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.

Reactive glia promote development of CD103+ CD69+ CD8+ T-cells through programmed cell death-ligand 1 (PD-L1).

PubMed

Prasad, Sujata; Hu, Shuxian; Sheng, Wen S; Chauhan, Priyanka; Lokensgard, James R

2018-06-01

Previous work from our laboratory has demonstrated in vivo persistence of CD103 + CD69 + brain resident memory CD8 + T-cells (bT RM ) following viral infection, and that the PD-1: PD-L1 pathway promotes development of these T RM cells within the brain. Although glial cells express low basal levels of PD-L1, its expression is upregulated upon IFN-γ-treatment, and they have been shown to modulate antiviral T-cell effector responses through the PD-1: PD-L1 pathway. We performed flow cytometric analysis of cells from co-cultures of mixed glia and CD8 + T-cells obtained from wild type mice to investigate the role of glial cells in the development of bT RM . In this study, we show that interactions between reactive glia and anti-CD3 Ab-stimulated CD8 + T-cells promote development of CD103 + CD69 + CD8 + T-cells through engagement of the PD-1: PD-L1 pathway. These studies used co-cultures of primary murine glial cells obtained from WT animals along with CD8 + T-cells obtained from either WT or PD-1 KO mice. We found that αCD3 Ab-stimulated CD8 + T-cells from WT animals increased expression of CD103 and CD69 when co-cultured with primary murine glial cells. In contrast, significantly reduced expression of CD103 and CD69 was observed using CD8 + T-cells from PD-1 KO mice. We also observed that reactive glia promoted high levels of CD127, a marker of memory precursor effector cells (MPEC), on CD69 + CD8 + T-cells, which promotes development of T RM cells. Interestingly, results obtained using T-cells from PD-1 KO animals showed significantly reduced expression of CD127 on CD69 + CD8 + cells. Additionally, blocking of glial PD-L1 resulted in decreased expression of CD103, along with reduced CD127 on CD69 + CD8 + T-cells. Taken together, these results demonstrate a role for activated glia in promoting development of bT RM through the PD-1: PD-L1 pathway. © 2018 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.

The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells

PubMed Central

Torreno-Pina, Juan A.; Manzo, Carlo; Salio, Mariolina; Aichinger, Michael C.; Oddone, Anna; Lakadamyali, Melike; Shepherd, Dawn; Besra, Gurdyal S.; Cerundolo, Vincenzo

2016-01-01

Invariant natural killer T (iNKT) cells recognize endogenous and exogenous lipid antigens presented in the context of CD1d molecules. The ability of iNKT cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. However, the mechanisms regulating such “tonic” activation of iNKT cells remain unclear. Here, we show that the spatiotemporal distribution of CD1d molecules on the surface of antigen-presenting cells (APCs) modulates activation of iNKT cells. By using superresolution microscopy, we show that CD1d molecules form nanoclusters at the cell surface of APCs, and their size and density are constrained by the actin cytoskeleton. Dual-color single-particle tracking revealed that diffusing CD1d nanoclusters are actively arrested by the actin cytoskeleton, preventing their further coalescence. Formation of larger nanoclusters occurs in the absence of interactions between CD1d cytosolic tail and the actin cytoskeleton and correlates with enhanced iNKT cell activation. Importantly and consistently with iNKT cell activation during inflammatory conditions, exposure of APCs to the Toll-like receptor 7/8 agonist R848 increases nanocluster density and iNKT cell activation. Overall, these results define a previously unidentified mechanism that modulates iNKT cell autoreactivity based on the tight control by the APC cytoskeleton of the sizes and densities of endogenous antigen-loaded CD1d nanoclusters. PMID:26798067

Human Lymphoid Tissues Harbor a Distinct CD69+CXCR6+ NK Cell Population.

PubMed

Lugthart, Gertjan; Melsen, Janine E; Vervat, Carly; van Ostaijen-Ten Dam, Monique M; Corver, Willem E; Roelen, Dave L; van Bergen, Jeroen; van Tol, Maarten J D; Lankester, Arjan C; Schilham, Marco W

2016-07-01

Knowledge of human NK cells is based primarily on conventional CD56(bright) and CD56(dim) NK cells from blood. However, most cellular immune interactions occur in lymphoid organs. Based on the coexpression of CD69 and CXCR6, we identified a third major NK cell subset in lymphoid tissues. This population represents 30-60% of NK cells in marrow, spleen, and lymph node but is absent from blood. CD69(+)CXCR6(+) lymphoid tissue NK cells have an intermediate expression of CD56 and high expression of NKp46 and ICAM-1. In contrast to circulating NK cells, they have a bimodal expression of the activating receptor DNAX accessory molecule 1. CD69(+)CXCR6(+) NK cells do not express the early markers c-kit and IL-7Rα, nor killer cell Ig-like receptors or other late-differentiation markers. After cytokine stimulation, CD69(+)CXCR6(+) NK cells produce IFN-γ at levels comparable to CD56(dim) NK cells. They constitutively express perforin but require preactivation to express granzyme B and exert cytotoxicity. After hematopoietic stem cell transplantation, CD69(+)CXCR6(+) lymphoid tissue NK cells do not exhibit the hyperexpansion observed for both conventional NK cell populations. CD69(+)CXCR6(+) NK cells constitute a separate NK cell population with a distinct phenotype and function. The identification of this NK cell population in lymphoid tissues provides tools to further evaluate the cellular interactions and role of NK cells in human immunity. Copyright © 2016 by The American Association of Immunologists, Inc.

Increase of CD69, CD161 and CD94 on NK cells in women with recurrent spontaneous abortion and in vitro fertilization failure.

PubMed

Ghafourian, Mehri; Karami, Najmeh; Khodadadi, Ali; Nikbakht, Roshan

2014-06-01

Recurrent spontaneous abortion (RSA) and in vitro fertilization (IVF) failure with unknown causes are the controversial issues that are probably related to the immune system. To compare circulating NK cells expressing activation and inhibition surface markers between patients with RSA and IVF failure with those of healthy multiparous and successful IVF control women, respectively. In this case-control study peripheral blood samples were collected from 43 patients who included 23 women with RSA and 20 with IVF failure, plus 43 healthy control women comprising of 36 normal multiparous women and seven women with successful IVF. The expression of CD69, CD94 and CD161 surface markers on CD56+NK cells were assessed using specific monoclonal antibodies by flowcytometry. The percentage of NK cells increased significantly in patients with RSA and in women with IVF failure in comparison to healthy multiparous and successful IVF control groups (p<0.001). The overall expression of CD69, CD94, CD161 were also increased significantly on NK cells in both patient groups compared to control groups (p<0.001). Elevated expression of CD69 and CD161 on NK cells can be considered as immunological risk markers in RSA and IVF failure. However, it is not clear if high expression of CD94 on peripheral blood NK cells is related to abnormal activity of endometrial NK cells.

T-Cell Receptor- and CD28-induced Vav1 activity is required for the accumulation of primed T cells into antigenic tissue

PubMed Central

David, Rachel; Ma, Liang; Ivetic, Aleksandar; Takesono, Aya; Ridley, Anne J.; Chai, Jian-Guo; Tybulewicz, Victor; Marelli-Berg, Federica M.

2016-01-01

Localization of primed T cells to antigenic tissue is essential for the development of effective immunity. Together with tissue-selective homing molecules, T-cell receptor (TCR)- and CD28-mediated signals have been shown to promote transendothelial migration of specific T cells into non-lymphoid antigen-rich tissue tissue. However, the cellular and molecular requirements for T-cell accumulation to target tissue following their recruitment are largely undefined. The guanine nucleotide exchange factor (GEF) Vav1 has an integral role in coupling TCR and CD28 to signalling pathways that regulate T cell activation and migration. Here, we have investigated the contribution of TCR- and CD28-induced Vav1 activity to the trafficking and localization of primed HY-specific CD4+ T cells to antigenic sites. Severe migratory defects displayed by Vav1-/- T cells in vitro were fully compensated by a combination of shear flow and chemokines, leading to normal recruitment of Vav1-/- T cells in vivo. In contrast, Vav1-/- T-cell retention into antigen-rich tissue was severely impaired, reflecting their inability to engage in sustained TCR- and CD28-mediated interactions with tissue-resident antigen-presenting cells (APCs). This novel function of APC-induced, TCR- and CD28-mediated Vav1 activity in the regulation of effector T-cell immunity highlights its potential as a therapeutic target in T-cell-mediated tissue damage. PMID:19060239

Inhibition of CD1 antigen presentation by human cytomegalovirus.

PubMed

Raftery, Martin J; Hitzler, Manuel; Winau, Florian; Giese, Thomas; Plachter, Bodo; Kaufmann, Stefan H E; Schönrich, Günther

2008-05-01

The betaherpesvirus human cytomegalovirus (HCMV) encodes several molecules that block antigen presentation by the major histocompatibility complex (MHC) proteins. Humans also possess one other family of antigen-presenting molecules, the CD1 family; however, the effect of HCMV on CD1 expression is unknown. The majority of CD1 molecules are classified on the basis of homology as group 1 CD1 and are present almost exclusively on professional antigen-presenting cells such as dendritic cells, which are a major target for HCMV infection and latency. We have determined that HCMV encodes multiple blocking strategies targeting group 1 CD1 molecules. CD1 transcription is strongly inhibited by the HCMV interleukin-10 homologue cmvIL-10. HCMV also blocks CD1 antigen presentation posttranscriptionally by the inhibition of CD1 localization to the cell surface. This function is not performed by a known HCMV MHC class I-blocking molecule and is substantially stronger than the blockage induced by herpes simplex virus type 1. Antigen presentation by CD1 is important for the development of the antiviral immune response and the generation of mature antigen-presenting cells. HCMV present in antigen-presenting cells thus blunts the immune response by the blockage of CD1 molecules.

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.

Cord blood derived CD4+ CD25(high) T cells become functional regulatory T cells upon antigen encounter.

PubMed

Mayer, Elisabeth; Bannert, Christina; Gruber, Saskia; Klunker, Sven; Spittler, Andreas; Akdis, Cezmi A; Szépfalusi, Zsolt; Eiwegger, Thomas

2012-01-01

Upon antigen exposure, cord blood derived T cells respond to ubiquitous environmental antigens by high proliferation. To date it remains unclear whether these "excessive" responses relate to different regulatory properties of the putative T regulatory cell (Treg) compartment or even expansion of the Treg compartment itself. Cord blood (>37 week of gestation) and peripheral blood (healthy controls) were obtained and different Treg cell subsets were isolated. The suppressive potential of Treg populations after antigen exposure was evaluated via functional inhibition assays ([(3)H]thymidine incorporation assay and CFSE staining) with or without allergen stimulation. The frequency and markers of CD4(+)CD25(high)FoxP3(+) T cells were characterized by mRNA analysis and flow cytometry. Cord blood derived CD4(+)CD25(high) cells did not show substantial suppressor capacity upon TCR activation, in contrast to CD4(+)CD25(high) cells freshly purified from adult blood. This could not be explained by a lower frequency of FoxP3(+)CD4(+)CD25(high)cells or FOXP3 mRNA expression. However, after antigen-specific stimulation in vitro, these cells showed strong proliferation and expansion and gained potent suppressive properties. The efficiency of their suppressive capacity can be enhanced in the presence of endotoxins. If T-cells were sorted according to their CD127 expression, a tiny subset of Treg cells (CD4(+)CD25(+)CD127(low)) is highly suppressive even without prior antigen exposure. Cord blood harbors a very small subset of CD4(+)CD25(high) Treg cells that requires antigen-stimulation to show expansion and become functional suppressive Tregs.

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.

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

Decreased expression of CD69 in chronic fatigue syndrome in relation to inflammatory markers: evidence for a severe disorder in the early activation of T lymphocytes and natural killer cells.

