells from such donors can be expanded from sorted memory, and sorted naïve, CD8+ T cells without need for autologous CD4+ T cells. Conclusions We propose that these pre-existing abacavir-reactive memory CD8+ T-cell responses must have been primed by earlier exposure to another foreign antigen and that these T cells cross-react with an abacavir-HLA-B*57:01-endogenous peptide ligand complex, in keeping with the model of heterologous immunity proposed in transplant rejection. PMID:25674793

Abacavir-reactive memory T cells are present in drug naïve individuals.

PubMed

Lucas, Andrew; Lucas, Michaela; Strhyn, Anette; Keane, Niamh M; McKinnon, Elizabeth; Pavlos, Rebecca; Moran, Ellen M; Meyer-Pannwitt, Viola; Gaudieri, Silvana; D'Orsogna, Lloyd; Kalams, Spyros; Ostrov, David A; Buus, Søren; Peters, Bjoern; Mallal, Simon; Phillips, Elizabeth

2015-01-01

Fifty-five percent of individuals with HLA-B*57:01 exposed to the antiretroviral drug abacavir develop a hypersensitivity reaction (HSR) that has been attributed to naïve T-cell responses to neo-antigen generated by the drug. Immunologically confirmed abacavir HSR can manifest clinically in less than 48 hours following first exposure suggesting that, at least in some cases, abacavir HSR is due to re-stimulation of a pre-existing memory T-cell population rather than priming of a high frequency naïve T-cell population. To determine whether a pre-existing abacavir reactive memory T-cell population contributes to early abacavir HSR symptoms, we studied the abacavir specific naïve or memory T-cell response using HLA-B*57:01 positive HSR patients or healthy controls using ELISpot assay, intra-cellular cytokine staining and tetramer labelling. Abacavir reactive CD8+ T-cell responses were detected in vitro in one hundred percent of abacavir unexposed HLA-B*57:01 positive healthy donors. Abacavir-specific CD8+ T cells from such donors can be expanded from sorted memory, and sorted naïve, CD8+ T cells without need for autologous CD4+ T cells. We propose that these pre-existing abacavir-reactive memory CD8+ T-cell responses must have been primed by earlier exposure to another foreign antigen and that these T cells cross-react with an abacavir-HLA-B*57:01-endogenous peptide ligand complex, in keeping with the model of heterologous immunity proposed in transplant rejection.

Induction of maturation and activation of human dendritic cells: a mechanism underlying the beneficial effect of Viscum album as complimentary therapy in cancer.

PubMed

Elluru, Sri Ramulu; Duong van Huyen, Jean-Paul; Delignat, Sandrine; Kazatchkine, Michel D; Friboulet, Alain; Kaveri, Srini V; Bayry, Jagadeesh

2008-06-04

Viscum album (VA) preparations have been used as a complimentary therapy in cancer. In addition to their cytotoxic properties, they have also been shown to have immunostimulatory properties. In the present study, we examine the hypothesis that the VA preparations induce activation of human DC that facilitates effective tumor regression. Four day old monocyte-derived immature DCs were treated with VA Qu Spez at 5, 10 and 15 microg/ml for 48 hrs. The expression of surface molecules was analyzed by flow cytometry. The ability of Qu Spez-educated DC to stimulate T cells was analyzed by allogeneic mixed lymphocyte reaction and activation of Melan-A/MART-1-specific M77-80 CD8+T cells. Cytokines in cell free culture supernatant was analyzed by cytokine bead array assay. VA Qu Spez stimulated DCs presented with increased expression of antigen presenting molecule HLA-DR and of co-stimulatory molecules CD40, CD80 and CD86. The VA Qu Spez also induced the secretion of inflammatory cytokines IL-6 and IL-8. Further, Qu Spez-educated DC stimulated CD4+T cells in a allogeneic mixed lymphocyte reaction and activated melanoma antigen Melan-A/MART-1-specific M77-80 CD8+T cells as evidenced by increased secretion of TNF-alpha and IFNgamma. The VA preparations stimulate the maturation and activation of human DCs, which may facilitate anti-tumoral immune responses. These results should assist in understanding the immunostimulatory properties of VA preparations and improving the therapeutic strategies.

Induction of maturation and activation of human dendritic cells: A mechanism underlying the beneficial effect of Viscum album as complimentary therapy in cancer

PubMed Central

Elluru, Sri Ramulu; van Huyen, Jean-Paul Duong; Delignat, Sandrine; Kazatchkine, Michel D; Friboulet, Alain; Kaveri, Srini V; Bayry, Jagadeesh

2008-01-01

Background Viscum album (VA) preparations have been used as a complimentary therapy in cancer. In addition to their cytotoxic properties, they have also been shown to have immunostimulatory properties. In the present study, we examine the hypothesis that the VA preparations induce activation of human DC that facilitates effective tumor regression. Methods Four day old monocyte-derived immature DCs were treated with VA Qu Spez at 5, 10 and 15 μg/ml for 48 hrs. The expression of surface molecules was analyzed by flow cytometry. The ability of Qu Spez-educated DC to stimulate T cells was analyzed by allogeneic mixed lymphocyte reaction and activation of Melan-A/MART-1-specific M77-80 CD8+T cells. Cytokines in cell free culture supernatant was analyzed by cytokine bead array assay. Results VA Qu Spez stimulated DCs presented with increased expression of antigen presenting molecule HLA-DR and of co-stimulatory molecules CD40, CD80 and CD86. The VA Qu Spez also induced the secretion of inflammatory cytokines IL-6 and IL-8. Further, Qu Spez-educated DC stimulated CD4+T cells in a allogeneic mixed lymphocyte reaction and activated melanoma antigen Melan-A/MART-1-specific M77-80 CD8+T cells as evidenced by increased secretion of TNF-α and IFNγ. Conclusion The VA preparations stimulate the maturation and activation of human DCs, which may facilitate anti-tumoral immune responses. These results should assist in understanding the immunostimulatory properties of VA preparations and improving the therapeutic strategies. PMID:18533025

Initial viral load determines the magnitude of the human CD8 T cell response to yellow fever vaccination.

PubMed

Akondy, Rama S; Johnson, Philip L F; Nakaya, Helder I; Edupuganti, Srilatha; Mulligan, Mark J; Lawson, Benton; Miller, Joseph D; Pulendran, Bali; Antia, Rustom; Ahmed, Rafi

2015-03-10

CD8 T cells are a potent tool for eliminating intracellular pathogens and tumor cells. Thus, eliciting robust CD8 T-cell immunity is the basis for many vaccines under development. However, the relationship between antigen load and the magnitude of the CD8 T-cell response is not well-described in a human immune response. Here we address this issue by quantifying viral load and the CD8 T-cell response in a cohort of 80 individuals immunized with the live attenuated yellow fever vaccine (YFV-17D) by sampling peripheral blood at days 0, 1, 2, 3, 5, 7, 9, 11, 14, 30, and 90. When the virus load was below a threshold (peak virus load < 225 genomes per mL, or integrated virus load < 400 genome days per mL), the magnitude of the CD8 T-cell response correlated strongly with the virus load (R(2) ∼ 0.63). As the virus load increased above this threshold, the magnitude of the CD8 T-cell responses saturated. Recent advances in CD8 T-cell-based vaccines have focused on replication-incompetent or single-cycle vectors. However, these approaches deliver relatively limited amounts of antigen after immunization. Our results highlight the requirement that T-cell-based vaccines should deliver sufficient antigen during the initial period of the immune response to elicit a large number of CD8 T cells that may be needed for protection.

Regulation of allergic airway inflammation by adoptive transfer of CD4+ T cells preferentially producing IL-10.

PubMed

Matsuda, Masaya; Doi, Kana; Tsutsumi, Tatsuya; Fujii, Shinya; Kishima, Maki; Nishimura, Kazuma; Kuroda, Ikue; Tanahashi, Yu; Yuasa, Rino; Kinjo, Toshihiko; Kuramoto, Nobuyuki; Mizutani, Nobuaki; Nabe, Takeshi

2017-10-05

Anti-inflammatory pharmacotherapy for asthma has mainly depended on the inhalation of glucocorticoids, which non-specifically suppress immune responses. If the anti-inflammatory cytokine interleukin (IL)-10 can be induced by a specific antigen, asthmatic airway inflammation could be suppressed when individuals are exposed to the antigen. The purpose of this study was to develop cellular immunotherapeutics for atopic diseases using IL-10-producing CD4 + T cells. Spleen cells isolated from ovalbumin (OVA)-sensitized mice were cultured with the antigen, OVA and growth factors, IL-21, IL-27 and TGF-β for 7 days. After the 7-day culture, the CD4 + T cells were purified using a murine CD4 magnetic beads system. When the induced CD4 + T cells were stimulated by OVA in the presence of antigen-presenting cells, IL-10 was preferentially produced in vitro. When CD4 + T cells were adoptively transferred to OVA-sensitized mice followed by intratracheal OVA challenges, IL-10 was preferentially produced in the serum and bronchoalveolar lavage fluid in vivo. IL-10 production coincided with the inhibition of eosinophilic airway inflammation and epithelial mucus plugging. Most of the IL-10-producing CD4 + T cells were negative for Foxp3 and GATA-3, transcription factors of naturally occurring regulatory T cells and Th2 cells, respectively, but double positive for LAG-3 and CD49b, surface markers of inducible regulatory T cells, Tr1 cells. Collectively, most of the induced IL-10-producing CD4 + T cells could be Tr1 cells, which respond to the antigen to produce IL-10, and effectively suppressed allergic airway inflammation. The induced Tr1 cells may be useful for antigen-specific cellular immunotherapy for atopic diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

High Cell Surface Expression of CD4 Allows Distinction of CD4+CD25+ Antigen-specific Effector T Cells from CD4+CD25+ Regulatory T Cells in Murine Experimental Autoimmune Encephalomyelitis

PubMed Central

Li, Jinzhu; Ridgway, William; Fathman, C. Garrison; Tse, Harley Y.; Shaw, Michael K.

2008-01-01

Analysis of T regulatory cells (Treg) and T effector cells (Teff) in experimental autoimmune encephalomyelitis is complicated by the fact that both cell types express CD4 and CD25. We demonstrate that encephalitogenic T cells, following antigen recognition, up regulate cell surface expression of CD4. The CD4high sub-population contains all of the antigen response as shown by proliferation and cytokine secretion, and only these cells are capable of transferring EAE to naive animals. On the other hand, a FACS separable CD25+ sub-population of cells displayed consistent levels of CD4 prior to and after antigen stimulation. These cells displayed characteristics of Treg, such as expressing high levels of the Foxp3 gene and the ability to suppress mitogenic T cell responses. PMID:17920698

CD1-Restricted T Cells at the Crossroad of Innate and Adaptive Immunity.