PubMed

Mihaylova, Ivana; DeRuyter, Marcel; Rummens, Jean-Luc; Bosmans, Eugene; Maes, Michael

2007-08-01

There is some evidence that patients with chronic fatigue syndrome (CFS) suffer from immune abnormalities, such as immune activation and decreased immune cell responsivity upon polyclonal stimili. This study was designed to evaluate lymphocyte activation in CFS by using a CD69 expression assay. CD69 acts as a costimulatory molecule for T- and natural killer (NK) cell activation. We collected whole blood from CFS patients, who met CDC criteria, and healthy volunteers. The blood samples were stimulated with mitogens during 18 h and the levels of activated T and NK cells expressing CD69 were measured on a Coulter Epics flow cytometer using a three color immunofluorescence staining protocol. The expression of the CD69 activation marker on T cells (CD3+, CD3+CD4+, and CD3+CD8+) and on NK cells (CD45+CD56+) was significantly lower in CFS patients than in healthy subjects. These differences were significant to the extent that a significant diagnostic performance was obtained, i.e. the area under the ROC curve was around 89%. No differences either in the number of leukocytes or in the number or percentage of lymphocytes, i.e. CD3, CD4, CD8 and CD19, could be found between CFS patients and the controls. Patients with CFS show defects in T- and NK cell activation. Since induction of CD69 surface expression is dependent on the activation of the protein kinase C (PKC) activation pathway, it is suggested that in CFS there is a disorder in the early activation of the immune system involving PKC.

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

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.

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.

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.

Antigen-Specific CD8+ T Cells Fail To Respond to Shigella flexneri ▿

PubMed Central

Jehl, Stephanie P.; Doling, Amy M.; Giddings, Kara S.; Phalipon, Armelle; Sansonetti, Philippe J.; Goldberg, Marcia B.; Starnbach, Michael N.

2011-01-01

CD8+ T lymphocytes often play a primary role in adaptive immunity to cytosolic microbial pathogens. Surprisingly, CD8+ T cells are not required for protective immunity to the enteric pathogen Shigella flexneri, despite the ability of Shigella to actively secrete proteins into the host cytoplasm, a location from which antigenic peptides are processed for presentation to CD8+ T cells. To determine why CD8+ T cells fail to play a role in adaptive immunity to S. flexneri, we investigated whether antigen-specific CD8+ T cells are primed during infection but are unable to confer protection or, alternatively, whether T cells fail to be primed. To test whether Shigella is capable of stimulating an antigen-specific CD8+ T-cell response, we created an S. flexneri strain that constitutively secretes a viral CD8+ T-cell epitope via the Shigella type III secretion system and characterized the CD8+ T-cell response to this strain both in mice and in cultured cells. Surprisingly, no T cells specific for the viral epitope were stimulated in mice infected with this strain, and cells infected with the recombinant strain were not targeted by epitope-specific T cells. Additionally, we found that the usually robust T-cell response to antigens artificially introduced into the cytoplasm of cultured cells was significantly reduced when the antigen-presenting cell was infected with Shigella. Collectively, these results suggest that antigen-specific CD8+ T cells are not primed during S. flexneri infection and, as a result, afford little protection to the host during primary or subsequent infection. PMID:21357720

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

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

A Lipid Based Antigen Delivery System Efficiently Facilitates MHC Class-I Antigen Presentation in Dendritic Cells to Stimulate CD8+ T Cells

NASA Astrophysics Data System (ADS)

Maji, Mithun; Mazumder, Saumyabrata; Bhattacharya, Souparno; Choudhury, Somsubhra Thakur; Sabur, Abdus; Shadab, Md.; Bhattacharya, Pradyot; Ali, Nahid

2016-06-01

The most effective strategy for protection against intracellular infections such as Leishmania is vaccination with live parasites. Use of recombinant proteins avoids the risks associated with live vaccines. However, due to low immunogenicity, they fail to trigger T cell responses particularly of CD8+ cells requisite for persistent immunity. Previously we showed the importance of protein entrapment in cationic liposomes and MPL as adjuvant for elicitation of CD4+ and CD8+ T cell responses for long-term protection. In this study we investigated the role of cationic liposomes on maturation and antigen presentation capacity of dendritic cells (DCs). We observed that cationic liposomes were taken up very efficiently by DCs and transported to different cellular sites. DCs activated with liposomal rgp63 led to efficient presentation of antigen to specific CD4+ and CD8+ T cells. Furthermore, lymphoid CD8+ T cells from liposomal rgp63 immunized mice demonstrated better proliferative ability when co-cultured ex vivo with stimulated DCs. Addition of MPL to vaccine enhanced the antigen presentation by DCs and induced more efficient antigen specific CD8+ T cell responses when compared to free and liposomal antigen. These liposomal formulations presented to CD8+ T cells through TAP-dependent MHC-I pathway offer new possibilities for a safe subunit vaccine.

CD21+ (B2 antigen+) cell decrement and CD4+CD29+ (helper-inducer) cell increment suggest an activation of cell immune reactivity in multiple sclerosis.

PubMed

Gambi, D; Porrini, A M; Giampietro, A; Macor, S

1991-08-01

Two-color flow cytometric analysis on peripheral blood lymphocytes of 35 untreated multiple sclerosis (MS) patients, 17 other medical disease (OMD) patients and 14 healthy control (HC) subjects was performed to evaluate the levels of different T and B cell subpopulations. In MS patients we observed an increase in CD4+CD29+ helper-inducer cells but this increase was not related to the different phases of the disease. We hypothesize that this change is related to the reduction of CD21+ cells expressing B2 antigen, a 140 kDa molecule disappearing after B cell activation. An increased level of CD4+CD45RA- (helper-inducer-like cells) and a reduction of CD4+CD29- (suppressor-inducer-like cells) were also present in our patients. These findings demonstrate an immune 'disequilibrium' in MS, which is linked with an increased level of CD25+ cells expressing the interleukin-2 (IL-2) receptor. IL-2, besides being a T cell growth factor, is also a B cell growth factor. These data let us hypothesize that an activation of the immune response is present in MS.

Inflammation-induced CD69+ Kupffer cell feedback inhibits T cell proliferation via membrane-bound TGF-β1.

PubMed

Zhang, Xiang; Jiang, Zhengping; Gu, Yan; Liu, Yanfang; Cao, Xuetao; Han, Yanmei

2016-12-01

Kupffer cells, tissue-resident macrophage lineage cell, are enriched in vertebrate liver. The mouse F4/80 + Kupffer cells have been subclassified into two subpopulations according to their phenotype and function: CD68 + subpopulation with potent reactive oxygen species (ROS) production and phagocytic capacities, and CD11b + subpopulation with a potent capacity to produce T helper 1 cytokines. In addition, CD11b + Kupffer cells/macrophages may be migrated from the bone marrow or spleen, especially in inflammatory conditions of the liver. For analyzing diverse Kupffer cell subsets, we infected mice with Listeria monocytogenes and analyzed the phenotype variations of hepatic Kupffer cells. During L. monocytogenes infection, hepatic CD69 + Kupffer cells were significantly induced and expanded, and CD69 + Kupffer cells expressed higher level of CD11b, and particularly high level of membrane-bound TGF-β1 (mTGF-β1) but lower level of F4/80. We also found that clodronate liposome administration did not eliminate hepatic CD69 + Kupffer cell subset. We consider the hepatic CD69 + Kupffer cell population corresponds to CD11b + Kupffer cells, the bone marrow-derived population. Hepatic CD69 + Kupffer cells suppressed Ag-nonspecific and OVA-specific CD4 T cell proliferation through mTGF-β1 both in vitro and in vivo, meanwhile, they did not interfere with activation of CD4 T cells. Thus, we have identified a new subset of inflammation-induced CD69 + Kupffer cells which can feedback inhibit CD4 T cell response via cell surface TGF-β1 at the late stage of immune response against infection. CD69 + Kupffer cells may contribute to protect host from pathological injure by preventing overactivation of immune response.

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

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.

Effector CD4+ T cells recognize intravascular antigen presented by patrolling monocytes.

PubMed

Westhorpe, Clare L V; Norman, M Ursula; Hall, Pam; Snelgrove, Sarah L; Finsterbusch, Michaela; Li, Anqi; Lo, Camden; Tan, Zhe Hao; Li, Songhui; Nilsson, Susan K; Kitching, A Richard; Hickey, Michael J

2018-02-21

Although effector CD4 + T cells readily respond to antigen outside the vasculature, how they respond to intravascular antigens is unknown. Here we show the process of intravascular antigen recognition using intravital multiphoton microscopy of glomeruli. CD4 + T cells undergo intravascular migration within uninflamed glomeruli. Similarly, while MHCII is not expressed by intrinsic glomerular cells, intravascular MHCII-expressing immune cells patrol glomerular capillaries, interacting with CD4 + T cells. Following intravascular deposition of antigen in glomeruli, effector CD4 + T-cell responses, including NFAT1 nuclear translocation and decreased migration, are consistent with antigen recognition. Of the MHCII + immune cells adherent in glomerular capillaries, only monocytes are retained for prolonged durations. These cells can also induce T-cell proliferation in vitro. Moreover, monocyte depletion reduces CD4 + T-cell-dependent glomerular inflammation. These findings indicate that MHCII + monocytes patrolling the glomerular microvasculature can present intravascular antigen to CD4 + T cells within glomerular capillaries, leading to antigen-dependent inflammation.

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.

Generation of anti-porcine CD69 monoclonal antibodies and their usefulness to evaluate early activation of cellular immunity by flow cytometric analysis.

PubMed

Hayashi, Yumiko; Okutani, Mie; Ogawa, Shohei; Tsukahara, Takamitsu; Inoue, Ryo

2018-05-01

T cell-mediated cellular immunity and humoral immunity are equally important for the prevention of diseases. To assess activation of human and mouse cellular immunity, early activation markers of lymphocytes are often used in flow cytometry targeting expression of CD69 molecules. Response of humoral immunity against infection or vaccination has been well investigated in pigs, but that of cellular immunity has been largely neglected due to lack of direct evaluation tools. Thus, in pig research a proper assay of antibody reacted with porcine CD69 is still unavailable. In the present study, two anti-porcine CD69 mAb-producing mouse hybridomas, 01-14-22-51 (IgG2b-κ) and 01-22-44-102 (IgG2a-κ), both showing fine reactivity with phorbol 12-myristate 13-acetate (PMA) and ionomycin-stimulated porcine peripheral blood lymphocytes in flow cytometry, were established. When porcine peripheral blood lymphocytes were activated with PMA and ionomycin and analyzed by flow cytometry, it was found that both mAbs generated in this study stained about 70% of lymphocytes. In contrast, after an identical procedure, only 5% and 13.5% of lymphocytes were stained with anti-interferon-γ mAb and anti-tumor necrosis factor-α mAb, respectively. These results indicate that evaluation of cellular immunity activation turns more sensitive after using our newly generated mAbs. © 2018 Japanese Society of Animal Science.

MyD88/CD40 Genetic Adjuvant Function in Cutaneous Atypical Antigen-Presenting Cells Contributes to DNA Vaccine Immunogenicity

PubMed Central

Slawin, Kevin M.; Levitt, Jonathan M.; Spencer, David M.

2016-01-01

Therapeutic DNA-based vaccines aim to prime an adaptive host immune response against tumor-associated antigens, eliminating cancer cells primarily through CD8+ cytotoxic T cell-mediated destruction. To be optimally effective, immunological adjuvants are required for the activation of tumor-specific CD8+ T cells responses by DNA vaccination. Here, we describe enhanced anti-tumor efficacy of an in vivo electroporation-delivered DNA vaccine by inclusion of a genetically encoded chimeric MyD88/CD40 (MC) adjuvant, which integrates both innate and adaptive immune signaling pathways. When incorporated into a DNA vaccine, signaling by the MC adjuvant increased antigen-specific CD8+ T cells and promoted elimination of pre-established tumors. Interestingly, MC-enhanced vaccine efficacy did not require direct-expression of either antigen or adjuvant by local antigen-presenting cells, but rather our data supports a key role for MC function in “atypical” antigen-presenting cells of skin. In particular, MC adjuvant-modified keratinocytes increased inflammatory cytokine secretion, upregulated surface MHC class I, and were able to increase in vitro and in vivo priming of antigen-specific CD8+ T cells. Furthermore, in the absence of critical CD8α+/CD103+ cross-priming dendritic cells, MC was still able to promote immune priming in vivo, albeit at a reduced level. Altogether, our data support a mechanism by which MC signaling activates an inflammatory phenotype in atypical antigen-presenting cells within the cutaneous vaccination site, leading to an enhanced CD8+ T cell response against DNA vaccine-encoded antigens, through both CD8α+/CD103+ dendritic cell-dependent and independent pathways. PMID:27741278

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.

Structure and expression of the human thymocyte antigens CD1a, CD1b, and CD1c

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

Martin, L.H.; Calabi, F.; Lefebvre, F.A.