PubMed

Pereira, Catia S; Macedo, M Fatima

2016-01-01

Lipid-specific T cells comprise a group of T cells that recognize lipids bound to the MHC class I-like CD1 molecules. There are four isoforms of CD1 that are expressed at the surface of antigen presenting cells and therefore capable of presenting lipid antigens: CD1a, CD1b, CD1c, and CD1d. Each one of these isoforms has distinct structural features and cellular localizations, which promotes binding to a broad range of different types of lipids. Lipid antigens originate from either self-tissues or foreign sources, such as bacteria, fungus, or plants and their recognition by CD1-restricted T cells has important implications in infection but also in cancer and autoimmunity. In this review, we describe the characteristics of CD1 molecules and CD1-restricted lipid-specific T cells, highlighting the innate-like and adaptive-like features of different CD1-restricted T cell subtypes.

T cell receptor αβ diversity inversely correlates with pathogen-specific antibody levels in human cytomegalovirus infection.

PubMed

Wang, George C; Dash, Pradyot; McCullers, Jonathan A; Doherty, Peter C; Thomas, Paul G

2012-04-04

A diverse T cell receptor (TCR) repertoire capable of recognizing a broad range of antigenic peptides is thought to be central to effective pathogen-specific immunity by counteracting escape mutations, selecting high-avidity T cells, and providing T cell specificities with comprehensive functional characteristics. However, evidence that TCR diversity is important for the successful control of human infections is limited. A single-cell strategy for the clonotypic analysis of human CD8⁺ TCRαβ repertoires was used to probe the diversity and magnitude of individual human cytomegalovirus (CMV)-specific CD8⁺ T cells recovered directly ex vivo. We found that CD8⁺ TCRαβ repertoire diversity, but not the size of the CD8⁺ T cell response, was inversely related to circulating CMV-specific antibody levels, a measure that has been correlated epidemiologically with differential mortality risks and found here to be higher in persons with detectable (versus undetectable) CMV viral loads. Overall, our findings indicate that CD8⁺ T cell diversity may be more important than T cell abundance in limiting the negative consequences of CMV persistence, demonstrate high prevalence of both TCRα and TCRβ public motif usage, and suggest that a highly diverse TCRαβ repertoire may be an important benchmark and target in the success of immunotherapeutic strategies.

Dendritic Cells Exposed to MVA-Based HIV-1 Vaccine Induce Highly Functional HIV-1-Specific CD8+ T Cell Responses in HIV-1-Infected Individuals

PubMed Central

Climent, Núria; Guerra, Susana; García, Felipe; Rovira, Cristina; Miralles, Laia; Gómez, Carmen Elena; Piqué, Núria; Gil, Cristina; Gatell, José María; Esteban, Mariano; Gallart, Teresa

2011-01-01

Currently, MVA virus vectors carrying HIV-1 genes are being developed as HIV-1/AIDS prophylactic/therapeutic vaccines. Nevertheless, little is known about the impact of these vectors on human dendritic cells (DC) and their capacity to present HIV-1 antigens to human HIV-specific T cells. This study aimed to characterize the interaction of MVA and MVA expressing the HIV-1 genes Env-Gag-Pol-Nef of clade B (referred to as MVA-B) in human monocyte-derived dendritic cells (MDDC) and the subsequent processes of HIV-1 antigen presentation and activation of memory HIV-1-specific T lymphocytes. For these purposes, we performed ex vivo assays with MDDC and autologous lymphocytes from asymptomatic HIV-infected patients. Infection of MDDC with MVA-B or MVA, at the optimal dose of 0.3 PFU/MDDC, induced by itself a moderate degree of maturation of MDDC, involving secretion of cytokines and chemokines (IL1-ra, IL-7, TNF-α, IL-6, IL-12, IL-15, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, IP-10, MIG, and IFN-α). MDDC infected with MVA or MVA-B and following a period of 48 h or 72 h of maturation were able to migrate toward CCL19 or CCL21 chemokine gradients. MVA-B infection induced apoptosis of the infected cells and the resulting apoptotic bodies were engulfed by the uninfected MDDC, which cross-presented HIV-1 antigens to autologous CD8+ T lymphocytes. MVA-B-infected MDDC co-cultured with autologous T lymphocytes induced a highly functional HIV-specific CD8+ T cell response including proliferation, secretion of IFN-γ, IL-2, TNF-α, MIP-1β, MIP-1α, RANTES and IL-6, and strong cytotoxic activity against autologous HIV-1-infected CD4+ T lymphocytes. These results evidence the adjuvant role of the vector itself (MVA) and support the clinical development of prophylactic and therapeutic anti-HIV vaccines based on MVA-B. PMID:21625608

Role of Metalloproteases in Vaccinia Virus Epitope Processing for Transporter Associated with Antigen Processing (TAP)-independent Human Leukocyte Antigen (HLA)-B7 Class I Antigen Presentation*

PubMed Central

Lorente, Elena; García, Ruth; Mir, Carmen; Barriga, Alejandro; Lemonnier, François A.; Ramos, Manuel; López, Daniel

2012-01-01

The transporter associated with antigen processing (TAP) translocates the viral proteolytic peptides generated by the proteasome and other proteases in the cytosol to the endoplasmic reticulum lumen. There, they complex with nascent human leukocyte antigen (HLA) class I molecules, which are subsequently recognized by the CD8+ lymphocyte cellular response. However, individuals with nonfunctional TAP complexes or tumor or infected cells with blocked TAP molecules are able to present HLA class I ligands generated by TAP-independent processing pathways. Herein, using a TAP-independent polyclonal vaccinia virus-polyspecific CD8+ T cell line, two conserved vaccinia-derived TAP-independent HLA-B*0702 epitopes were identified. The presentation of these epitopes in normal cells occurs via complex antigen-processing pathways involving the proteasome and/or different subsets of metalloproteinases (amino-, carboxy-, and endoproteases), which were blocked in infected cells with specific chemical inhibitors. These data support the hypothesis that the abundant cellular proteolytic systems contribute to the supply of peptides recognized by the antiviral cellular immune response, thereby facilitating immunosurveillance. These data may explain why TAP-deficient individuals live normal life spans without any increased susceptibility to viral infections. PMID:22298786

Nanostructure and force spectroscopy analysis of human peripheral blood CD4+ T cells using atomic force microscopy.

PubMed

Hu, Mingqian; Wang, Jiongkun; Cai, Jiye; Wu, Yangzhe; Wang, Xiaoping

2008-09-12

To date, nanoscale imaging of the morphological changes and adhesion force of CD4(+) T cells during in vitro activation remains largely unreported. In this study, we used atomic force microscopy (AFM) to study the morphological changes and specific binding forces in resting and activated human peripheral blood CD4(+) T cells. The AFM images revealed that the volume of activated CD4(+) T cells increased and the ultrastructure of these cells also became complex. Using a functionalized AFM tip, the strength of the specific binding force of the CD4 antigen-antibody interaction was found to be approximately three times that of the unspecific force. The adhesion forces were not randomly distributed over the surface of a single activated CD4(+) T cell, indicated that the CD4 molecules concentrated into nanodomains. The magnitude of the adhesion force of the CD4 antigen-antibody interaction did not change markedly with the activation time. Multiple bonds involved in the CD4 antigen-antibody interaction were measured at different activation times. These results suggest that the adhesion force involved in the CD4 antigen-antibody interaction is highly selective and of high affinity.

Antibodies Against Immune Checkpoint Molecules Restore Functions of Tumor-Infiltrating T Cells in Hepatocellular Carcinomas.

PubMed

Zhou, Guoying; Sprengers, Dave; Boor, Patrick P C; Doukas, Michail; Schutz, Hannah; Mancham, Shanta; Pedroza-Gonzalez, Alexander; Polak, Wojciech G; de Jonge, Jeroen; Gaspersz, Marcia; Dong, Haidong; Thielemans, Kris; Pan, Qiuwei; IJzermans, Jan N M; Bruno, Marco J; Kwekkeboom, Jaap

2017-10-01

Ligand binding to inhibitory receptors on immune cells, such as programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte associated protein 4 (CTLA4), down-regulates the T-cell-mediated immune response (called immune checkpoints). Antibodies that block these receptors increase antitumor immunity in patients with melanoma, non-small-cell lung cancer, and renal cell cancer. Tumor-infiltrating CD4 + and CD8 + T cells in patients with hepatocellular carcinoma (HCC) have been found to be functionally compromised. We analyzed HCC samples from patients to determine if these inhibitory pathways prevent T-cell responses in HCCs and to find ways to restore their antitumor functions. We collected HCC samples from 59 patients who underwent surgical resection from November 2013 through May 2017, along with tumor-free liver tissues (control tissues) and peripheral blood samples. We isolated tumor-infiltrating lymphocytes (TIL) and intra-hepatic lymphocytes. We used flow cytometry to quantify expression of the inhibitory receptors PD-1, hepatitis A virus cellular receptor 2 (TIM3), lymphocyte activating 3 (LAG3), and CTLA4 on CD8 + and CD4 + T cells from tumor, control tissue, and blood; we studied the effects of antibodies that block these pathways in T-cell activation assays. Expression of PD-1, TIM3, LAG3, and CTLA4 was significantly higher on CD8 + and CD4 + T cells isolated from HCC tissue than control tissue or blood. Dendritic cells, monocytes, and B cells in HCC tumors expressed ligands for these receptors. Expression of PD-1, TIM3, and LAG3 was higher on tumor-associated antigen (TAA)-specific CD8 + TIL, compared with other CD8 + TIL. Compared with TIL that did not express these inhibitory receptors, CD8 + and CD4 + TIL that did express these receptors had higher levels of markers of activation, but similar or decreased levels of granzyme B and effector cytokines. Antibodies against CD274 (PD-ligand1 [PD-L1]), TIM3, or LAG3 increased proliferation of CD8 + and CD4 + TIL and cytokine production in response to stimulation with polyclonal antigens or TAA. Importantly, combining antibody against PD-L1 with antibodies against TIM3, LAG3, or CTLA4 further increased TIL functions. The immune checkpoint inhibitory molecules PD-1, TIM3, and LAG3 are up-regulated on TAA-specific T cells isolated from human HCC tissues, compared with T cells from tumor-free liver tissues or blood. Antibodies against PD-L1, TIM3, or LAG3 restore responses of HCC-derived T cells to tumor antigens, and combinations of the antibodies have additive effects. Strategies to block PD-L1, TIM3, and LAG3 might be developed for treatment of primary liver cancer. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Bystander hyperactivation of preimmune CD8+ T cells in chronic HCV patients