1987-12-01

The CD1 human antigens are a family of at least three components, CD1a, CD1b, and CD1c, that are characteristic of the cortical stage of thymocyte maturation. CD1a was originally named HTA1 or T6 and thought to be the human equivalent of mouse Tla. The genes coding for all three have not been identified by transfection into mouse cells. The transfectants express the surface antigens that can then be recognized by the corresponding cluster of monoclonal antibodies used to define the three members of CD1. The full sequence of the genomic DNA is described for all three. The intron-exon structure ofmore » CD1a is deduced by comparison with a near-full-length cDNA clone. Similar structures are proposed for the other two, largely based on sequence homology. An unusually long 5'-untranslated exon (280 bases long) is highly conserved between the three genes, suggesting an important but unknown function. CD1c has a duplicated form of this exon that is thought to be spliced out. The major homology between the three antigens is in the ..beta../sub 2/-microglobulin-binding-domain. The general relatedness to major histocompatibility complex class I and class II molecules is significant but low, with no section of higher homology to mouse Tla.« less

Mycobacterial Antigen Driven Activation of CD14++CD16− Monocytes Is a Predictor of Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome

PubMed Central

Andrade, Bruno B.; Singh, Amrit; Narendran, Gopalan; Schechter, Melissa E.; Nayak, Kaustuv; Subramanian, Sudha; Anbalagan, Selvaraj; Jensen, Stig M. R.; Porter, Brian O.; Antonelli, Lis R.; Wilkinson, Katalin A.; Wilkinson, Robert J.; Meintjes, Graeme; van der Plas, Helen; Follmann, Dean; Barber, Daniel L.; Swaminathan, Soumya; Sher, Alan; Sereti, Irini

2014-01-01

Paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an aberrant inflammatory response occurring in a subset of TB-HIV co-infected patients initiating anti-retroviral therapy (ART). Here, we examined monocyte activation by prospectively quantitating pro-inflammatory plasma markers and monocyte subsets in TB-HIV co-infected patients from a South Indian cohort at baseline and following ART initiation at the time of IRIS, or at equivalent time points in non-IRIS controls. Pro-inflammatory biomarkers of innate and myeloid cell activation were increased in plasma of IRIS patients pre-ART and at the time of IRIS; this association was confirmed in a second cohort in South Africa. Increased expression of these markers correlated with elevated antigen load as measured by higher sputum culture grade and shorter duration of anti-TB therapy. Phenotypic analysis revealed the frequency of CD14++CD16− monocytes was an independent predictor of TB-IRIS, and was closely associated with plasma levels of CRP, TNF, IL-6 and tissue factor during IRIS. In addition, production of inflammatory cytokines by monocytes was higher in IRIS patients compared to controls pre-ART. These data point to a major role of mycobacterial antigen load and myeloid cell hyperactivation in the pathogenesis of TB-IRIS, and implicate monocytes and monocyte-derived cytokines as potential targets for TB-IRIS prevention or treatment. PMID:25275318

A Novel Method Linking Antigen Presentation by Human Monocyte-Derived Macrophages to CD8+ T Cell Polyfunctionality

PubMed Central

Short, Kirsty R.; Grant, Emma J.; Vissers, Marloes; Reading, Patrick C.; Diavatopoulos, Dimitri A.; Kedzierska, Katherine

2013-01-01

To understand the interactions between innate and adaptive immunity, and specifically how virally infected macrophages impact T cell function, novel assays examining the ability of macrophages to present antigen to CD8+ T cells are needed. In the present study, we have developed a robust in vitro assay to measure how antigen presentation by human monocyte-derived macrophages (MDMs) affects the functional capacity of autologous CD8+ T cells. The assay is based on the polyfunctional characteristics of antigen-specific CD8+ T cells, and is thus called a Mac-CD8 Polyfunctionality Assay. Following purification of monocytes and their maturation to MDMs, MDMs were pulsed with an antigenic peptide to be presented to CD8+ T cells. Peptide-pulsed MDMs were then incubated with antigen-specific CD8+ T cells in order to assess the efficacy of antigen presentation to T cells. CD8+ T cell polyfunctionality was assessed by staining with mAbs to IFN-γ, TNF-α, and CD107a in a multi-color intracellular cytokine staining assay. To highlight the utility of the Mac-CD8 Polyfunctionality Assay, we assessed the effects of influenza infection on the ability of human macrophages to present antigen to CD8+ T cells. We found that influenza infection of human MDMs can alter the effector efficacy of MDMs to activate more CD8+ T cells with cytotoxic capacity. This has important implications for understanding how the virus-infected macrophages affect adaptive immunity at the site of infection. PMID:24312096

[CD69 expression on T cell surface in patients with obstructive sleep apnea hypopnea syndrome].

PubMed

Chen, Y H; Wang, P F; Wang, H J; Hu, X; Li, Z Y; Xiong, H Q

2017-02-20

Objective: To investigate the detection and significance of T cell CD69 expression in peripheral blood of patients with obstructive sleep apnea hypopnea syndrome (OSAHS). Method: According to AHI, 81 OSAHS patients diagnosed by PSG were divided into 3 groups: light, medium and heavy, with 27 cases in each group; 27 patients without OSAHS as control group. Flow cytometry was used to detect the expression rate of CD69 in T cells, to analyze the correlation between the expression rate of CD69 on T cells and the gender, age, BMI, and PSG index. Result: ①The CD69 expression rate of T cells in peripheral blood of OSAHS patients with snoring degree increases gradually ( P < 0.05); Comparison between the two shows that there was no significant difference in CD69 expression rate on T cells between the control group and the mild group ( t = 1.649, P > 0.05); there were significant differences between the other groups ( P < 0.05). ②The CD69 expression rate of T cells in peripheral blood of OSAHS patients has no correlation with BMI, age and gender ( P > 0.05), were positively correlated with AHI, negatively correlated with LSaO₂ ( P < 0.01). ③The CD69 expression rate of T cells and AHI in 27 cases of severe OSAHS patients with a comprehensive treatment has significantly reduced, LSaO₂ increased significantly ( P < 0.01). Conclusion: Increased expression of CD69 in peripheral blood T cells may be one of the mechanisms of OSAHS complicated with cardiovascular disease. Detection of CD69 expression rate in T cells for reflecting the degree of disease in patients with OSAHS, assessment of risk of cardiovascular damage, have certain clinical significance. Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.

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.

Expression of a CD20-specific chimeric antigen receptor enhances cytotoxic activity of NK cells and overcomes NK-resistance of lymphoma and leukemia cells.

PubMed

Müller, Tina; Uherek, Christoph; Maki, Guitta; Chow, Kai Uwe; Schimpf, Annemarie; Klingemann, Hans-Georg; Tonn, Torsten; Wels, Winfried S

2008-03-01

Despite the clinical success of CD20-specific antibody rituximab, malignancies of B-cell origin continue to present a major clinical challenge, in part due to an inability of the antibody to activate antibody-dependent cell-mediated cytotoxicity (ADCC) in some patients, and development of resistance in others. Expression of chimeric antigen receptors in effector cells operative in ADCC might allow to bypass insufficient activation via FcgammaRIII and other resistance mechanisms that limit natural killer (NK)-cell activity. Here we have generated genetically modified NK cells carrying a chimeric antigen receptor that consists of a CD20-specific scFv antibody fragment, via a flexible hinge region connected to the CD3zeta chain as a signaling moiety. As effector cells we employed continuously growing, clinically applicable human NK-92 cells. While activity of the retargeted NK-92 against CD20-negative targets remained unchanged, the gene modified NK cells displayed markedly enhanced cytotoxicity toward NK-sensitive CD20 expressing cells. Importantly, in contrast to parental NK-92, CD20-specific NK cells efficiently lysed CD20 expressing but otherwise NK-resistant established and primary lymphoma and leukemia cells, demonstrating that this strategy can overcome NK-cell resistance and might be suitable for the development of effective cell-based therapeutics for the treatment of B-cell malignancies.

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.

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

Fuzhengpaidu granule regulates immune activation molecules CD38 and human leukocyte antigen-D related on CD4+ and CD8+ T cells in patients with acquired immunodeficiency syndrome/human immunodeficiency virus.

PubMed

Jiang, Feng; Zhang, Rongxin; Gu, Zhenfang; Zhang, Huailing; Guo, Huijun; Deng, Xin; Liang, Jian

2013-08-01

To evaluate the effect of Fuzhengpaidu granule (FZPDG) on immune activation molecules CD38 and human leukocyte antigen-D related (HLA-DR) on CD4+ and CD8+ cells in HIV/AIDS patients, and to explore the underlying mechanism of this therapy. Plasma changes in CD3+, CD4+, CD8+, CD3 + CD4 + CD38 +, CD3 + CD4 + HLA-DR+, CD3 + CD8+CD38+, and CD3+CD8+HLA-DR+ levels in HIV/ AIDS patients treated with FZPDG for six months were examined by flow cytometry and compared with levels in healthy controls. The clinical trial included 34 outpatients with HIV/AIDS. Before treatment, plasma levels of CD38+ and HLA-DR+ on CD4/CD8 cells were higher than those in 28 health controls (P < 0.05). There were no significant changes in serum levels of CD3+, CD4+, and CD8+ T cells between pretreatment baseline versus after treatment, which were 82.85% +/- 5.41%, 14.57% +/- 10.31% and 54.55% +/- 11.43% before treatment and 79.15% +/- 8.21%, 19.96% +/- 9.58% and 56.36% +/- 11.67% after treatment, respectively (P > 0.05). Plasma levels of CD3+ CD4+CD38+ and CD3+CD4+HLA-DR+ were 2.3% +/-2.2% and 7.8% +/- 5.5% before treatment and 1.2% +/-0.8% and 2.6% +/- 1.0% after treatment, respectively. Plasma levels of CD3+CD8+CD38+ and CD3+CD8+ HLA-DR+ were 41.4% +/- 13.4% and 17.8% +/- 11.3% before treatment, which changed to 27.1% +/- 10.2% and 3.8% +/- 2.4% after treatment, respectively (P < 0.05). HIV/AIDS patients exhibited an immune activation profile following FZPDG treatment. A potential mechanism of action for FZPDG appears to lie in its ability to up-regulate CD38 and HLA-DR levels on CD4+ T cells, and down-regulate them on CD8+ cells, thereby modulating immune activation of CD4+and CD8+T cells.

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.

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

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.

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

Ex vivo tetramer staining and cell surface phenotyping for early activation markers CD38 and HLA-DR to enumerate and characterize malaria antigen-specific CD8+ T-cells induced in human volunteers immunized with a Plasmodium falciparum adenovirus-vectored malaria vaccine expressing AMA1.

PubMed

Schwenk, Robert; Banania, Glenna; Epstein, Judy; Kim, Yohan; Peters, Bjoern; Belmonte, Maria; Ganeshan, Harini; Huang, Jun; Reyes, Sharina; Stryhn, Anette; Ockenhouse, Christian F; Buus, Soren; Richie, Thomas L; Sedegah, Martha

2013-10-29

Malaria is responsible for up to a 600,000 deaths per year; conveying an urgent need for the development of a malaria vaccine. Studies with whole sporozoite vaccines in mice and non-human primates have shown that sporozoite-induced CD8+ T cells targeting liver stage antigens can mediate sterile protection. There is a need for a direct method to identify and phenotype malaria vaccine-induced CD8+ T cells in humans. Fluorochrome-labelled tetramers consisting of appropriate MHC class I molecules in complex with predicted binding peptides derived from Plasmodium falciparum AMA-1 were used to label ex vivo AMA-1 epitope specific CD8+ T cells from research subjects responding strongly to immunization with the NMRC-M3V-Ad-PfCA (adenovirus-vectored) malaria vaccine. The identification of these CD8+ T cells on the basis of their expression of early activation markers was also investigated. Analyses by flow cytometry demonstrated that two of the six tetramers tested: TLDEMRHFY: HLA-A*01:01 and NEVVVKEEY: HLA-B*18:01, labelled tetramer-specific CD8+ T cells from two HLA-A*01:01 volunteers and one HLA-B*18:01 volunteer, respectively. By contrast, post-immune CD8+ T cells from all six of the immunized volunteers exhibited enhanced expression of the CD38 and HLA-DRhi early activation markers. For the three volunteers with positive tetramer staining, the early activation phenotype positive cells included essentially all of the tetramer positive, malaria epitope- specific CD8+ T cells suggesting that the early activation phenotype could identify all malaria vaccine-induced CD8+ T cells without prior knowledge of their exact epitope specificity. The results demonstrated that class I tetramers can identify ex vivo malaria vaccine antigen-specific CD8+ T cells and could therefore be used to determine their frequency, cell surface phenotype and transcription factor usage. The results also demonstrated that vaccine antigen-specific CD8+ T cells could be identified by activation markers

CD1a presentation of endogenous antigens by group 2 innate lymphoid cells.

PubMed

Hardman, Clare S; Chen, Yi-Ling; Salimi, Maryam; Jarrett, Rachael; Johnson, David; Järvinen, Valtteri J; Owens, Raymond J; Repapi, Emmanouela; Cousins, David J; Barlow, Jillian L; McKenzie, Andrew N J; Ogg, Graham

2017-12-22

Group 2 innate lymphoid cells (ILC2) are effectors of barrier immunity, with roles in infection, wound healing, and allergy. A proportion of ILC2 express MHCII (major histocompatibility complex II) and are capable of presenting peptide antigens to T cells and amplifying the subsequent adaptive immune response. Recent studies have highlighted the importance of CD1a-reactive T cells in allergy and infection, activated by the presentation of endogenous neolipid antigens and bacterial components. Using a human skin challenge model, we unexpectedly show that human skin-derived ILC2 can express CD1a and are capable of presenting endogenous antigens to T cells. CD1a expression is up-regulated by TSLP (thymic stromal lymphopoietin) at levels observed in the skin of patients with atopic dermatitis, and the response is dependent on PLA2G4A. Furthermore, this pathway is used to sense Staphylococcus aureus by promoting Toll-like receptor-dependent CD1a-reactive T cell responses to endogenous ligands. These findings define a previously unrecognized role for ILC2 in lipid surveillance and identify shared pathways of CD1a- and PLA2G4A-dependent ILC2 inflammation amenable to therapeutic intervention. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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

Expression of interleukin-2 (IL-2) receptor alpha and CD45RO antigen on T-lymphocytes cultured with measles virus antigens, compared with humoral immunity in measles vaccinees.

PubMed

Toyoda, M; Ihara, T; Nakano, T; Ito, M; Kamiya, H

1999-03-17

In response to two types of measles virus (MV) antigens, a vaccine strain CAM and a wild strain isolated in 1994, the expression of IL-2 receptor alpha (CD25)(+)CD45RO(+)CD4(+) T-lymphocytes (T-cell activation) was analyzed by flow cytometry. In 75 healthy subjects with measles hemagglutination inhibition tests > or =1:16, the percentage of T-cell activation was significantly increased compared with that in seronegative individuals (p) < 0.05). Moreover, the T-cell expression was not significantly different among the vaccinated (n = 38), the naturally infected (n = 28) and the subclinically infected (exposed with wild type without history of measles infection and HI titers > or =1:16) (n = 10) groups. T-cell activation stimulated with MV antigens and HI antibody titers persisted for almost 30 years in the vaccinated group. These results suggest that cell-mediated immunity persists for long periods after vaccination and does not be influenced by antigenic drift.

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.

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

The simultaneous ex vivo detection of low-frequency antigen-specific CD4+ and CD8+ T-cell responses using overlapping peptide pools.

PubMed

Singh, Satwinder Kaur; Meyering, Maaike; Ramwadhdoebe, Tamara H; Stynenbosch, Linda F M; Redeker, Anke; Kuppen, Peter J K; Melief, Cornelis J M; Welters, Marij J P; van der Burg, Sjoerd H

2012-11-01

The ability to measure antigen-specific T cells at the single-cell level by intracellular cytokine staining (ICS) is a promising immunomonitoring tool and is extensively applied in the evaluation of immunotherapy of cancer. The protocols used to detect antigen-specific CD8+ T-cell responses generally work for the detection of antigen-specific T cells in samples that have undergone at least one round of in vitro pre-stimulation. Application of a common protocol but now using long peptides as antigens was not suitable to simultaneously detect antigen-specific CD8+ and CD4+ T cells directly ex vivo in cryopreserved samples. CD8 T-cell reactivity to monocytes pulsed with long peptides as antigens ranged between 5 and 25 % of that observed against monocytes pulsed with a direct HLA class I fitting minimal CTL peptide epitope. Therefore, we adapted our ICS protocol and show that the use of tenfold higher concentration of long peptides to load APC, the use of IFN-α and poly(I:C) to promote antigen processing and improve T-cell stimulation, does allow for the ex vivo detection of low-frequency antigen-specific CD8+ and CD4+ T cells in an HLA-independent setting. While most of the improvements were related to increasing the ability to measure CD8+ T-cell reactivity following stimulation with long peptides to at least 50 % of the response detected when using a minimal peptide epitope, the final analysis of blood samples from vaccinated patients successfully showed that the adapted ICS protocol also increases the ability to ex vivo detect low-frequency p53-specific CD4+ T-cell responses in cryopreserved PBMC samples.

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.

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.

Increased CD69 Expression on Peripheral Eosinophils from Patients with Food Protein-Induced Enterocolitis Syndrome.

PubMed

Wada, Taizo; Matsuda, Yusuke; Toma, Tomoko; Koizumi, Eiko; Okamoto, Hiroyuki; Yachie, Akihiro

2016-01-01

Food protein-induced enterocolitis syndrome (FPIES) is an uncommon, non-IgE-mediated food allergy. We recently described a significant increase in fecal eosinophil-derived neurotoxin (EDN) after ingestion of the causative food. However, little is known about the activation status of circulating eosinophils in patients with an acute FPIES reaction. Surface CD69 expression was assessed by flow cytometry on peripheral eosinophils from 5 patients with FPIES before and after ingestion of the causative food. Fecal EDN was measured by enzyme-linked immunosorbent assay. No eosinophil activation was observed before ingestion; however, a significant increase in CD69 expression on eosinophils after an acute FIPES reaction was demonstrated in all of the patients. There was no significant change in absolute eosinophil counts in the peripheral blood. The levels of fecal EDN increased on the day after ingestion of the causative food in all patients. These results suggest that circulating eosinophils as well as eosinophils in the intestinal mucosal tissue are activated in acute FPIES reactions and might be associated with systemic immune events in FPIES. © 2016 S. Karger AG, Basel.

Antigen presentation by small intestinal epithelial cells uniquely enhances IFN-γ secretion from CD4{sup +} intestinal intraepithelial lymphocytes

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

Hatano, Ryo; Yamada, Kiyoshi; Iwamoto, Taku

2013-06-14

Highlights: •Small intestinal epithelial cells (sIECs). •sIECs are able to induce antigen specific proliferation of CD4{sup +} IELs. •sIECs induce markedly enhanced IFN-γ secretion by CD4{sup +} IELs. •Induction of enhanced IFN-γ secretion by sIECs is uniquely observed in CD4{sup +} IELs. -- Abstract: Small intestinal epithelial cells (sIECs) express major histocompatibility complex class II molecules even in a normal condition, and are known to function as antigen presenting cells (APCs) at least in vitro. These findings raised the possibility that sIECs play an important role in inducing immune responses against luminal antigens, especially those of intestinal intraepithelial lymphocytes (IELs)more » and lamina propria lymphocytes (LPLs). We herein showed that antigenic stimulation with sIECs induced markedly greater secretion of interferon-gamma (IFN-γ) by CD4{sup +} IELs, but not interleukin (IL)-4, IL-10 and IL-17 although the proliferative response was prominently lower than that with T cell-depleted splenic APCs. In contrast, no enhanced IFN-γ secretion by CD4{sup +} LPLs and primed splenic CD4{sup +} T cells was observed when stimulated with sIECs. Taken together, these results suggest that sIECs uniquely activate CD4{sup +} IELs and induce remarkable IFN-γ secretion upon antigenic stimulation in vivo.« less

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.

Turned on by danger: activation of CD1d-restricted invariant natural killer T cells

PubMed Central

Lawson, Victoria

2012-01-01

CD1d-restricted invariant natural killer T (iNKT) cells bear characteristics of innate and adaptive lymphocytes, which allow them to bridge the two halves of the immune response and play roles in many disease settings. Recent work has characterized precisely how their activation is initiated and regulated. Novel antigens from important pathogens have been identified, as has an abundant self-antigen, β-glucopyranosylcaramide, capable of mediating an iNKT-cell response. Studies of the iNKT T-cell receptor (TCR)–antigen–CD1d complex show how docking between CD1d–antigen and iNKT TCR is highly conserved, and how small sequence differences in the TCR establish intrinsic variation in iNKT TCR affinity. The sequence of the TCR CDR3β loop determines iNKT TCR affinity for ligand–CD1d, independent of ligand identity. CD1d ligands can promote T helper type 1 (Th1) or Th2 biased cytokine responses, depending on the composition of their lipid tails. Ligands loaded into CD1d on the cell surface promote Th2 responses, whereas ligands with long hydrophobic tails are loaded endosomally and promote Th1 responses. This information is informing the design of synthetic iNKT-cell antigens. The iNKT cells may be activated by exogenous antigen, or by a combination of dendritic cell-derived interleukin-12 and iNKT TCR–self-antigen–CD1d engagement. The iNKT-cell activation is further modulated by recent foreign or self-antigen encounter. Activation of dendritic cells through pattern recognition receptors alters their antigen presentation and cytokine production, strongly influencing iNKT-cell activation. In a range of bacterial infections, dendritic cell-dependent innate activation of iNKT cells through interleukin-12 is the dominant influence on their activity. PMID:22734667

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

Activating and inhibitory receptors on synovial fluid natural killer cells of arthritis patients: role of CD94/NKG2A in control of cytokine secretion

PubMed Central

de Matos, Cristina Teixeira; Berg, Louise; Michaëlsson, Jakob; Felländer-Tsai, Li; Kärre, Klas; Söderström, Kalle

2007-01-01

Natural killer (NK) cells are activated early during inflammatory events and contribute to the shaping of the ensuing adaptive immune response. To further understand the role for NK cells in inflammation, we investigated the phenotype and function of synovial fluid (SF) NK cells from patients with chronic joint inflammation, as well as from patients with transient inflammation of the knee following trauma. We confirm that synovial NK cells are similar to the well-characterized CD56bright peripheral blood (PB) NK-cell subset present in healthy individuals. However, compared to this PB subset the synovial NK cells express a higher degree of activation markers including CD69 and NKp44, the latter being up-regulated also on CD56bright NK cells in the PB of patients. Activated synovial NK cells produced interferon-γ and tumour necrosis factor, and the production was further up-regulated by antibody masking of CD94/NKG2A, and down-regulated by target cells expressing human leucocyte antigen-E in complex with peptides known to engage CD94/NKG2A. We conclude that synovial NK cells have an activated phenotype and that CD94/NKG2A is a key regulator of synovial NK-cell cytokine synthesis. PMID:17521371

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

AFM force measurements of the gp120-sCD4 and gp120 or CD4 antigen-antibody interactions

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

Chen, Yong, E-mail: dr_yongchen@hotmail.com; Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612; Zeng, Gucheng

2011-04-08

Highlights: {yields} The unbinding force of sCD4-gp120 interaction was 25.45 {+-} 20.46 pN. {yields} The unbinding force of CD4 antigen-antibody interaction was 51.22 {+-} 34.64 pN. {yields} The unbinding force of gp120 antigen-antibody interaction was 89.87 {+-} 44.63 pN. {yields} The interaction forces between various HIV inhibitors and the target molecules are significantly different. {yields} Functionalizing on AFM tip or substrate of an interaction pair caused different results. -- Abstract: Soluble CD4 (sCD4), anti-CD4 antibody, and anti-gp120 antibody have long been regarded as entry inhibitors in human immunodeficiency virus (HIV) therapy. However, the interactions between these HIV entry inhibitors andmore » corresponding target molecules are still poorly understood. In this study, atomic force microscopy (AFM) was utilized to investigate the interaction forces among them. We found that the unbinding forces of sCD4-gp120 interaction, CD4 antigen-antibody interaction, and gp120 antigen-antibody interaction were 25.45 {+-} 20.46, 51.22 {+-} 34.64, and 89.87 {+-} 44.63 pN, respectively, which may provide important mechanical information for understanding the effects of viral entry inhibitors on HIV infection. Moreover, we found that the functionalization of an interaction pair on AFM tip or substrate significantly influenced the results, implying that we must perform AFM force measurement and analyze the data with more caution.« less

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.