PubMed Central

Alanio, Cécile; Nicoli, Francesco; Sultanik, Philippe; Flecken, Tobias; Perot, Brieuc; Duffy, Darragh; Bianchi, Elisabetta; Lim, Annick; Clave, Emmanuel; van Buuren, Marit M; Schnuriger, Aurélie; Johnsson, Kerstin; Boussier, Jeremy; Garbarg-Chenon, Antoine; Bousquet, Laurence; Mottez, Estelle; Schumacher, Ton N; Toubert, Antoine; Appay, Victor; Heshmati, Farhad; Thimme, Robert; Pol, Stanislas; Mallet, Vincent; Albert, Matthew L

2015-01-01

Chronic infection perturbs immune homeostasis. While prior studies have reported dysregulation of effector and memory cells, little is known about the effects on naïve T cell populations. We performed a cross-sectional study of chronic hepatitis C (cHCV) patients using tetramer-associated magnetic enrichment to study antigen-specific inexperienced CD8+ T cells (i.e., tumor or unrelated virus-specific populations in tumor-free and sero-negative individuals). cHCV showed normal precursor frequencies, but increased proportions of memory-phenotype inexperienced cells, as compared to healthy donors or cured HCV patients. These observations could be explained by low surface expression of CD5, a negative regulator of TCR signaling. Accordingly, we demonstrated TCR hyperactivation and generation of potent CD8+ T cell responses from the altered T cell repertoire of cHCV patients. In sum, we provide the first evidence that naïve CD8+ T cells are dysregulated during cHCV infection, and establish a new mechanism of immune perturbation secondary to chronic infection. DOI: http://dx.doi.org/10.7554/eLife.07916.001 PMID:26568315

Immunogenicity and protective efficacy of DMT liposome-adjuvanted tuberculosis subunit CTT3H vaccine

PubMed Central

Teng, Xindong; Tian, Maopeng; Li, Jianrong; Tan, Songwei; Yuan, Xuefeng; Yu, Qi; Jing, Yukai; Zhang, Zhiping; Yue, Tingting; Zhou, Lei; Fan, Xionglin

2015-01-01

Different strategies have been proposed for the development of protein subunit vaccine candidates for tuberculosis (TB), which shows better safety than other types of candidates and the currently used Bacillus Calmette-Guérin (BCG) vaccine. In order to develop more effective protein subunits depending on the mechanism of cell-mediated immunity against TB, a polyprotein CTT3H, based on 5 immunodominant antigens (CFP10, TB10.4, TB8.4, Rv3615c, and HBHA) with CD8+ epitopes of Mycobacterium tuberculosis, was constructed in this study. We vaccinated C57BL/6 mice with a TB subunit CTT3H protein in an adjuvant of dimethyldioctadecylammonium/monophosphoryl lipid A/trehalose 6,6′-dibehenate (DDA/MPL/TDB, DMT) liposome to investigate the immunogenicity and protective efficacy of this novel vaccine. Our results demonstrated that DMT liposome-adjuvanted CTT3H vaccine not only induced an antigen-specific CD4+ Th1 response, but also raised the number of PPD- and CTT3H-specific IFN-γ+ CD8+ T cells and elicited strong CTL responses against TB10.4, which provided more effective protection against a 60 CFU M. tuberculosis aerosol challenge than PBS control and DMT adjuvant alone. Our findings indicate that DMT-liposome is an effective adjuvant to stimulate CD8+ T cell responses and the DMT-adjuvanted subunit CTT3H vaccine is a promising candidate for the next generation of TB vaccine. PMID:25905680

Immunogenicity and protective efficacy of DMT liposome-adjuvanted tuberculosis subunit CTT3H vaccine.

PubMed

Teng, Xindong; Tian, Maopeng; Li, Jianrong; Tan, Songwei; Yuan, Xuefeng; Yu, Qi; Jing, Yukai; Zhang, Zhiping; Yue, Tingting; Zhou, Lei; Fan, Xionglin

2015-01-01

Different strategies have been proposed for the development of protein subunit vaccine candidates for tuberculosis (TB), which shows better safety than other types of candidates and the currently used Bacillus Calmette-Guérin (BCG) vaccine. In order to develop more effective protein subunits depending on the mechanism of cell-mediated immunity against TB, a polyprotein CTT3H, based on 5 immunodominant antigens (CFP10, TB10.4, TB8.4, Rv3615c, and HBHA) with CD8(+) epitopes of Mycobacterium tuberculosis, was constructed in this study. We vaccinated C57BL/6 mice with a TB subunit CTT3H protein in an adjuvant of dimethyldioctadecylammonium/monophosphoryl lipid A/trehalose 6,6'-dibehenate (DDA/MPL/TDB, DMT) liposome to investigate the immunogenicity and protective efficacy of this novel vaccine. Our results demonstrated that DMT liposome-adjuvanted CTT3H vaccine not only induced an antigen-specific CD4(+) Th1 response, but also raised the number of PPD- and CTT3H-specific IFN-γ(+) CD8(+) T cells and elicited strong CTL responses against TB10.4, which provided more effective protection against a 60 CFU M. tuberculosis aerosol challenge than PBS control and DMT adjuvant alone. Our findings indicate that DMT-liposome is an effective adjuvant to stimulate CD8(+) T cell responses and the DMT-adjuvanted subunit CTT3H vaccine is a promising candidate for the next generation of TB vaccine.

Transgenic antigen-specific, HLA-A*02:01-allo-restricted cytotoxic T cells recognize tumor-associated target antigen STEAP1 with high specificity

PubMed Central

Schirmer, David; Grünewald, Thomas G. P.; Klar, Richard; Schmidt, Oxana; Wohlleber, Dirk; Rubío, Rebeca Alba; Uckert, Wolfgang; Thiel, Uwe; Bohne, Felix; Busch, Dirk H.; Krackhardt, Angela M.; Burdach, Stefan; Richter, Günther H. S.

2016-01-01

ABSTRACT Pediatric cancers, including Ewing sarcoma (ES), are only weakly immunogenic and the tumor-patients' immune system often is devoid of effector T cells for tumor elimination. Based on expression profiling technology, targetable tumor-associated antigens (TAA) are identified and exploited for engineered T-cell therapy. Here, the specific recognition and lytic potential of transgenic allo-restricted CD8+ T cells, directed against the ES-associated antigen 6-transmembrane epithelial antigen of the prostate 1 (STEAP1), was examined. Following repetitive STEAP1130 peptide-driven stimulations with HLA-A*02:01+ dendritic cells (DC), allo-restricted HLA-A*02:01− CD8+ T cells were sorted with HLA-A*02:01/peptide multimers and expanded by limiting dilution. After functional analysis of suitable T cell clones via ELISpot, flow cytometry and xCELLigence assay, T cell receptors' (TCR) α- and β-chains were identified, cloned into retroviral vectors, codon optimized, transfected into HLA-A*02:01− primary T cell populations and tested again for specificity and lytic capacity in vitro and in a Rag2−/−γc−/− mouse model. Initially generated transgenic T cells specifically recognized STEAP1130-pulsed or transfected cells in the context of HLA-A*02:01 with minimal cross-reactivity as determined by specific interferon-γ (IFNγ) release, lysed cells and inhibited growth of HLA-A*02:01+ ES lines more effectively than HLA-A*02:01− ES lines. In vivo tumor growth was inhibited more effectively with transgenic STEAP1130-specific T cells than with unspecific T cells. Our results identify TCRs capable of recognizing and inhibiting growth of STEAP1-expressing HLA-A*02:01+ ES cells in vitro and in vivo in a highly restricted manner. As STEAP1 is overexpressed in a wide variety of cancers, we anticipate these STEAP1-specific TCRs to be potentially useful for immunotherapy of other STEAP1-expressing tumors. PMID:27471654

Increase in TGF-β Secreting CD4+CD25+ FOXP3+ T Regulatory Cells in Anergic Lepromatous Leprosy Patients

PubMed Central

Saini, Chaman; Ramesh, Venkatesh; Nath, Indira

2014-01-01

Background Lepromatous leprosy caused by Mycobacterium leprae is associated with antigen specific T cell unresponsiveness/anergy whose underlying mechanisms are not fully defined. We investigated the role of CD25+FOXP3+ regulatory T cells in both skin lesions and M.leprae stimulated PBMC cultures of 28 each of freshly diagnosed patients with borderline tuberculoid (BT) and lepromatous leprosy (LL) as well as 7 healthy household contacts of leprosy patients and 4 normal skin samples. Methodology/Principle Findings Quantitative reverse transcribed PCR (qPCR), immuno-histochemistry/flowcytometry and ELISA were used respectively for gene expression, phenotype characterization and cytokine levels in PBMC culture supernatants. Both skin lesions as well as in vitro antigen stimulated PBMC showed increased percentage/mean fluorescence intensity of cells and higher gene expression for FOXP3+, TGF-β in lepromatous (p<0.01) as compared to tuberculoid leprosy patients. CD4+CD25+FOXP3+ T cells (Tregs) were increased in unstimulated basal cultures (p<0.0003) and showed further increase in in vitro antigen but not mitogen (phytohemaglutinin) stimulated PBMC (iTreg) in lepromatous as compared to tuberculoid leprosy patients (p<0.002). iTregs of lepromatous patients showed intracellular TGF-β which was further confirmed by increase in TGF-β in culture supernatants (p<0.003). Furthermore, TGF-β in iTreg cells was associated with phosphorylation of STAT5A. TGF-β was seen in CD25+ cells of the CD4+ but not that of CD8+ T cell lineage in leprosy patients. iTregs did not show intracellular IFN-γ or IL-17 in lepromatous leprosy patients. Conclusions/Significance Our results indicate that FOXP3+ iTregs with TGF-β may down regulate T cell responses leading to the antigen specific anergy associated with lepromatous leprosy. PMID:24454972

Virological Outcome after Structured Interruption of Antiretroviral Therapy for Human Immunodeficiency Virus Infection Is Associated with the Functional Profile of Virus-Specific CD8+ T Cells▿

PubMed Central

Daucher, Marybeth; Price, David A.; Brenchley, Jason M.; Lamoreaux, Laurie; Metcalf, Julia A.; Rehm, Catherine; Nies-Kraske, Elizabeth; Urban, Elizabeth; Yoder, Christian; Rock, Diane; Gumkowski, Julie; Betts, Michael R.; Dybul, Mark R.; Douek, Daniel C.