The reliability of cytoplasmic CD3 and CD22 antigen expression in the immunodiagnosis of acute leukemia: a study of 500 cases.

PubMed

Janossy, G; Coustan-Smith, E; Campana, D

1989-03-01

Current views about the origin of acute lymphoid leukemia (ALL) emphasize the importance of maturation arrest at a precursor cell level. Recently, the CD22 antigen has been identified in the cytoplasm of normal bone marrow-borne immature B lineage cells, while the CD3 antigen (epsilon chain) has been detected within normal immature thymic blasts. In the first part our study performed on 100 cases of known acute leukemias, the expression of such cytoplasmic molecules, referred to as cCD22 and cCD3, was analyzed together with their appearance in the leukemic cells' membrane (mCD22 and mCD3). The presence of cCD22 in B-lineage ALL and that of cCD3 in T-ALL has indeed fully confirmed the diagnosis reached by other markers, and mCD22 and mCD3 were expressed on only a few cases of B- and T-lineage ALL, also revealing a degree of developmental asynchrony within leukemic blasts. In the subsequent analysis both cCD22 and cCD3 have been included in a standard panel of diagnostic reagents applied on 500 consecutive cases of acute leukemia. Here the aim was to analyze both the diagnostic precision of individual markers and the heterogeneity of various leukemic types in terms of the expression of membrane and intracellular antigens and their cytochemical features (Sudan Black B and esterases). It has been found that cCD22 and cCD3 are exquisitely specific for B-precursor ALL (TdT+, CD19+) and T-ALL (TdT+, CD7+), respectively, while both markers are absent in acute myeloblastic leukemia (AML) and acute myelomonocytic and monocytic leukemia (AMML/AMoL). These observations contrast the findings which demonstrate that 31% of cases among nonlymphoid acute leukemia (including AML and AMML) express CD7 and/or TdT. The study of myeloid antigens detected by CD13, CD33, and CD14 is also informative and complementary, both in diagnosing and subdividing the AML and AMML/AMoL groups. The peculiar main observation of this study is that only with the combined use of these markers in a

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.

Anergic self-reactive B cells present self antigen and respond normally to CD40-dependent T-cell signals but are defective in antigen-receptor-mediated functions.

PubMed Central

Eris, J M; Basten, A; Brink, R; Doherty, K; Kehry, M R; Hodgkin, P D

1994-01-01

B-cell tolerance to soluble protein self antigens such as hen egg lysozyme (HEL) is mediated by clonal anergy. Anergic B cells fail to mount antibody responses even in the presence of carrier-primed T cells, suggesting an inability to activate or respond to T helper cells. To investigate the nature of this defect, B cells from tolerant HEL/anti-HEL double-transgenic mice were incubated with a membrane preparation from activated T-cell clones expressing the CD40 ligand. These membranes, together with interleukin 4 and 5 deliver the downstream antigen-independent CD40-dependent B-cell-activating signals required for productive T-B collaboration. Anergic B cells responded to this stimulus by proliferating and secreting antibody at levels comparable to or better than control B cells. Furthermore, anergic B cells presented HEL acquired in vivo and could present the unrelated antigen, conalbumin, targeted for processing via surface IgD. In contrast, the low immunoglobulin receptor levels on anergic B cells were associated with reduced de novo presentation of HEL and a failure to upregulate costimulatory ligands for CD28. These defects in immunoglobulin-receptor-mediated functions could be overcome in vivo, suggesting a number of mechanisms for induction of autoantibody responses. Images PMID:7514304

Dietary Fructo-Oligosaccharides Attenuate Early Activation of CD4+ T Cells Which Produce both Th1 and Th2 Cytokines in the Intestinal Lymphoid Tissues of a Murine Food Allergy Model.

PubMed

Tsuda, Masato; Arakawa, Haruka; Ishii, Narumi; Ubukata, Chihiro; Michimori, Mana; Noda, Masanari; Takahashi, Kyoko; Kaminogawa, Shuichi; Hosono, Akira

2017-01-01

Fructo-oligosaccharides (FOS) are prebiotic agents with immunomodulatory effects involving improvement of the intestinal microbiota and metabolome. In this study, we investigated the cellular mechanisms through which FOS modulate intestinal antigen-specific CD4+ T cell responses in food allergy, using OVA23-3 mice. OVA23-3 mice were fed an experimental diet containing either ovalbumin (OVA) or OVA and FOS for 1 week. Body weight and mucosal mast cell protease 1 in the serum were measured as the indicator of intestinal inflammation. Single-cell suspensions were prepared from intestinal and systemic lymphoid tissues for cellular analysis. Cytokine production was measured by ELISA. Activation markers and intracellular cytokines in CD4+ T cells were analyzed by flow cytometry. Activated CD4+ T cells were purified to examine cytokine production. Dietary intake of FOS provided moderate protection from the intestinal inflammation induced by the OVA-containing diet. FOS significantly reduced food allergy-induced Th2 cytokine responses in intestinal tissues but not in systemic tissues. FOS decreased OVA diet-induced IFN-γ+IL-4+ double-positive CD4+ T cells and early-activated CD45RBhighCD69+CD4+ T cells in the mesenteric lymph nodes. Furthermore, we confirmed that these CD45RBhighCD69+CD4+ T cells are able to produce high levels of IFN-γ and moderate level of IL-4, IL-10, and IL-13. Dietary intake of FOS during the development of food allergy attenuates the induction of intestinal Th2 cytokine responses by regulating early activation of naïve CD4+ T cells, which produce both Th1 and Th2 cytokines. Our results suggest FOS might be a potential food agent for the prevention of food allergy by modulating oral sensitization to food antigens. © 2017 S. Karger AG, Basel.

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

Activation status of mucosal-associated invariant T cells reflects disease activity and pathology of systemic lupus erythematosus.

PubMed

Chiba, Asako; Tamura, Naoto; Yoshikiyo, Kazunori; Murayama, Goh; Kitagaichi, Mie; Yamaji, Ken; Takasaki, Yoshinari; Miyake, Sachiko

2017-03-14

Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes constituting a large proportion of peripheral blood T cells expressing αβ T-cell receptor in humans. In this study, we aimed to investigate their involvement in systemic lupus erythematosus (SLE). Peripheral blood MAIT cells from patients with SLE were assessed for their frequency, activation markers, and cell death by flow cytometry. The correlation between plasma cytokine levels and CD69 expression on MAIT cells was analyzed. The major histocompatibility complex class I-related protein MR1-restricted antigen-presenting capacity of antigen-presenting cells was investigated. Cytokine-mediated activation of MAIT cells in the absence of exogenous antigens was also examined. The frequency of MAIT cells was markedly reduced in SLE. The reduced number of MAIT cells was not attributable to the downregulation of surface markers, but it was partially due to the enhanced cell death of MAIT cells, possibly by activation-induced cell death. The CD69 expression levels on MAIT cells in SLE correlated with disease activity. Moreover, monocytes from patients with SLE exhibited increased ability to induce MAIT cell activation. The plasma concentration of interleukin (IL)-6, IL-18, and interferon (IFN)-α positively correlated with the expression levels of CD69 on MAIT cells in SLE. MAIT cells were activated by cytokines, including IFN-α, IL-15, and IL-12 plus IL-18, in the absence of exogenous antigens. These results suggest that MAIT cells reflect the pathological condition of SLE and that their activated status correlates with presence of disease.

Monocyte:T cell interaction regulates human T cell activation through a CD28/CD46 crosstalk

PubMed Central

Charron, Lauren; Doctrinal, Axelle; Choileain, Siobhan Ni; Astier, Anne L.

2015-01-01

T cell activation requires engagement of the T cell receptor and of at least one costimulatory molecule. The key role of CD28 in inducing T cell activation has been reported several decades ago and the molecular mechanisms involved well described. The complement regulator CD46 also acts as a costimulatory molecule for T cells but, in contrast to CD28, has the ability to drive T cell differentiation from producing some IFNγ to secreting some potent anti-inflammatory IL-10, acquiring a so-called Type I regulatory phenotype (Tr1). Proteolytic cleavage of CD46 occurs upon costimulation and is important for T cell activation and IL-10 production. The observation that CD46 cleavage was reduced when PBMC were costimulated compared to purified naive T cells led us to hypothesize that interactions between different cell types within the PBMC were able to modulate the CD46 pathway. We show that CD46 downregulation is also reduced when CD4+ T cells are co-cultured with autologous monocytes. Indeed, monocyte:T cell co-cultures impaired CD46–mediated T cell differentiation and coactivation, by reducing downregulation of surface CD46, lowering induction of the early activation marker CD69, as well as reducing the levels of IL-10 secretion. Blocking of CD86 could partly restore CD69 expression and cytokine secretion, demonstrating that the CD28-CD86 pathway regulates CD46 activation. Direct concomitant ligation of CD28 and CD46 on CD4+ T cells also modulated CD46 expression and regulated cytokine production. These data identify a crosstalk between two main costimulatory pathways and provide novel insights into the regulation of human T cell activation. PMID:25787182

CD1d-restricted immunoglobulin G formation to GPI-anchored antigens mediated by NKT cells.

PubMed

Schofield, L; McConville, M J; Hansen, D; Campbell, A S; Fraser-Reid, B; Grusby, M J; Tachado, S D

1999-01-08

Immunoglobulin G (IgG) responses require major histocompatibility complex (MHC)-restricted recognition of peptide fragments by conventional CD4(+) helper T cells. Immunoglobulin G responses to glycosylphosphatidylinositol (GPI)- anchored protein antigens, however, were found to be regulated in part through CD1d-restricted recognition of the GPI moiety by thymus-dependent, interleukin-4-producing CD4(+), natural killer cell antigen 1.1 [(NK1.1)+] helper T cells. The CD1-NKT cell pathway regulated immunogobulin G responses to the GPI-anchored surface antigens of Plasmodium and Trypanosoma and may be a general mechanism for rapid, MHC-unrestricted antibody responses to diverse pathogens.

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

Differential cellular internalization of anti-CD19 and -CD22 immunotoxins results in different cytotoxic activity.

PubMed

Du, Xing; Beers, Richard; Fitzgerald, David J; Pastan, Ira

2008-08-01

B-cell malignancies routinely express surface antigens CD19 and CD22. Immunotoxins against both antigens have been evaluated, and the immunotoxins targeting CD22 are more active. To understand this disparity in cytotoxicity and guide the screening of therapeutic targets, we compared two immunotoxins, FMC63(Fv)-PE38-targeting CD19 and RFB4(Fv)-PE38 (BL22)-targeting CD22. Six lymphoma cell lines have 4- to 9-fold more binding sites per cell for CD19 than for CD22, but BL22 is 4- to 140-fold more active than FMC63(Fv)-PE38, although they have a similar cell binding affinity (Kd, approximately 7 nmol/L). In 1 hour, large amounts of BL22 are internalized (2- to 3-fold more than the number of CD22 molecules on the cell surface), whereas only 5.2% to 16.6% of surface-bound FMC63(Fv)-PE38 is internalized. The intracellular reservoir of CD22 decreases greatly after immunotoxin internalization, indicating that it contributes to the uptake of BL22. Treatment of cells with cycloheximide does not reduce the internalization of BL22. Both internalized immunotoxins are located in the same vesicles. Our results show that the rapid internalization of large amounts of BL22 bound to CD22 makes CD22 a better therapeutic target than CD19 for immunotoxins and probably for other immunoconjugates that act inside cells.