2008-01-01

A clear understanding of the antiviral effects of CD8+ T cells in the context of chronic human immunodeficiency virus (HIV) infection is critical for the development of prophylactic vaccines and therapeutics designed to support T-cell-mediated immunity. However, defining the potential correlates of effective CD8+ T-cell immunity has proven difficult; notably, comprehensive analyses have demonstrated that the size and shape of the CD8+ T-cell response are not necessarily indicative of efficacy determined by measures of plasma viral load. Here, we conducted a detailed quantitative and qualitative analysis of CD8+ T-cell responses to autologous virus in a cohort of six HIV-infected individuals with a history of structured interruption of antiretroviral therapy (ART) (SIT). The magnitude and breadth of the HIV-specific response did not, by themselves, explain the changes observed in plasma virus levels after the cessation of ART. Furthermore, mutational escape from targeted epitopes could not account for the differential virological outcomes in this cohort. However, the functionality of HIV-specific CD8+ T-cell populations upon antigen encounter, determined by the simultaneous and independent measurement of five CD8+ T-cell functions (degranulation and gamma interferon, macrophage inflammatory protein 1β, tumor necrosis factor alpha, and interleukin-2 levels) reflected the emergent level of plasma virus, with multiple functions being elicited in those individuals with lower levels of viremia after SIT. These data show that the quality of the HIV-specific CD8+ T-cell response, rather than the quantity, is associated with the dynamics of viral replication in the absence of ART and suggest that the effects of SIT can be assessed by measuring the functional profile of HIV-specific CD8+ T cells. PMID:18234797

Cross-Priming of Naive Cd8 T Cells against Melanoma Antigens Using Dendritic Cells Loaded with Killed Allogeneic Melanoma Cells

PubMed Central

Berard, Frederic; Blanco, Patrick; Davoust, Jean; Neidhart-Berard, Eve-Marie; Nouri-Shirazi, Mahyar; Taquet, Nicolas; Rimoldi, Donata; Cerottini, Jean Charles; Banchereau, Jacques; Palucka, A. Karolina

2000-01-01

The goal of tumor immunotherapy is to elicit immune responses against autologous tumors. It would be highly desirable that such responses include multiple T cell clones against multiple tumor antigens. This could be obtained using the antigen presenting capacity of dendritic cells (DCs) and cross-priming. That is, one could load the DC with tumor lines of any human histocompatibility leukocyte antigen (HLA) type to elicit T cell responses against the autologous tumor. In this study, we show that human DCs derived from monocytes and loaded with killed melanoma cells prime naive CD45RA+CD27+CD8+ T cells against the four shared melanoma antigens: MAGE-3, gp100, tyrosinase, and MART-1. HLA-A201+ naive T cells primed by DCs loaded with HLA-A201− melanoma cells are able to kill several HLA-A201+ melanoma targets. Cytotoxic T lymphocyte priming towards melanoma antigens is also obtained with cells from metastatic melanoma patients. This demonstration of cross-priming against shared tumor antigens builds the basis for using allogeneic tumor cell lines to deliver tumor antigens to DCs for vaccination protocols. PMID:11104796

Murine Polyomavirus Virus-Like Particles Carrying Full-Length Human PSA Protect BALB/c Mice from Outgrowth of a PSA Expressing Tumor

PubMed Central

Eriksson, Mathilda; Andreasson, Kalle; Weidmann, Joachim; Lundberg, Kajsa; Tegerstedt, Karin

2011-01-01

Virus-like particles (VLPs) consist of capsid proteins from viruses and have been shown to be usable as carriers of protein and peptide antigens for immune therapy. In this study, we have produced and assayed murine polyomavirus (MPyV) VLPs carrying the entire human Prostate Specific Antigen (PSA) (PSA-MPyVLPs) for their potential use for immune therapy in a mouse model system. BALB/c mice immunized with PSA-MPyVLPs were only marginally protected against outgrowth of a PSA-expressing tumor. To improve protection, PSA-MPyVLPs were co-injected with adjuvant CpG, either alone or loaded onto murine dendritic cells (DCs). Immunization with PSA-MPyVLPs loaded onto DCs in the presence of CpG was shown to efficiently protect mice from tumor outgrowth. In addition, cellular and humoral immune responses after immunization were examined. PSA-specific CD4+ and CD8+ cells were demonstrated, but no PSA-specific IgG antibodies. Vaccination with DCs loaded with PSA-MPyVLPs induced an eight-fold lower titre of anti-VLP antibodies than vaccination with PSA-MPyVLPs alone. In conclusion, immunization of BALB/c mice with PSA-MPyVLPs, loaded onto DCs and co-injected with CpG, induces an efficient PSA-specific tumor protective immune response, including both CD4+ and CD8+ cells with a low induction of anti-VLP antibodies. PMID:21858228

Treating B-cell cancer with T cells expressing anti-CD19 chimeric antigen receptors.

PubMed

Kochenderfer, James N; Rosenberg, Steven A

2013-05-01

Most B-cell malignancies express CD19, and a majority of patients with B-cell malignancies are not cured by current standard therapies. Chimeric antigen receptors (CARs) are fusion proteins consisting of antigen recognition moieties and T-cell activation domains. T cells can be genetically modified to express CARs, and adoptive transfer of anti-CD19 CAR T cells is now being tested in clinical trials. Effective clinical treatment with anti-CD19 CAR T cells was first reported in 2010 after a patient with advanced-stage lymphoma treated at the NCI experienced a partial remission of lymphoma and long-term eradication of normal B cells. Additional patients have subsequently obtained long-term remissions of advanced-stage B-cell malignancies after infusions of anti-CD19 CAR T cells. Long-term eradication of normal CD19(+) B cells from patients receiving infusions of anti-CD19 CAR T cells demonstrates the potent antigen-specific activity of these T cells. Some patients treated with anti-CD19 CAR T cells have experienced acute adverse effects, which were associated with increased levels of serum inflammatory cytokines. Although anti-CD19 CAR T cells are at an early stage of development, the potent antigen-specific activity observed in patients suggests that infusions of anti-CD19 CAR T cells might become a standard therapy for some B-cell malignancies.

Ber-H2 (CD30) Immunohistochemical Staining of Human Fetal Tissues

PubMed Central

2005-01-01

OBJECTIVE: CD30 antigen has long been considered to be restricted to the tumour cells of Hodgkin's disease and of anaplastic large cell lymphoma as well as to T and B activated lymphocytes. It is now apparent that the range of normal and neoplastic cells, which may express CD30 antigen, is much wider than was at first thought. In order to gain insight into the physiological function of CD30 antigen, we studied the distribution of its expression in the tissues of fetuses from week 8th to week 16th. MATERIALS AND METHODS: We investigated the immunohistochemical expression of CD30 antigen in paraffin-embedded tissue samples representing all systems from 30 fetuses after therapeutic abortion at 8th to 10th and 12th to 16th week of gestation, respectively, using the monoclonal antibody Ber-H2. RESULTS: Our results demonstrated that CD30 is expressed early in human fetal development (8th to 10th week of gestation) in several fetal tissues derived from all three germ layers (gastrointestinal tract, special glands of the postpharyngeal foregut, urinary, musculoskeletal, reproductive, nervous, endocrine systems), with the exception of the skin and hematolymphoid system (thymus), in which the antigen is expressed later on (10th week onwards). Expression of CD30 was restricted to the hematolymphoid system in the 12-16 weeks of gestation. No expression of the marker was observed in the respiratory and cardiovascular systems during the entire period examined. CONCLUSIONS: CD30 antigen is of importance in cell development, and proliferation. It is also pathway-related to terminal differentiation in many fetal tissues and organs. PMID:16244703

IL-15 induces antigen-independent expansion and differentiation of human naive CD8+ T cells in vitro.

PubMed

Alves, Nuno L; Hooibrink, Berend; Arosa, Fernando A; van Lier, René A W

2003-10-01

Recent studies in mice have shown that although interleukin 15 (IL-15) plays an important role in regulating homeostasis of memory CD8+ T cells, it has no apparent function in controlling homeostatic proliferation of naive T cells. We here assessed the influence of IL-15 on antigen-independent expansion and differentiation of human CD8+ T cells. Both naive and primed human T cells divided in response to IL-15. In this process, naive CD8+ T cells successively down-regulated CD45RA and CD28 but maintained CD27 expression. Concomitant with these phenotypic changes, naive cells acquired the ability to produce interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha), expressed perforin and granzyme B, and acquired cytotoxic properties. Primed CD8+ T cells, from both noncytotoxic (CD45RA-CD27+) and cytotoxic (CD45RA+CD27-) subsets, responded to IL-15 and yielded ample numbers of cytokine-secreting and cytotoxic effector cells. In summary, all human CD8+ T-cell subsets had the ability to respond to IL-15, which suggests a generic influence of this cytokine on CD8+ T-cell homeostasis in man.

CD4+ T cell-mediated rejection of MHC class II-positive tumor cells is dependent on antigen secretion and indirect presentation on host APCs.

PubMed

Haabeth, Ole Audun Werner; Fauskanger, Marte; Manzke, Melanie; Lundin, Katrin U; Corthay, Alexandre; Bogen, Bjarne; Tveita, Anders Aune

2018-05-11

Tumor-specific CD4+ T cells have been shown to mediate efficient anti-tumor immune responses against cancer. Such responses can occur through direct binding to MHC class II (MHC II)-expressing tumor cells or indirectly via activation of professional antigen-presenting cells (APC) that take up and present the tumor antigen. We have previously shown that CD4+ T cells reactive against an epitope within the Ig light chain variable region of a murine B cell lymphoma can reject established tumors. Given the presence of MHC II molecules at the surface of lymphoma cells, we investigated whether MHC II-restricted antigen presentation on tumor cells alone was required for rejection. Variants of the A20 B lymphoma cell line that either secreted or intracellularly retained different versions of the tumor-specific antigen revealed that antigen secretion by the MHC II-expressing tumor cells was essential both for the priming and effector phase of CD4+ T cell-driven anti-tumor immune responses. Consistent with this, genetic ablation of MHC II in tumor cells, both in the case of B lymphoma and B16 melanoma, did not preclude rejection of tumors by tumor antigen-specific CD4+ T cells in vivo. These findings demonstrate that MHC class II expression on tumor cells themselves is not required for CD4+ T cell-mediated rejection, and that indirect display on host APC is sufficient for effective tumor elimination. These results support the importance of tumor-infiltrating APC as mediators of tumor cell killing by CD4+ T cells. Copyright ©2018, American Association for Cancer Research.

Beta-Amyloid Peptides Enhance the Proliferative Response of Activated CD4+CD28+ Lymphocytes from Alzheimer Disease Patients and from Healthy Elderly

PubMed Central

Jóźwik, Agnieszka; Landowski, Jerzy; Bidzan, Leszek; Fülop, Tamas; Bryl, Ewa; Witkowski, Jacek M.

2012-01-01

Alzheimer's disease (AD) is the most frequent form of dementia among elderly. Despite the vast amount of literature on non-specific immune mechanisms in AD there is still little information about the potential antigen-specific immune response in this pathology. It is known that early stages of AD include β-amyloid (Aβ)- reactive antibodies production and inflammatory response. Despite some evidence gathered proving cellular immune response background in AD pathology, the specific reactions of CD4+ and CD8+ cells remain unknown as the previous investigations yielded conflicting results. Here we investigated the CD4+CD28+ population of human peripheral blood T cells and showed that soluble β-amyloids alone were unable to stimulate these cells to proliferate significantly, resulting only in minor, probably antigen-specific, proliferative response. On the other hand, the exposure of in vitro pre-stimulated lymphocytes to soluble Aβ peptides significantly enhanced the proliferative response of these cells which had also lead to increased levels of TNF, IL-10 and IL-6. We also proved that Aβ peptide-enhanced proliferative response of CD4+CD28+ cells is autonomous and independent from disease status while being associated with the initial, ex vivo activation status of the CD4+ cells. In conclusion, we suggest that the effect of Aβ peptides on the immune system of AD patients does not depend on the specific reactivity to Aβ epitope(s), but is rather a consequence of an unspecific modulation of the cell cycle dynamics and cytokine production by T cells, occurring simultaneously in a huge proportion of Aβ peptide-exposed T lymphocytes and affecting the immune system performance. PMID:22428008

Niches for the Long-Term Maintenance of Tissue-Resident Memory T Cells

PubMed Central

Takamura, Shiki

2018-01-01

Tissue-resident memory T cells (TRM cells) are a population of immune cells that reside in the lymphoid and non-lymphoid organs without recirculation through the blood. These important cells occupy and utilize unique anatomical and physiological niches that are distinct from those for other memory T cell populations, such as central memory T cells in the secondary lymphoid organs and effector memory T cells that circulate through the tissues. CD8+ TRM cells typically localize in the epithelial layers of barrier tissues where they are optimally positioned to act as sentinels to trigger antigen-specific protection against reinfection. CD4+ TRM cells typically localize below the epithelial layers, such as below the basement membrane, and cluster in lymphoid structures designed to optimize interactions with antigen-presenting cells upon reinfection. A key feature of TRM populations is their ability to be maintained in barrier tissues for prolonged periods of time. For example, skin CD8+ TRM cells displace epidermal niches originally occupied by γδ T cells, thereby enabling their stable persistence for years. It is also clear that the long-term maintenance of TRM cells in different microenvironments is dependent on multiple tissue-specific survival cues, although the specific details are poorly understood. However, not all TRM persist over the long term. Recently, we identified a new spatial niche for the maintenance of CD8+ TRM cells in the lung, which is created at the site of tissue regeneration after injury [termed repair-associated memory depots (RAMD)]. The short-lived nature of RAMD potentially explains the short lifespans of CD8+ TRM cells in this particular tissue. Clearly, a better understanding of the niche-dependent maintenance of TRM cells will be important for the development of vaccines designed to promote barrier immunity. In this review, we discuss recent advances in our understanding of the properties and nature of tissue-specific niches that maintain TRM cells in different tissues. PMID:29904388

Flow cytometric determination of quantitative immunophenotypes

NASA Astrophysics Data System (ADS)

Redelman, Douglas; Ensign, Wayne; Roberts, Don

2001-05-01

Immunofluorescent flow cytometric analysis of peripheral blood leucocytes is most commonly used to identify and enumerate cells defined by one or more clusters of differentiation (CD) antigens. Although less widely employed, quantitative tests that measure the amounts of CD antigens expressed per cell are used in some situations such as the characterization of lymphomas and leukocytes or the measurement of CD38 on CD3plu8pluT cells in HIV infected individuals. The CD antigens used to identify leukocyte populations are functionally important molecules and it is known that under- or over-expression of some CD antigens can affect cellular responses. For example, high or low expression of CD19 on B cells is associated with autoimmune conditions or depressed antibody responses, respectively. In the current studies, the quantitative expression of CD antigens on T cells, B cells and monocytes was determined in a group of age and sex-matched Marines at several times before and after training exercises. There was substantial variation among these individuals in the quantitative expression of CD antigens and in the number of cells in various populations. However, there was relatively little variation within individuals during the two months they were examined. Thus, the number of cells in leukocyte sub-populations and the amount of CD antigens expressed per cell appear to comprise a characteristic quantitative immunophenotype.

Vaccine strategies against Babesia bovis based on prime-boost immunizations in mice with modified vaccinia Ankara vector and recombinant proteins.

PubMed

Jaramillo Ortiz, José Manuel; Del Médico Zajac, María Paula; Zanetti, Flavia Adriana; Molinari, María Paula; Gravisaco, María José; Calamante, Gabriela; Wilkowsky, Silvina Elizabeth

2014-08-06

In this study, a recombinant modified vaccinia virus Ankara vector expressing a chimeric multi-antigen was obtained and evaluated as a candidate vaccine in homologous and heterologous prime-boost immunizations with a recombinant protein cocktail. The chimeric multi-antigen comprises immunodominant B and T cell regions of three Babesia bovis proteins. Humoral and cellular immune responses were evaluated in mice to compare the immunogenicity induced by different immunization schemes. The best vaccination scheme was achieved with a prime of protein cocktail and a boost with the recombinant virus. This scheme induced high level of specific IgG antibodies and secreted IFN and a high degree of activation of IFNγ(+) CD4(+) and CD8(+) specific T cells. This is the first report in which a novel vaccine candidate was constructed based on a rationally designed multi-antigen and evaluated in a prime-boost regime, optimizing the immune response necessary for protection against bovine babesiosis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Chimeric Antigen Receptor-Engineered T Cells for Immunotherapy of Cancer

PubMed Central

Cartellieri, Marc; Bachmann, Michael; Feldmann, Anja; Bippes, Claudia; Stamova, Slava; Wehner, Rebekka; Temme, Achim; Schmitz, Marc

2010-01-01

CD4+ and CD8+ T lymphocytes are powerful components of adaptive immunity, which essentially contribute to the elimination of tumors. Due to their cytotoxic capacity, T cells emerged as attractive candidates for specific immunotherapy of cancer. A promising approach is the genetic modification of T cells with chimeric antigen receptors (CARs). First generation CARs consist of a binding moiety specifically recognizing a tumor cell surface antigen and a lymphocyte activating signaling chain. The CAR-mediated recognition induces cytokine production and tumor-directed cytotoxicity of T cells. Second and third generation CARs include signal sequences from various costimulatory molecules resulting in enhanced T-cell persistence and sustained antitumor reaction. Clinical trials revealed that the adoptive transfer of T cells engineered with first generation CARs represents a feasible concept for the induction of clinical responses in some tumor patients. However, further improvement is required, which may be achieved by second or third generation CAR-engrafted T cells. PMID:20467460

Immunotherapy with an HIV-DNA Vaccine in Children and Adults

PubMed Central

Palma, Paolo; Gudmundsdotter, Lindvi; Finocchi, Andrea; Eriksson, Lars E.; Mora, Nadia; Santilli, Veronica; Aquilani, Angela; Manno, Emma C.; Zangari, Paola; Romiti, Maria Luisa; Montesano, Carla; Grifoni, Alba; Brave, Andreas; Ljungberg, Karl; Blomberg, Pontus; Bernardi, Stefania; Sandström, Eric; Hejdeman, Bo; Rossi, Paolo; Wahren, Britta

2014-01-01

Therapeutic HIV immunization is intended to induce new HIV-specific cellular immune responses and to reduce viral load, possibly permitting extended periods without antiretroviral drugs. A multigene, multi-subtype A, B, C HIV-DNA vaccine (HIVIS) has been used in clinical trials in both children and adults with the aim of improving and broadening the infected individuals’ immune responses. Despite the different country locations, different regimens and the necessary variations in assays performed, this is, to our knowledge, the first attempt to compare children’s and adults’ responses to a particular HIV vaccine. Ten vertically HIV-infected children aged 4–16 years were immunized during antiretroviral therapy (ART). Another ten children were blindly recruited as controls. Both groups continued their antiretroviral treatment during and after vaccinations. Twelve chronically HIV-infected adults were vaccinated, followed by repeated structured therapy interruptions (STI) of their antiretroviral treatment. The adult group included four controls, receiving placebo vaccinations. The HIV-DNA vaccine was generally well tolerated, and no serious adverse events were registered in any group. In the HIV-infected children, an increased specific immune response to Gag and RT proteins was detected by antigen-specific lymphoproliferation. Moreover, the frequency of HIV-specific CD8+ T-cell lymphocytes releasing perforin was significantly higher in the vaccinees than the controls. In the HIV-infected adults, increased CD8+ T-cell responses to Gag, RT and viral protease peptides were detected. No augmentation of HIV-specific lymphoproliferative responses were detected in adults after vaccination. In conclusion, the HIV-DNA vaccine can elicit new HIV-specific cellular immune responses, particularly to Gag antigens, in both HIV-infected children and adults. Vaccinated children mounted transient new HIV-specific immune responses, including both CD4+ T-cell lymphoproliferation and late CD8+ T-cell responses. In the adult cohort, primarily CD8+ T-cell responses related to MHC class I alleles were noted. However, no clinical benefits with respect to viral load reduction were ascribable to the vaccinations alone. No severe adverse effects related to the vaccine were found in either cohort, and no virological failures or drug resistances were detected. PMID:26344746

Functional Heterogeneity in the CD4+ T Cell Response to Murine γ-Herpesvirus 68

PubMed Central

Hu, Zhuting; Blackman, Marcia A.; Kaye, Kenneth M.; Usherwood, Edward J.