Superior Therapeutic Index in Lymphoma Therapy: CD30+ CD34+ Hematopoietic Stem Cells Resist a Chimeric Antigen Receptor T-cell Attack

PubMed Central

Hombach, Andreas A; Görgens, André; Chmielewski, Markus; Murke, Florian; Kimpel, Janine; Giebel, Bernd; Abken, Hinrich

2016-01-01

Recent clinical trials with chimeric antigen receptor (CAR) redirected T cells targeting CD19 revealed particular efficacy in the treatment of leukemia/lymphoma, however, were accompanied by a lasting depletion of healthy B cells. We here explored CD30 as an alternative target, which is validated in lymphoma therapy and expressed by a broad variety of Hodgkin's and non-Hodgkin's lymphomas. As a safty concern, however, CD30 is also expressed by lymphocytes and hematopoietic stem and progenitor cells (HSPCs) during activation. We revealed that HRS3scFv-derived CAR T cells are superior since they were not blocked by soluble CD30 and did not attack CD30+ HSPCs while eliminating CD30+ lymphoma cells. Consequently, normal hemato- and lymphopoiesis was not affected in the long-term in the humanized mouse; the number of blood B and T cells remained unchanged. We provide evidence that the CD30+ HSPCs are protected against a CAR T-cell attack by substantially lower CD30 levels than lymphoma cells and higher levels of the granzyme B inactivating SP6/PI9 serine protease, which furthermore increased upon activation. Taken together, adoptive cell therapy with anti-CD30 CAR T cells displays a superior therapeutic index in the treatment of CD30+ malignancies leaving healthy activated lymphocytes and HSPCs unaffected. PMID:27112062

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 presenting cells (APCs) from thermally injured and/or septic rats modulate CD4+ T cell responses of naive rat.

PubMed

Fazal, Nadeem; Raziuddin, Syed; Khan, Mehdi; Al-Ghoul, Walid M

2006-01-01

Regulation of immune response is marked by complex interactions among the cells that recognize and present antigens. Antigen presenting cells (APCs), the antigen presenting cell component of the innate immune response plays an important role in effector CD4+ T cell response. Thermal injury and/or superimposed sepsis in rats' leads to suppressed CD4+ T cell functions. We investigated modulations of CD4+ T cell function by APCs (purified non-T cells) from thermally injured and/or septic rats. Rats were subjected to 30% total body surface area scald burn or exposed to 37 degrees C water (Sham burn) and sepsis was induced by cecal-ligation and puncture (CLP) method. At day 3 post-injury animals were sacrificed and CD4+ T cells and APCs from mesenteric lymph nodes (MLN) were obtained using magnetic microbead isolation procedure. APCs from injured rats were co-cultured with sham rat MLN CD4+ T cells and proliferative responses (thymidine incorporation), phenotypic changes (Flow cytometry), IL-2 production (ELISA) and CTLA-4 mRNA (RT-PCR) were determined in naive rat CD4+ T cells. The data indicate that APCs from thermally injured and/or septic rats when co-cultured with CD4+ T cells suppressed CD4+ T cell effector functions. This lack of CD4+ T cell activation was accompanied with altered co-stimulatory molecules, i.e., CD28 and/or CTLA-4 (CD152). In conclusion, our studies indicated that defective APCs from thermally injured and/or septic rats modulate CD4+ T cell functions via changes in co-stimulatory molecules expressed on naive CD4+ T cells. This altered APC: CD4+ T cell interaction leads to suppressed CD4+ T cell activation of healthy animals.

Mass Spectrometry-Based Identification Of The Tumor Antigen UN1 as the Transmembrane CD43 Sialoglycoprotein*

PubMed Central

de Laurentiis, Annamaria; Gaspari, Marco; Palmieri, Camillo; Falcone, Cristina; Iaccino, Enrico; Fiume, Giuseppe; Massa, Ornella; Masullo, Mariorosario; Tuccillo, Franca Maria; Roveda, Laura; Prati, Ubaldo; Fierro, Olga; Cozzolino, Immacolata; Troncone, Giancarlo; Tassone, Pierfrancesco; Scala, Giuseppe; Quinto, Ileana

2011-01-01

The UN1 monoclonal antibody recognized the UN1 antigen as a heavily sialylated and O-glycosylated protein with the apparent molecular weight of 100–120 kDa; this antigen was peculiarly expressed in fetal tissues and several cancer tissues, including leukemic T cells, breast, and colon carcinomas. However, the lack of primary structure information has limited further investigation on the role of the UN1 antigen in neoplastic transformation. In this study, we have identified the UN1 antigen as CD43, a transmembrane sialoglycoprotein involved in cell adhesion, differentiation, and apoptosis. Indeed, mass spectrometry detected two tryptic peptides of the membrane-purified UN1 antigen that matched the amino acidic sequence of the CD43 intracellular domain. Immunological cross-reactivity, migration pattern in mono- and bi-dimensional electrophoresis, and CD43 gene-dependent expression proved the CD43 identity of the UN1 antigen. Moreover, the monosaccharide GalNAc-O-linked to the CD43 peptide core was identified as an essential component of the UN1 epitope by glycosidase digestion of specific glycan branches. UN1-type CD43 glycoforms were detected in colon, sigmoid colon, and breast carcinomas, whereas undetected in normal tissues from the same patients, confirming the cancer-association of the UN1 epitope. Our results highlight UN1 monoclonal antibody as a suitable tool for cancer immunophenotyping and analysis of CD43 glycosylation in tumorigenesis. PMID:21372249

High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4+ T cells in human melanoma.

PubMed

Linnemann, Carsten; van Buuren, Marit M; Bies, Laura; Verdegaal, Els M E; Schotte, Remko; Calis, Jorg J A; Behjati, Sam; Velds, Arno; Hilkmann, Henk; Atmioui, Dris El; Visser, Marten; Stratton, Michael R; Haanen, John B A G; Spits, Hergen; van der Burg, Sjoerd H; Schumacher, Ton N M

2015-01-01

Tumor-specific neo-antigens that arise as a consequence of mutations are thought to be important for the therapeutic efficacy of cancer immunotherapies. Accumulating evidence suggests that neo-antigens may be commonly recognized by intratumoral CD8+ T cells, but it is unclear whether neo-antigen-specific CD4+ T cells also frequently reside within human tumors. In view of the accepted role of tumor-specific CD4+ T-cell responses in tumor control, we addressed whether neo-antigen-specific CD4+ T-cell reactivity is a common property in human melanoma.

CD81 Controls Sustained T Cell Activation Signaling and Defines the Maturation Stages of Cognate Immunological Synapses

PubMed Central

Rocha-Perugini, V.; Zamai, M.; González-Granado, J. M.; Barreiro, O.; Tejera, E.; Yañez-Mó, M.; Caiolfa, V. R.

2013-01-01

In this study, we investigated the dynamics of the molecular interactions of tetraspanin CD81 in T lymphocytes, and we show that CD81 controls the organization of the immune synapse (IS) and T cell activation. Using quantitative microscopy, including fluorescence recovery after photobleaching (FRAP), phasor fluorescence lifetime imaging microscopy-Föster resonance energy transfer (phasorFLIM-FRET), and total internal reflection fluorescence microscopy (TIRFM), we demonstrate that CD81 interacts with ICAM-1 and CD3 during conjugation between T cells and antigen-presenting cells (APCs). CD81 and ICAM-1 exhibit distinct mobilities in central and peripheral areas of early and late T cell-APC contacts. Moreover, CD81–ICAM-1 and CD81-CD3 dynamic interactions increase over the time course of IS formation, as these molecules redistribute throughout the contact area. Therefore, CD81 associations unexpectedly define novel sequential steps of IS maturation. Our results indicate that CD81 controls the temporal progression of the IS and the permanence of CD3 in the membrane contact area, contributing to sustained T cell receptor (TCR)-CD3-mediated signaling. Accordingly, we find that CD81 is required for proper T cell activation, regulating CD3ζ, ZAP-70, LAT, and extracellular signal-regulated kinase (ERK) phosphorylation; CD69 surface expression; and interleukin-2 (IL-2) secretion. Our data demonstrate the important role of CD81 in the molecular organization and dynamics of the IS architecture that sets the signaling threshold in T cell activation. PMID:23858057

Accumulation of CD69+ tissue‑resident memory T cells in the nasal polyps of patients with chronic rhinosinusitis.

PubMed

Ickrath, Pascal; Kleinsasser, Norbert; Ding, Xin; Ginzkey, Christian; Beyersdorf, Niklas; Hagen, Rudolf; Kerkau, Thomas; Hackenberg, Stephan

2018-08-01

In patients with chronic rhinosinusitis with nasal polyps (CRSwNP), a relative accumulation of cluster of differentiation (CD)8+ T cells over CD4+ T cells occurs in nasal polyps compared with the peripheral blood. Nasal CD8+ T cells and CD4+ T cells predominantly present an effector memory phenotype. Immunological studies have reported that memory T cells recirculate from the tissues to the peripheral blood and a high percentage of these T cells persist within the tissue. The aim of the present study was to characterize CD69+ sphingosine‑1‑phosphate receptor 1 (S1PR1)‑ tissue resident memory T cells (Trm) in the polyps of patients with CRSwNP. Tissue and blood samples were collected from 10 patients undergoing nasal sinus surgery. Expression of specific extra‑ and intracellular molecules were analyzed using multicolor flow cytometry. A significantly higher level of CD8+ T cells than CD4+ T cells was present in nasal polyps, while significantly more CD4+ T cells than CD8+ T cells were detected in the peripheral blood of patients with CRSwNP. The frequency of CD69+ T cells was significantly higher in CD8+ and CD4+ T cells in nasal polyps compared with the peripheral blood. The frequency of CD69+ S1PR1‑ Trm was also significantly higher in CD4+ and CD8+ T cells from nasal polyps compared with the peripheral blood. Within polyps, the frequency of CD69+ S1PR1‑ Trm was again significantly higher in CD8+ compared with CD4+ T cells. In summary, a significantly higher frequency of CD69+ S1PR1‑ T cells was observed in the nasal polyps compared with the peripheral blood in patients with CRSwNP. The results of the present study suggest that local regulation of the immune response occurs within nasal polyps. As such, Trm should be considered a potential stimulus in the pathogenesis of nasal polyps. However, the role of Trm in nasal polyps as a pathogenic trigger of the local inflammatory reaction requires further investigation.

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

Enhanced ADCC Activity of Affinity Maturated and Fc-Engineered Mini-Antibodies Directed against the AML Stem Cell Antigen CD96

PubMed Central

Kellner, Christian; Bräutigam, Joachim; Staudinger, Matthias; Schub, Natalie; Peipp, Matthias; Gramatzki, Martin; Humpe, Andreas

2012-01-01

CD96, a cell surface antigen recently described to be preferentially expressed on acute myeloid leukemia (AML) leukemic stem cells (LSC) may represent an interesting target structure for the development of antibody-based therapeutic approaches. The v-regions from the CD96-specific hybridoma TH-111 were isolated and used to generate a CD96-specific single chain fragment of the variable regions (scFv). An affinity maturated variant resulting in 4-fold enhanced CD96-binding was generated by random mutagenesis and stringent selection using phage display. The affinity maturated scFv CD96-S32F was used to generate bivalent mini-antibodies by genetically fusing an IgG1 wild type Fc region or a variant with enhanced CD16a binding. Antibody dependent cell-mediated cytotoxicity (ADCC) experiments revealed that Fc engineering was essential to trigger significant effector cell-mediated lysis when the wild type scFv was used. The mini-antibody variant generated by fusing the affinity-maturated scFv with the optimized Fc variant demonstrated the highest ADCC activity (2.3-fold enhancement in efficacy). In conclusion, our data provide proof of concept that CD96 could serve as a target structure for effector cell-mediated lysis and demonstrate that both enhancing affinity for CD96 and for CD16a resulted in mini-antibodies with the highest cytolytic potential. PMID:22879978

IL-10 down-regulates T cell activation by antigen-presenting liver sinusoidal endothelial cells through decreased antigen uptake via the mannose receptor and lowered surface expression of accessory molecules.

PubMed

Knolle, P A; Uhrig, A; Hegenbarth, S; Löser, E; Schmitt, E; Gerken, G; Lohse, A W

1998-12-01

Our study demonstrates that antigen-presenting liver sinusoidal endothelial cells (LSEC) induce production of interferon-gamma (IFN-gamma) from cloned Th1 CD4+ T cells. We show that LSEC used the mannose receptor for antigen uptake, which further strengthened the role of LSEC as antigen-presenting cell (APC) population in the liver. The ability of LSEC to activate cloned CD4+ T cells antigen-specifically was down-regulated by exogenous prostaglandin E2 (PGE2) and by IL-10. We identify two separate mechanisms by which IL-10 down-regulated T cell activation through LSEC. IL-10 decreased the constitutive surface expression of MHC class II as well as of the accessory molecules CD80 and CD86 on LSEC. Furthermore, IL-10 diminished mannose receptor activity in LSEC. Decreased antigen uptake via the mannose receptor and decreased expression of accessory molecules may explain the down-regulation of T cell activation through IL-10. Importantly, the expression of low numbers of antigen on MHC II in the absence of accessory signals on LSEC may lead to induction of anergy in T cells. Because PGE2 and IL-10 are released from LSEC or Kupffer cells (KC) in response to those concentrations of endotoxin found physiologically in portal venous blood, it is possible that the continuous presence of these mediators and their negative effect on the local APC may explain the inability of the liver to induce T cell activation and to clear chronic infections. Our results support the notion that antigen presentation by LSEC in the hepatic microenvironment contributes to the observed inability to mount an effective cell-mediated immune response in the liver.