2015-01-01

CD4+ T cells are critical for the control of virus infections, T cell memory and immune surveillance. Here we studied the differentiation and function of murine γ-herpesvirus 68 (MHV-68)-specific CD4+ T cells using gp150-specific TCR transgenic mice. This allowed a more detailed study of the characteristics of the CD4+ T cell response than previously available approaches for this virus. Most gp150-specific CD4+ T cells expressed T-bet and produced IFN-γ, indicating MHV-68 infection triggered differentiation of CD4+ T cells largely into the Th1 subset, whereas some became TFH and Foxp3+ regulatory T cells. These CD4+ T cells were protective against MHV-68 infection, in the absence of CD8+ T cells and B cells, and protection depended on IFN-γ secretion. Marked heterogeneity was observed in the CD4+ T cells, based on Ly6C expression. Ly6C expression positively correlated with IFN-γ, TNF-α and granzyme B production, T-bet and KLRG1 expression, proliferation and CD4+ T cell-mediated cytotoxicity. Ly6C expression inversely correlated with survival, CCR7 expression and secondary expansion potential. Ly6C+ and Ly6C− gp150-specific CD4+ T cells were able to interconvert in a bidirectional manner upon secondary antigen exposure in vivo. These results indicate that Ly6C expression is closely associated with antiviral activity in effector CD4+ T cells, but inversely correlated with memory potential. Interconversion between Ly6C+ and Ly6C− cells may maintain a balance between the two antigen-specific CD4+ T cell populations during MHV-68 infection. These findings have significant implications for Ly6C as a surface marker to distinguish functionally distinct CD4+ T cells during persistent virus infection. PMID:25662997

In vivo blockade of the PD-1 pathway using soluble rPD-1-Fc enhances CD4+ and CD8+ T cell responses but has limited clinical benefit

PubMed Central

Amancha, Praveen K.; Hong, Jung Joo; Rogers, Kenneth; Ansari, Aftab A.; Villinger, Francois

2013-01-01

The PD-1/PD-Ligand pathway has been shown to limit cell mediated effector functions during chronic viral infections impeding clearance of pathogens. As a strategy to reverse this exhaustion and increase T cell poly-functionality, PD-1 ligands were blocked in vivo using a recombinant macaque PD1-Fc fusion protein (rPD-1-Fc) in SIVmac239 infected rhesus macaques during the early chronic phase of infection, either alone or in combination with ART. In vitro blockade showed improvement of antigen specific CD4+ and CD8+ T cells from monkeys chronically infected with SIV. Of note, a prolonged 5-day blockade in culture was beneficial for both gag specific CD4+ and CD8+ T cells based on proliferation and dual cytokine production. While the in vivo administration of a recombinant rhesus PD-1 Fc fusion protein (rPD-1-Fc) induced enhanced SIV specific CD4 and CD8 T cell proliferation both in the blood and gut, it failed to alter plasma viremia. However, rPD-1-Fc administration in the context of ART interruption induced a significant delay of viral load rebound. In addition, rPD-1-Fc administration in MamuA*001+ monkeys led to both an increase in the frequencies and Ki67 expression of GagCM9+ CD8+ T cells in the blood and rectal mucosa and poly-functionality of GagCM9+ CD8+ T cells in blood. In conclusion, however, our data suggest that PD-1/PD-Ligand blockade using soluble rPD-1-Fc instead of anti-PD1 Mab, while effective in rescuing the effector function of SIV-specific CD4+ and CD8+ T cells during the early chronic phase of infection, has limited clinical benefit. PMID:24227774

B and T lymphocyte attenuator mediates inhibition of tumor-reactive CD8+ T cells in patients after allogeneic stem cell transplantation.

PubMed

Hobo, Willemijn; Norde, Wieger J; Schaap, Nicolaas; Fredrix, Hanny; Maas, Frans; Schellens, Karen; Falkenburg, J H Frederik; Korman, Alan J; Olive, Daniel; van der Voort, Robbert; Dolstra, Harry

2012-07-01

Allogeneic stem cell transplantation (allo-SCT) can cure hematological malignancies by inducing alloreactive T cell responses targeting minor histocompatibility antigens (MiHA) expressed on malignant cells. Despite induction of robust MiHA-specific T cell responses and long-term persistence of alloreactive memory T cells specific for the tumor, often these T cells fail to respond efficiently to tumor relapse. Previously, we demonstrated the involvement of the coinhibitory receptor programmed death-1 (PD-1) in suppressing MiHA-specific CD8(+) T cell immunity. In this study, we investigated whether B and T lymphocyte attenuator (BTLA) plays a similar role in functional impairment of MiHA-specific T cells after allo-SCT. In addition to PD-1, we observed higher BTLA expression on MiHA-specific CD8(+) T cells compared with that of the total population of CD8(+) effector-memory T cells. In addition, BTLA's ligand, herpes virus entry mediator (HVEM), was found constitutively expressed by myeloid leukemia, B cell lymphoma, and multiple myeloma cells. Interference with the BTLA-HVEM pathway, using a BTLA blocking Ab, augmented proliferation of BTLA(+)PD-1(+) MiHA-specific CD8(+) T cells by HVEM-expressing dendritic cells. Notably, we demonstrated that blocking of BTLA or PD-1 enhanced ex vivo proliferation of MiHA-specific CD8(+) T cells in respectively 7 and 9 of 11 allo-SCT patients. Notably, in 3 of 11 patients, the effect of BTLA blockade was more prominent than that of PD-1 blockade. Furthermore, these expanded MiHA-specific CD8(+) T cells competently produced effector cytokines and degranulated upon Ag reencounter. Together, these results demonstrate that BTLA-HVEM interactions impair MiHA-specific T cell functionality, providing a rationale for interfering with BTLA signaling in post-stem cell transplantation therapies.

Dendritic cells cross-present HIV antigens from live as well as apoptotic infected CD4+ T lymphocytes

NASA Astrophysics Data System (ADS)

Marañón, Concepción; Desoutter, Jean-François; Hoeffel, Guillaume; Cohen, William; Hanau, Daniel; Hosmalin, Anne

2004-04-01

A better understanding of the antigen presentation pathways that lead to CD8+ T cell recognition of HIV epitopes in vivo is needed to achieve better immune control of HIV replication. Here, we show that cross-presentation of very small amounts of HIV proteins from apoptotic infected CD4+ T lymphocytes by dendritic cells to CD8+ T cells is much more efficient than other known HIV presentation pathways, i.e., direct presentation of infectious virus or cross-presentation of defective virus. Unexpectedly, dendritic cells also take up actively antigens into endosomes from live infected CD4+ T lymphocytes and cross-present them as efficiently as antigens derived from apoptotic infected cells. Moreover, live infected CD4+ T cells costimulate cross-presenting dendritic cells in the process. Therefore, dendritic cells can present very small amounts of viral proteins from infected T cells either after apoptosis, which is frequent during HIV infection, or not. Thus, if HIV expression is transiently induced while costimulation is enhanced (for instance after IL-2 and IFN immune therapy), this HIV antigen presentation pathway could be exploited to eradicate latently infected reservoirs, which are poorly recognized by patients' immune systems.

HLA-A02:01-Restricted Epitopes Identified from the Herpes Simplex Virus Tegument Protein VP11/12 Preferentially Recall Polyfunctional Effector Memory CD8+ T Cells from Seropositive Asymptomatic Individuals and Protect “Humanized” HLA-A*02:01 Transgenic Mice Against Ocular Herpes

PubMed Central

Srivastava, Ruchi; Khan, Arif A.; Spencer, Doran; Vahed, Hawa; Lopes, Patricia P.; Thai, Nhi Thi Uyen; Wang, Christine; Pham, Thanh T.; Huang, Jiawei; Scarfone, Vanessa M.; Nesburn, Anthony B.; Wechsler, Steven L.; BenMohamed, Lbachir

2014-01-01

The Herpes Simplex Virus type 1 virion tegument phosphoprotein 11/12 (HSV-1 VP11/12) is a major antigen targeted by CD8+ T cells from HSV-seropositive individuals. However, whether and which VP11/12-epitope-specific CD8+ T cells play a role in the “natural” protection seen in seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we used multiple prediction computer-assisted algorithms to identify 10 potential HLA-A*02:01-restricted CD8+ T cell epitopes from the 716 amino acids sequence of VP11/12. Three out of ten epitopes exhibited high to moderate binding affinity to HLA-A*02:01 molecules. In ten sequentially studied HLA-A*02:01 positive and HSV-1-seropositive ASYMP individuals, the most frequent, robust and polyfunctional effector CD8+ T-cell responses, as assessed by a combination of tetramer frequency, granzyme B, granzyme K, perforin, CD107a/b cytotoxic degranulation, IFN-γ and multiplex cytokines assays, were predominantly directed against three epitopes: VP11/1266–74, VP11/12220–228 and VP11/12702–710. Interestingly, ASYMP individuals had significantly higher proportion of CD45RAlowCCR7lowCD44highCD62LlowCD27lowCD28lowCD8+ effector memory T cells (TEM) specific to the three epitopes, compared to symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent ocular herpetic disease). Moreover, immunization of HLA-A*02:01 transgenic mice with the three ASYMP CD8+ TEM cell epitopes induced robust and polyfunctional epitope-specific CD8+ TEM cells that were associated with a strong protective immunity against ocular herpes infection and disease. Our findings outline phenotypic and functional features of protective HSV-specific CD8+ T cells that should guide the development of an effective T-cell-based herpes vaccine. PMID:25617474

Human immunodeficiency virus infection of helper T cell clones. Early proliferative defects despite intact antigen-specific recognition and interleukin 4 secretion.