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

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.

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

Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma.

PubMed

Drent, Esther; Groen, Richard W J; Noort, Willy A; Themeli, Maria; Lammerts van Bueren, Jeroen J; Parren, Paul W H I; Kuball, Jürgen; Sebestyen, Zsolt; Yuan, Huipin; de Bruijn, Joost; van de Donk, Niels W C J; Martens, Anton C M; Lokhorst, Henk M; Mutis, Tuna

2016-05-01

Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38(+) fractions of CD34(+) hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38(+) malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies. Copyright© Ferrata Storti Foundation.

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

Activated T cell subsets in human type 1 diabetes: evidence for expansion of the DR+ CD30+ subpopulation in new-onset disease

PubMed Central

Baker, C; Chang, L; Elsegood, K A; Bishop, A J; Gannon, D H; Narendran, P; Leech, N J; Dayan, C M

2007-01-01

An important limitation in T cell studies of human autoimmune (type 1) diabetes is lack of direct access to cells infiltrating the pancreas. We hypothesized that cells recently released from the pancreas into the blood might express a characteristic combination of markers of activation. We therefore examined the recently activated circulating T cell population [CD3+, human leucocyte antigen D-related (HLA-DR+)] using cytokine production and 10 additional subset markers [CD69, CD25, CD122, CD30, CD44v6, CD57, CD71, CCR3 (CD193), CCR5 (CD195) or CXCR3 (CD183)], comparing newly diagnosed adult (ND) (age 18–40 years) patients (n = 19) to patients with diabetes for > 10 years [long-standing (LS), n = 19] and HLA-matched controls (C, n = 16). CD3+ DR+ cells were enriched by two-step immunomagnetic separation. No differences in basal or stimulated production of interleukin (IL)-4, IL-10, IL-13 or interferon (IFN)-γ by CD3+ DR+ enriched cells were observed between the different groups of subjects. However, among the CD3+ DR+ population, significant expansions appeared to be present in the very small CD30+, CD69+ and CD122+ subpopulations. A confirmatory study was then performed using new subjects (ND = 26, LS = 15), three-colour flow cytometry, unseparated cells and three additional subset markers (CD38, CD134, CD4/CD25). This confirmed the expansion of the CD3+ DR+ CD30+ subpopulation in ND subjects. We conclude that a relative expansion in the T cell subpopulation with the activated phenotype CD3+ DR+ CD30+ is seen in the peripheral blood of subjects with newly diagnosed type 1 diabetes. This subpopulation represents less than 0·7% of circulating T cells and may provide a rich source of disease-specific T cells that can be isolated from blood. PMID:17302896

Cyclosporine-resistant, Rab27a-independent Mobilization of Intracellular Preformed CD40L Mediates Antigen-specific T Cell Help In Vitro

PubMed Central

Koguchi, Yoshinobu; Gardell, Jennifer L.; Thauland, Timothy J.; Parker, David C.

2011-01-01

CD40L is critically important for the initiation and maintenance of adaptive immune responses. It is generally thought that CD40L expression in CD4+ T cells is regulated transcriptionally and made from new mRNA following antigen recognition. However, recent studies with two-photon microscopy revealed that the majority of cognate interactions between effector CD4+ T cells and APCs are too short for de novo synthesis of CD40L. Given that effector and memory CD4+ T cells store preformed CD40L (pCD40L) in lysosomal compartments and that pCD40L comes to the cell surface within minutes of antigenic stimulation, we and others have proposed that pCD40L might mediate T cell-dependent activation of cognate APCs during brief encounters in vivo. However, it has not been shown that this relatively small amount of pCD40L is sufficient to activate APCs, owing to the difficulty of separating the effects of pCD40L from those of de novo CD40L and other cytokines in vitro. Here we show that pCD40L surface mobilization is resistant to cyclosporine or FK506 treatment, while de novo CD40L and cytokine expression are completely inhibited. These drugs thus provide a tool to dissect the role of pCD40L in APC activation. We find that pCD40L mediates selective activation of cognate but not bystander APCs in vitro and that mobilization of pCD40L does not depend on Rab27a, which is required for mobilization of lytic granules. Therefore, effector CD4+ T cells deliver pCD40L specifically to APCs on the same time scale as the lethal hit of CTLs but with distinct molecular machinery. PMID:21677130

Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia.

PubMed

Haso, Waleed; Lee, Daniel W; Shah, Nirali N; Stetler-Stevenson, Maryalice; Yuan, Constance M; Pastan, Ira H; Dimitrov, Dimiter S; Morgan, Richard A; FitzGerald, David J; Barrett, David M; Wayne, Alan S; Mackall, Crystal L; Orentas, Rimas J

2013-02-14

Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3 constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL.

Differential Dynamics of CD4+ and CD8+ T-Lymphocyte Proliferation and Activation in Acute Simian Immunodeficiency Virus Infection

PubMed Central

Kaur, Amitinder; Hale, Corrina L.; Ramanujan, Saroja; Jain, Rakesh K.; Johnson, R. Paul

2000-01-01

Although lymphocyte turnover in chronic human immunodeficiency virus and simian immunodeficiency virus (SIV) infection has been extensively studied, there is little information on turnover in acute infection. We carried out a prospective kinetic analysis of lymphocyte proliferation in 13 rhesus macaques inoculated with pathogenic SIV. A short-lived dramatic increase in circulating Ki-67+ lymphocytes observed at 1 to 4 weeks was temporally related to the onset of SIV replication. A 5- to 10-fold increase in Ki-67+ CD8+ T lymphocytes and a 2- to 3-fold increase in Ki-67+ CD3− CD8+ natural killer cells accounted for >85% of proliferating lymphocytes at peak proliferation. In contrast, there was little change in the percentage of Ki-67+ CD4+ T lymphocytes during acute infection, although transient increases in Ki-67− and Ki-67+ CD4+ T lymphocytes expressing CD69, Fas, and HLA-DR were observed. A two- to fourfold decline in CD4+ T lymphocytes expressing CD25 and CD69 was seen later in SIV infection. The majority of Ki-67+ CD8+ T lymphocytes were phenotypically CD45RA− CD49dhi Fashi CD25− CD69− CD28− HLA-DR− and persisted at levels twofold above baseline 6 months after SIV infection. Increased CD8+ T-lymphocyte proliferation was associated with cell expansion, paralleled the onset of SIV-specific cytotoxic T-lymphocyte activity, and had an oligoclonal component. Thus, divergent patterns of proliferation and activation are exhibited by CD4+ and CD8+ T lymphocytes in early SIV infection and may determine how these cells are differentially affected in AIDS. PMID:10954541

Endogenous antigen processing drives the primary CD4+ T cell response to influenza

PubMed Central

Miller, Michael A.; Ganesan, Asha Purnima V.; Luckashenak, Nancy; Mendonca, Mark; Eisenlohr, Laurence C.

2015-01-01

By convention, CD4+ T lymphocytes recognize foreign and self peptides derived from internalized antigens in combination with MHC class II molecules. Alternative pathways of epitope production have been identified but their contributions to host defense have not been established. We show here in a mouse infection model that the CD4+ T cell response to influenza, critical for durable protection from the virus, is driven principally by unconventional processing of antigen synthesized within the infected antigen-presenting cell, not by classical processing of endocytosed virions or material from infected cells. Investigation of the cellular components involved, including the H2-M molecular chaperone, the proteasome, and gamma-interferon inducible lysosomal thiol reductase revealed considerable heterogeneity in the generation of individual epitopes, an arrangement that ensures peptide diversity and broad CD4+ T cell engagement. These results could fundamentally revise strategies for rational vaccine design and may lead to key insights into the induction of autoimmune and anti-tumor responses. PMID:26413780

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.

An evaluation of serum soluble CD30 levels and serum CD26 (DPPIV) enzyme activity as markers of type 2 and type 1 cytokines in HIV patients receiving highly active antiretroviral therapy

PubMed Central

Keane, N M; Price, P; Lee, S; Stone, S F; French, M A

2001-01-01

This study evaluates serum CD26 (dipeptidyl peptidase IV, DPPIV) enzyme activity and serum levels of soluble CD30 as markers of T1 and T2 cytokine environments in HIV patients who achieved immune reconstitution after highly active antiretroviral therapy (HAART). Patients who had experienced inflammatory disease associated with pre-existent opportunistic infections after HAART (immune restoration diseases, IRD) were considered separately. Serum sCD30 levels and CD26 (DPPIV) enzyme activity were compared with IFN-γ production by PBMC cultured with cytomegalovirus (CMV) antigen in controls and patient groups. High sCD30 levels were associated with low IFN-γ production after antigenic stimulation in control subjects and, to a lesser extent, in immune reconstituted HIV patients. There was no association between serum CD26 (DPPIV) enzyme activity and IFN-γ production or sCD30 levels. Serum sCD30 levels and CD26 (DPPIV) enzyme activity were significantly increased in immune reconstituted patients with high HIV viral loads. Patients who had experienced CMV retinitis as an IRD had significantly higher sCD30 levels than all other patient groups. Hence, high sCD30 levels may be a marker of a T2 cytokine environment in HIV patients with immune reconstitution and are associated with higher HIV viral loads and a history of CMV associated IRD. PMID:11678906

Progesterone impairs antigen-non-specific immune protection by CD8 T memory cells via interferon-γ gene hypermethylation.

PubMed

Yao, Yushi; Li, Hui; Ding, Jie; Xia, Yixin; Wang, Lei

2017-11-01

Pregnant women and animals have increased susceptibility to a variety of intracellular pathogens including Listeria monocytogenes (LM), which has been associated with significantly increased level of sex hormones such as progesterone. CD8 T memory(Tm) cell-mediated antigen-non-specific IFN-γ responses are critically required in the host defense against LM. However, whether and how increased progesterone during pregnancy modulates CD8 Tm cell-mediated antigen-non-specific IFN-γ production and immune protection against LM remain poorly understood. Here we show in pregnant women that increased serum progesterone levels are associated with DNA hypermethylation of IFN-γ gene promoter region and decreased IFN-γ production in CD8 Tm cells upon antigen-non-specific stimulation ex vivo. Moreover, IFN-γ gene hypermethylation and significantly reduced IFN-γ production post LM infection in antigen-non-specific CD8 Tm cells are also observed in pregnant mice or progesterone treated non-pregnant female mice, which is a reversible phenotype following demethylation treatment. Importantly, antigen-non-specific CD8 Tm cells from progesterone treated mice have impaired anti-LM protection when adoptive transferred in either pregnant wild type mice or IFN-γ-deficient mice, and demethylation treatment rescues the adoptive protection of such CD8 Tm cells. These data demonstrate that increased progesterone impairs immune protective functions of antigen-non-specific CD8 Tm cells via inducing IFN-γ gene hypermethylation. Our findings thus provide insights into a new mechanism through which increased female sex hormone regulate CD8 Tm cell functions during pregnancy.

Progesterone impairs antigen-non-specific immune protection by CD8 T memory cells via interferon-γ gene hypermethylation

PubMed Central

Yao, Yushi; Li, Hui; Ding, Jie; Xia, Yixin

2017-01-01

Pregnant women and animals have increased susceptibility to a variety of intracellular pathogens including Listeria monocytogenes (LM), which has been associated with significantly increased level of sex hormones such as progesterone. CD8 T memory(Tm) cell-mediated antigen-non-specific IFN-γ responses are critically required in the host defense against LM. However, whether and how increased progesterone during pregnancy modulates CD8 Tm cell-mediated antigen-non-specific IFN-γ production and immune protection against LM remain poorly understood. Here we show in pregnant women that increased serum progesterone levels are associated with DNA hypermethylation of IFN-γ gene promoter region and decreased IFN-γ production in CD8 Tm cells upon antigen-non-specific stimulation ex vivo. Moreover, IFN-γ gene hypermethylation and significantly reduced IFN-γ production post LM infection in antigen-non-specific CD8 Tm cells are also observed in pregnant mice or progesterone treated non-pregnant female mice, which is a reversible phenotype following demethylation treatment. Importantly, antigen-non-specific CD8 Tm cells from progesterone treated mice have impaired anti-LM protection when adoptive transferred in either pregnant wild type mice or IFN-γ-deficient mice, and demethylation treatment rescues the adoptive protection of such CD8 Tm cells. These data demonstrate that increased progesterone impairs immune protective functions of antigen-non-specific CD8 Tm cells via inducing IFN-γ gene hypermethylation. Our findings thus provide insights into a new mechanism through which increased female sex hormone regulate CD8 Tm cell functions during pregnancy. PMID:29155896

Evaluation of the effect of phosphodiesterase on equine platelet activation and the effect of antigen challenge on platelet phosphodiesterase activity in horses with recurrent airway obstruction.