PubMed Central

Laurence, J; Friedman, S M; Chartash, E K; Crow, M K; Posnett, D N

1989-01-01

HIV selectively inhibited the proliferative response of clonal CD4+ T lymphocytes to alloantigen while other alloantigen-dependent responses were unperturbed. Specifically, impaired blastogenesis could be dissociated from alloantigen-specific induction of the B cell activation molecule CD23, IL-4 release, and inositol lipid hydrolysis. In addition, membrane expression of pertinent T cell receptor molecules, including CD2, CD3, and T cell antigen receptor (Ti), remained intact. Using two MHC class II-specific human CD4+ helper T cell clones, the proliferative defect was shown to be an early consequence of HIV infection, occurring within 4 d of viral inoculation and preceding increases in mature virion production. It was generalizable to three distinct methods of T cell activation, all independent of antigen-presenting cells: anti-CD3 mediated cross-linking of the CD3/Ti complex; anti-CD2 and phorbol 12-myristic 13-acetate (PMA); and anti-CD28 plus PMA. These abnormalities were not mitigated by addition of exogenous IL-2, even though expression of the IL-2 receptor (CD25) was unaltered. These studies define a selective blockade in T cell function early after HIV exposure that could serve as a model for certain in vivo manifestations of AIDS. PMID:2470786

The CD1 family: serving lipid antigens to T cells since the Mesozoic era.

PubMed

Zajonc, Dirk M

2016-08-01

Class I-like CD1 molecules are in a family of antigen-presenting molecules that bind lipids and lipopeptides, rather than peptides for immune surveillance by T cells. Since CD1 lacks the high degree of polymorphism found in their major histocompatibility complex (MHC) class I molecules, different species express different numbers of CD1 isotypes, likely to be able to present structurally diverse classes of lipid antigens. In this review, we will present a historical overview of the structures of the different human CD1 isotypes and also discuss species-specific adaptations of the lipid-binding groove. We will discuss how single amino acid changes alter the shape and volume of the CD1 binding groove, how these minor changes can give rise to different numbers of binding pockets, and how these pockets affect the lipid repertoire that can be presented by any given CD1 protein. We will compare the structures of various lipid antigens and finally, we will discuss recognition of CD1-presented lipid antigens by antigen receptors on T cells (TCRs).

The CD1 family: serving lipid antigens to T cells since the Mesozoic era

PubMed Central

Zajonc, Dirk M.

2016-01-01

Class I-like CD1 molecules are in a family of antigen-presenting molecules that bind lipids and lipopeptides, rather than peptides for immune surveillance by T cells. Since CD1 lacks the high degree of polymorphism found in their major histocompatibility complex (MHC) class I molecules, different species express different numbers of CD1 isotypes, likely to be able to present structurally diverse classes of lipid antigens. In this review, we will present a historical overview of the structures of the different human CD1 isotypes and also discuss species-specific adaptations of the lipid-binding groove. We will discuss how single amino acid changes alter the shape and volume of the CD1 binding groove, how these minor changes can give rise to different numbers of binding pockets, and how these pockets affect the lipid repertoire that can be presented by any given CD1 protein. We will compare the structures of various lipid antigens and finally, we will discuss recognition of CD1-presented lipid antigens by antigen receptors on T cells (TCRs). PMID:27368414

Synergism of Cytotoxic T Lymphocyte–Associated Antigen 4 Blockade and Depletion of Cd25+ Regulatory T Cells in Antitumor Therapy Reveals Alternative Pathways for Suppression of Autoreactive Cytotoxic T Lymphocyte Responses

PubMed Central

Sutmuller, Roger P.M.; van Duivenvoorde, Leonie M.; van Elsas, Andrea; Schumacher, Ton N.M.; Wildenberg, Manon E.; Allison, James P.; Toes, Rene E.M.; Offringa, Rienk; Melief, Cornelis J.M.

2001-01-01

Therapeutic efficacy of a tumor cell–based vaccine against experimental B16 melanoma requires the disruption of either of two immunoregulatory mechanisms that control autoreactive T cell responses: the cytotoxic T lymphocyte–associated antigen (CTLA)-4 pathway or the CD25+ regulatory T (Treg) cells. Combination of CTLA-4 blockade and depletion of CD25+ Treg cells results in maximal tumor rejection. Efficacy of the antitumor therapy correlates with the extent of autoimmune skin depigmentation as well as with the frequency of tyrosinase-related protein 2180–188–specific CTLs detected in the periphery. Furthermore, tumor rejection is dependent on the CD8+ T cell subset. Our data demonstrate that the CTL response against melanoma antigens is an important component of the therapeutic antitumor response and that the reactivity of these CTLs can be augmented through interference with immunoregulatory mechanisms. The synergism in the effects of CTLA-4 blockade and depletion of CD25+ Treg cells indicates that CD25+ Treg cells and CTLA-4 signaling represent two alternative pathways for suppression of autoreactive T cell immunity. Simultaneous intervention with both regulatory mechanisms is therefore a promising concept for the induction of therapeutic antitumor immunity. PMID:11560997

CD137 is a Useful Marker for Identifying CD4+ T Cell Responses to Mycobacterium tuberculosis.

PubMed

Yan, Z-H; Zheng, X-F; Yi, L; Wang, J; Wang, X-J; Wei, P-J; Jia, H-Y; Zhou, L-J; Zhao, Y-L; Zhang, H-T

2017-05-01

Upregulation of CD137 on recently activated CD8 + T cells has been used to identify rare viral and tumour antigen-specific T cells from the peripheral blood. We aimed to evaluate the accuracy of CD137 for identifying Mycobacterium tuberculosis (Mtb)-reactive CD4 + T cells in the peripheral blood of infected individuals by flow cytometry and to investigate the characteristics of these CD137 + CD4 + T cells. We initially enrolled 31 active tuberculosis (TB) patients, 31 individuals with latent TB infection (LTBI) and 25 healthy donors. The intracellular CD137 and interferon-γ (IFN-γ) production by CD4 + T cells was simultaneously detected under unstimulated and CFP10-stimulated (culture filtrate protein 10, a Mtb-specific antigen) conditions. In unstimulated CD4 + T cells, we found that the CD137 expression in the TB group was significantly higher than that in the LTBI group. Stimulation with CFP10 largely increased the CD4 + T cell CD137 expression in both the TB and LTBI groups. After CFP10 stimulation, the frequency of CD137 + CD4 + T cells was higher than that of IFN-γ + CD4 + T cells in both the TB and LTBI groups. Most of the CFP10-activated IFN-γ-secreting cells were CD137-positive, but only a small fraction of the CD137-positive cells expressed IFN-γ. An additional 20 patients with TB were enrolled to characterize the CD45RO + CCR7 + , CD45RO + CCR7 - and CD45RO - subsets in the CD137 + CD4 + T cell populations. The Mtb-specific CD137 + CD4 + T cells were mainly identified as having an effector memory phenotype. In conclusion, CD137 is a useful marker that can be used for identifying Mtb-reactive CD4 + T cells by flow cytometry. © 2017 The Foundation for the Scandinavian Journal of Immunology.

Natural killer cell-type body cavity lymphoma following chronic active Epstein-Barr virus infection.

PubMed

Ogata, Masao; Imamura, Tomoyuki; Mizunoe, Syunji; Ohtsuka, Eiichi; Kikuchi, Hiroshi; Nasu, Masaru

2003-06-01

We describe a 69-year-old female who developed natural killer cell-type body cavity lymphoma following chronic active Epstein-Barr virus (CAEBV) infection. Examination of the patient's pleural effusion revealed large abnormal lymphocytes, which were CD2(+), CD7(+), CD30(+), CD56(+), CD3(-), and CD4(-). No rearrangement of T cell receptor genes was detected. Clonal proliferation of Epstein-Barr virus (EBV)-infected cells in pleural effusion was demonstrated by Southern blot hybridization analysis. Human herpesvirus type-8 (HHV-8) DNA was not detected in these cells. The patient achieved a complete remission with combination chemotherapy. Prior to the clinical onset of lymphoma, high fever of unknown origin had persisted for 21 months. IgG antibodies to EBV-viral capsid antigen and to EBV-early antigens, types D and R were not high (1:160 and less than 1:10, respectively). Two months after the onset of fever, however, retrospective quantitative PCR assay revealed a high EBV DNA load in plasma, indicating that CAEBV infection had been the cause of the patient's recurrent fever. The remarkable features of this case are (i) the development of lymphoma following CAEBV infection that demonstrated a normal pattern of EBV-specific antibodies, (ii) the development of HHV-8-negative body cavity lymphoma, and (iii) the effectiveness of combination chemotherapy. Copyright 2003 Wiley-Liss, Inc.

Cellular immune responses in patients with hepatitis B surface antigen seroclearance induced by antiviral therapy

PubMed Central

2011-01-01

Background The mechanisms by which chronic hepatitis B is completely resolved through antiviral therapy are unknown, and the contribution of acquired T cell immunity to hepatitis B surface antigen (HBsAg) seroclearance has not been investigated. Therefore, we measured the T-cell responses to core and envelope antigens in patients with HBsAg seroclearance. Methods Fourteen subjects with HBsAg seroclearance following antiviral treatment for chronic hepatitis B, 7 HBeAg-positive immunotolerant HBV carriers and 9 HBeAg-negative inactive HBsAg carriers were recruited. HBV-specific T-cell responses to recombinant HBV core (rHBcAg) and envelope (rHBsAg) proteins and pools of core and envelope peptides were measured using an ELISPOT assay detecting interferon-gamma and intracellular cytokine staining (ICS) assays detecting interferon-gamma or interleukin 2. Results Interferon-gamma ELISPOT assays showed a low frequency of weak responses to the rHBsAg and S peptide pool in the HBsAg seroclearance group, and the response frequency to the rHBcAg and the C peptide pool was higher than to the rHBsAg (P < 0.001) and S peptide pool (P = 0.001) respectively. A higher response frequency to C than S peptide pools was confirmed in the interferon-gamma ICS assays for both CD4+ (P = 0.033) and CD8+ (P = 0.040) T cells in the HBsAg seroclearance group. The responses to C and S antigens in the inactive carriers were similar. Conclusions There was a low frequency of CD4+ and CD8+ T cell immune responses to envelope antigens in Chinese subjects with HBsAg seroclearance following antiviral therapy. It is unlikely that these immune responses are responsible for HBsAg seroclearance in these subjects. PMID:21320337

Antitumor immune responses mediated by dendritic cells

PubMed Central

Spel, Lotte; Boelens, Jaap-Jan; Nierkens, Stefan; Boes, Marianne

2013-01-01

Dendritic cells (DCs) are essential for the induction of adaptive immune responses against malignant cells by virtue of their capacity to effectively cross-present exogenous antigens to T lymphocytes. Dying cancer cells are indeed a rich source of antigens that may be harnessed for the development of DC-based vaccines. In particular, malignant cells succumbing to apoptosis, rather than necrosis, appear to release antigens in a manner that allows for the elicitation of adaptive immune responses. In this review, we describe the processes that mediate the cross-presentation of antigens released by apoptotic cancer cells to CD8+ T lymphocytes, resulting in the activation of protective tumor-specific immune responses. PMID:24482744

Strategy for monitoring T cell responses to NY-ESO-1 in patients with any HLA class I allele

PubMed Central

Gnjatic, Sacha; Nagata, Yasuhiro; Jäger, Elke; Stockert, Elisabeth; Shankara, Srinivas; Roberts, Bruce L.; Mazzara, Gail P.; Lee, Sang Yull; Dunbar, P. Rod; Dupont, Bo; Cerundolo, Vincenzo; Ritter, Gerd; Chen, Yao-Tseng; Knuth, Alexander; Old, Lloyd J.

2000-01-01

NY-ESO-1 elicits frequent antibody responses in cancer patients, accompanied by strong CD8+ T cell responses against HLA-A2-restricted epitopes. To broaden the range of cancer patients who can be assessed for immunity to NY-ESO-1, a general method was devised to detect T cell reactivity independent of prior characterization of epitopes. A recombinant adenoviral vector encoding the full cDNA sequence of NY-ESO-1 was used to transduce CD8-depleted peripheral blood lymphocytes as antigen-presenting cells. These modified antigen-presenting cells were then used to restimulate memory effector cells against NY-ESO-1 from the peripheral blood of cancer patients. Specific CD8+ T cells thus sensitized were assayed on autologous B cell targets infected with a recombinant vaccinia virus encoding NY-ESO-1. Strong polyclonal responses were observed against NY-ESO-1 in antibody-positive patients, regardless of their HLA profile. Because the vectors do not cross-react immunologically, only responses to NY-ESO-1 were detected. The approach described here allows monitoring of CD8+ T cell responses to NY-ESO-1 in the context of various HLA alleles and has led to the definition of NY-ESO-1 peptides presented by HLA-Cw3 and HLA-Cw6 molecules. PMID:11005863

Trypanosoma cruzi vaccine candidate antigens Tc24 and TSA-1 recall memory immune response associated with HLA-A and -B supertypes in Chagasic chronic patients from Mexico.

PubMed

Villanueva-Lizama, Liliana E; Cruz-Chan, Julio V; Aguilar-Cetina, Amarú Del C; Herrera-Sanchez, Luis F; Rodriguez-Perez, Jose M; Rosado-Vallado, Miguel E; Ramirez-Sierra, Maria J; Ortega-Lopez, Jaime; Jones, Kathryn; Hotez, Peter; Bottazzi, Maria Elena; Dumonteil, Eric

2018-01-01

Trypanosoma cruzi antigens TSA-1 and Tc24 have shown promise as vaccine candidates in animal studies. We evaluated here the recall immune response these antigens induce in Chagasic patients, as a first step to test their immunogenicity in humans. We evaluated the in vitro cellular immune response after stimulation with recombinant TSA-1 (rTSA-1) or recombinant Tc24 (rTc24) in mononuclear cells of asymptomatic Chagasic chronic patients (n = 20) compared to healthy volunteers (n = 19) from Yucatan, Mexico. Proliferation assays, intracellular cytokine staining, cytometric bead arrays, and memory T cell immunophenotyping were performed by flow cytometry. Peripheral blood mononuclear cells (PBMC) from Chagasic patients showed significant proliferation after stimulation with rTc24 and presented a phenotype of T effector memory cells (CD45RA-CCR7-). These cells also produced IFN-γ and, to a lesser extent IL10, after stimulation with rTSA-1 and rTc24 proteins. Overall, both antigens recalled a broad immune response in some Chagasic patients, confirming that their immune system had been primed against these antigens during natural infection. Analysis of HLA-A and HLA-B allele diversity by PCR-sequencing indicated that HLA-A03 and HLA-B07 were the most frequent supertypes in this Mexican population. Also, there was a significant difference in the frequency of HLA-A01 and HLA-A02 supertypes between Chagasic patients and controls, while the other alleles were evenly distributed. Some aspects of the immune response, such as antigen-induced IFN-γ production by CD4+ and CD8+ T cells and CD8+ proliferation, showed significant association with specific HLA-A supertypes, depending on the antigen considered. In conclusion, our results confirm the ability of both TSA-1 and Tc24 recombinant proteins to recall an immune response induced by the native antigens during natural infection in at least some patients. Our data support the further development of these antigens as therapeutic vaccine against Chagas disease.

Suppression of Antigen-Specific T Cell Responses by the Kaposi's Sarcoma-Associated Herpesvirus Viral OX2 Protein and Its Cellular Orthologue, CD200

PubMed Central

Misstear, Karen; Chanas, Simon A.; Rezaee, S. A. Rahim; Colman, Rachel; Quinn, Laura L.; Long, Heather M.; Goodyear, Oliver; Lord, Janet M.; Hislop, Andrew D.

2012-01-01

Regulating appropriate activation of the immune response in the healthy host despite continual immune surveillance dictates that immune responses must be either self-limiting and therefore negatively regulated following their activation or prevented from developing inappropriately. In the case of antigen-specific T cells, their response is attenuated by several mechanisms, including ligation of CTLA-4 and PD-1. Through the study of the viral OX2 (vOX2) immunoregulator encoded by Kaposi's sarcoma-associated herpesvirus (KSHV), we have identified a T cell-attenuating role both for this protein and for CD200, a cellular orthologue of the viral vOX2 protein. In vitro, antigen-presenting cells (APC) expressing either native vOX2 or CD200 suppressed two functions of cognate antigen-specific T cell clones: gamma interferon (IFN-γ) production and mobilization of CD107a, a cytolytic granule component and measure of target cell killing ability. Mechanistically, vOX2 and CD200 expression on APC suppressed the phosphorylation of ERK1/2 mitogen-activated protein kinase in responding T cells. These data provide the first evidence for a role of both KSHV vOX2 and cellular CD200 in the negative regulation of antigen-specific T cell responses. They suggest that KSHV has evolved to harness the host CD200-based mechanism of attenuation of T cell responses to facilitate virus persistence and dissemination within the infected individual. Moreover, our studies define a new paradigm in immune modulation by viruses: the provision of a negative costimulatory signal to T cells by a virus-encoded orthologue of CD200. PMID:22491458

Cytokine response to selected MTB antigens in Ghanaian TB patients, before and at 2 weeks of anti-TB therapy is characterized by high expression of IFN-γ and Granzyme B and inter- individual variation.

PubMed

Mensah, Gloria Ivy; Addo, Kennedy Kwasi; Tetteh, John Amissah; Sowah, Sandra; Loescher, Thomas; Geldmacher, Christof; Jackson-Sillah, Dolly

2014-09-10

There has been a long held belief that patients with drug-susceptible TB are non-infectious after two weeks of therapy. Recent microbiological and epidemiological evidence has challenged this dogma, however, the nature of the Mtb-specific cellular immune response during this period has not been adequately investigated. This knowledge could be exploited in the development of immunological biomarkers of early treatment response. Cellular response to four Mtb infection phase-dependent antigens, ESAT-6/CFP-10 fusion protein and three DosR encoded proteins (Rv1733c, Rv2029c, Rv2628) were evaluated in a Ghanaian TB cohort (n=20) before and after 2 weeks of anti TB therapy. After 6-days in vitro stimulation, Peripheral blood mononuclear cell (PBMC) culture supernatant was harvested and the concentration of IFN-γ, Granzyme B, IL-10, IL-17, sIL2Rα and TNF-α were determined in a 6-plex Luminex assay. Frequencies of IFN-γ + CD4 and CD8 T cells were also determined in an intracellular cytokine assay. All antigens induced higher levels of IFN-γ, followed by Granzyme B, TNF-α and IL-17 and low levels of IL-10 and sIL-2R-α in PBMC before treatment and after 2 weeks of treatment. Median cytokine levels of IFN-γ, Granzyme B, IL-17 and sIL-2R-α increased during week two, but it was significant for only Rv1733-specific production of Granzyme B (P = 0. 013). The median frequency of antigen specific IFN-γ + CD4 T cells increased at week two; however, only the increase in the ESAT-6/CFP-10-specific response was significant (P = 0. 0008). In contrast, the median frequency of ESAT-6/CFP-10- specific IFN-γ + CD8 T cell responses declined during week two (P = 0. 0024). Additionally, wide inter-individual variation with three distinct patterns were observed; increase in all cytokine levels, decrease in all cytokine levels and fluctuating cytokine levels after 2 weeks of treatment. The second week of effective chemotherapy was characterized by a general increase in cytokine response to Mtb-specific antigens suggestive of an improvement in cellular response with therapy. However, the wide inter-individual variation observed would limit the utility of cytokine biomarkers during this period.

Depletion of Regulatory T Cells Augments a Vaccine-Induced T Effector Cell Response against the Liver-Stage of Malaria but Fails to Increase Memory

PubMed Central

Espinoza Mora, Maria del Rosario; Steeg, Christiane; Tartz, Susanne; Heussler, Volker; Sparwasser, Tim; Link, Andreas; Fleischer, Bernhard; Jacobs, Thomas

2014-01-01

Regulatory T cells (Treg) have been shown to restrict vaccine-induced T cell responses in different experimental models. In these studies CD4+CD25+ Treg were depleted using monoclonal antibodies against CD25, which might also interfere with CD25 on non-regulatory T cell populations and would have no effect on Foxp3+CD25− Treg. To obtain more insights in the specific function of Treg during vaccination we used mice that are transgenic for a bacterial artificial chromosome expressing a diphtheria toxin (DT) receptor-eGFP fusion protein under the control of the foxp3 gene locus (depletion of regulatory T cell mice; DEREG). As an experimental vaccine-carrier recombinant Bordetella adenylate cyclase toxoid fused with a MHC-class I-restricted epitope of the circumsporozoite protein (ACT-CSP) of Plasmodium berghei (Pb) was used. ACT-CSP was shown by us previously to introduce the CD8+ epitope of Pb-CSP into the MHC class I presentation pathway of professional antigen-presenting cells (APC). Using this system we demonstrate here that the number of CSP-specific T cells increases when Treg are depleted during prime but also during boost immunization. Importantly, despite this increase of T effector cells no difference in the number of antigen-specific memory cells was observed. PMID:25115805