PubMed

Dunkel, Bettina; Rickards, Karen J; Werling, Dirk; Page, Clive P; Cunningham, Fiona M

2010-05-01

To determine whether expression of equine platelet activation-dependent surface markers is influenced by phospodiesterase (PDE) isoenzyme activity and whether antigen challenge alters platelet PDE activity in horses with recurrent airway obstruction (RAO). 16 horses. 7 healthy horses were used for in vitro experiments, 6 horses with RAO were used for antigen challenge, and 6 healthy horses were used as control animals. Three of the healthy horses had also been used in the in vitro experiments. Effects of PDE inhibition and activation of adenylyl cyclase on CD41/61 and CD62P expression on platelets and platelet-neutrophil aggregate formation in vitro were investigated via flow cytometry. Platelet PDE activity and sensitivity to inhibition of PDE3 and PDE5 isoenzymes were examined in horses with RAO and control horses before and after antigen challenge. Inhibition of PDE or activation of adenylyl cyclase significantly inhibited stimulus-induced expression of CD41/61 and CD62P (by approx 94% and 40%, respectively) and percentage of CD62P positive cells (by approx 30%). Only the PDE3 inhibitor, trequinsin, caused a significant (53%) reduction in platelet-neutrophil aggregate formation. Platelet PDE activity decreased following antigen challenge in RAO-affected horses and control horses. In horses with RAO, a significant increase in sensitivity of platelet PDE to inhibition by the PDE5 inhibitor zaprinast was observed after 5 hours. Results provided further evidence that PDE3 is an important regulator of equine platelet activation and suggested that changes in regulation of platelet PDE5 may contribute to antigen-induced response in horses with RAO.

Comprehensive Analysis of the Activation and Proliferation Kinetics and Effector Functions of Human Lymphocytes, and Antigen Presentation Capacity of Antigen-Presenting Cells in Xenogeneic Graft-Versus-Host Disease.

PubMed

Kawasaki, Yasufumi; Sato, Kazuya; Hayakawa, Hiroko; Takayama, Norihito; Nakano, Hirofumi; Ito, Ryoji; Mashima, Kiyomi; Oh, Iekuni; Minakata, Daisuke; Yamasaki, Ryoko; Morita, Kaoru; Ashizawa, Masahiro; Yamamoto, Chihiro; Hatano, Kaoru; Fujiwara, Shin-Ichiro; Ohmine, Ken; Muroi, Kazuo; Kanda, Yoshinobu

2018-04-17

Xenogeneic graft-versus-host disease (GVHD) models in highly immunodeficient mice are currently being used worldwide to investigate human immune responses against foreign antigens in vivo. However, the individual roles of CD4 + and CD8 + T cells, and donor/host hematopoietic and nonhematopoietic antigen-presenting cells (APCs) in the induction and development of GVHD have not been fully investigated. In the present study, we comprehensively investigated the immune responses of human T cells and the antigen presentation capacity of donor/host hematopoietic and nonhematopoietic APCs in xenogeneic GVHD models using nonobese diabetic/Shi-scid-IL2rg null mice. CD4 + T cells and, to a lesser extent, CD8 + T cells individually mediated potentially lethal GVHD. In addition to inflammatory cytokine production, CD4 + T cells also supported the activation and proliferation of CD8 + T cells. Using bone marrow chimeras, we demonstrated that host hematopoietic, but not nonhematopoietic, APCs play a critical role in the development of CD4 + T cell-mediated GVHD. During early GVHD, we detected 2 distinct populations in memory CD4 + T cells. One population was highly activated and proliferated in major histocompatibility complex antigen (MHC) +/+ mice but not in MHC -/- mice, indicating alloreactive T cells. The other population showed a less activated and slowly proliferative status regardless of host MHC expression, and was associated with higher susceptibility to apoptosis, indicating nonalloreactive T cells in homeostasis-driven proliferation. These observations are clinically relevant to donor T cell response after allogeneic hematopoietic stem cell transplantation. Our findings provide a better understanding of the immunobiology of humanized mice and support the development of novel options for the prevention and treatment for GVHD. Copyright © 2018. Published by Elsevier Inc.

Strategy for eliciting antigen-specific CD8+ T cell-mediated immune response against a cryptic CTL epitope of merkel cell polyomavirus large T antigen

PubMed Central

2012-01-01

Background Merkel cell carcinoma (MCC) is a relatively new addition to the expanding category of oncovirus-induced cancers. Although still comparably rare, the number of cases has risen dramatically in recent years. Further complicating this trend is that MCC is an extremely aggressive neoplasm with poor patient prognosis and limited treatment options for advanced disease. The causative agent of MCC has been identified as the merkel cell polyomavirus (MCPyV). The MCPyV-encoded large T (LT) antigen is an oncoprotein that is theorized to be essential for virus-mediated tumorigenesis and is therefore, an excellent MCC antigen for the generation of antitumor immune responses. As a foreign antigen, the LT oncoprotein avoids the obstacle of immune tolerance, which normally impedes the development of antitumor immunity. Ergo, it is an excellent target for anti-MCC immunotherapy. Since tumor-specific CD8+ T cells lead to better prognosis for MCC and numerous other cancers, we have generated a DNA vaccine that is capable of eliciting LT-specific CD8+ T cells. The DNA vaccine (pcDNA3-CRT/LT) encodes the LT antigen linked to a damage-associated molecular pattern, calreticulin (CRT), as it has been demonstrated that the linkage of CRT to antigens promotes the induction of antigen-specific CD8+ T cells. Results The present study shows that DNA vaccine-induced generation of LT-specific CD8+ T cells is augmented by linking CRT to the LT antigen. This is relevant since the therapeutic effects of the pcDNA3-CRT/LT DNA vaccine is mediated by LT-specific CD8+ T cells. Mice vaccinated with the DNA vaccine produced demonstrably more LT-specific CD8+ T cells. The DNA vaccine was also able to confer LT-specific CD8+ T cell-mediated protective and therapeutic effects to prolong the survival of mice with LT-expressing tumors. In the interest of determining the LT epitope which most MCC-specific CD8+ T cells recognize, we identified the amino acid sequence of the immunodominant LT epitope

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

Phenotypic and Functional Properties of Human Steady State CD14+ and CD1a+ Antigen Presenting Cells and Epidermal Langerhans Cells.

PubMed

Fehres, Cynthia M; Bruijns, Sven C M; Sotthewes, Brigit N; Kalay, Hakan; Schaffer, Lana; Head, Steven R; de Gruijl, Tanja D; Garcia-Vallejo, Juan J; van Kooyk, Yvette

2015-01-01

Cutaneous antigen presenting cells (APCs) are critical for the induction and regulation of skin immune responses. The human skin contains phenotypically and functionally distinct APCs subsets that are present at two separated locations. While CD1ahigh LCs form a dense network in the epidermis, the CD14+ and CD1a+ APCs reside in the dermal compartment. A better understanding of the biology of human skin APC subsets is necessary for the improvement of vaccine strategies that use the skin as administration route. In particular, progress in the characterization of uptake and activatory receptors will certainly improve APC-targeting strategies in vaccination. Here we performed a detailed analysis of the expression and function of glycan-binding and pattern-recognition receptors in skin APC subsets. The results demonstrate that under steady state conditions human CD1a+ dermal dendritic cells (DCs) were phenotypically most mature as measured by the expression of CD83 and CD86, whereas the CD14+ cells showed a higher expression of the CLRs DC-SIGN, mannose receptor and DCIR and had potent antigen uptake capacity. Furthermore, steady state LCs showed superior antigen cross-presentation as compared to the dermal APC subsets. Our results also demonstrate that the TLR3 ligand polyribosinic-polyribocytidylic acid (pI:C) was the most potent stimulator of cytokine production by both LCs and dDCs. These studies warrant further exploration of human CD1a+ dDCs and LCs as target cells for cancer vaccination to induce anti-tumor immune responses.

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.

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

Molecular basis of mycobacterial lipid antigen presentation by CD1c and its recognition by αβ T cells

PubMed Central

Roy, Sobhan; Ly, Dalam; Li, Nan-Sheng; Altman, John D.; Piccirilli, Joseph A.; Moody, D. Branch; Adams, Erin J.

2014-01-01

CD1c is a member of the group 1 CD1 family of proteins that are specialized for lipid antigen presentation. Despite high cell surface expression of CD1c on key antigen-presenting cells and the discovery of its mycobacterial lipid antigen presentation capability, the molecular basis of CD1c recognition by T cells is unknown. Here we present a comprehensive functional and molecular analysis of αβ T-cell receptor (TCR) recognition of CD1c presenting mycobacterial phosphomycoketide antigens. Our structure of CD1c with the mycobacterial phosphomycoketide (PM) shows similarities to that of CD1c-mannosyl-β1-phosphomycoketide in that the A' pocket accommodates the mycoketide alkyl chain; however, the phosphate head-group of PM is shifted ∼6 Å in relation to that of mannosyl-β1-PM. We also demonstrate a bona fide interaction between six human TCRs and CD1c-mycoketide complexes, measuring high to moderate affinities. The crystal structure of the DN6 TCR and mutagenic studies reveal a requirement of five complementarity determining region (CDR) loops for CD1c recognition. Furthermore, mutagenesis of CD1c reveals residues in both the α1 and α2 helices involved in TCR recognition, yet not entirely overlapping among the examined TCRs. Unlike patterns for MHC I, no archetypical binding footprint is predicted to be shared by CD1c-reactive TCRs, even when recognizing the same or similar antigens. PMID:25298532

Anti-CD22–chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia

PubMed Central

Haso, Waleed; Lee, Daniel W.; Shah, Nirali N.; Stetler-Stevenson, Maryalice; Yuan, Constance M.; Pastan, Ira H.; Dimitrov, Dimiter S.; Morgan, Richard A.; FitzGerald, David J.; Barrett, David M.; Wayne, Alan S.; Mackall, Crystal L.

2013-01-01

Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3ζ constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL. PMID:23243285

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

Cis-Acting Pathways Selectively Enforce the Non-Immunogenicity of Shed Placental Antigen for Maternal CD8 T Cells

PubMed Central

Tay, Chin-Siean; Tagliani, Elisa; Collins, Mary K.; Erlebacher, Adrian

2013-01-01

Maternal immune tolerance towards the fetus and placenta is thought to be established in part by pathways that attenuate T cell priming to antigens released from the placenta into maternal blood. These pathways remain largely undefined and their existence, at face value, seems incompatible with a mother's need to maintain a functional immune system during pregnancy. A particular conundrum is evident if we consider that maternal antigen presenting cells, activated in order to prime T cells to pathogen-derived antigens, would also have the capacity to prime T cells to co-ingested placental antigens. Here, we address this paradox using a transgenic system in which placental membranes are tagged with a strong surrogate antigen (ovalbumin). We find that although a remarkably large quantity of acellular ovalbumin-containing placental material is released into maternal blood, splenic CD8 T cells in pregnant mice bearing unmanipulated T cell repertoires are not primed to ovalbumin even if the mice are intravenously injected with adjuvants. This failure was largely independent of regulatory T cells, and instead was linked to the intrinsic characteristics of the released material that rendered it selectively non-immunogenic, potentially by sequestering it from CD8α+ dendritic cells. The release of ovalbumin-containing placental material into maternal blood thus had no discernable impact on CD8 T cell priming to soluble ovalbumin injected intravenously during pregnancy, nor did it induce long-term tolerance to ovalbumin. Together, these results outline a major pathway governing the maternal immune response to the placenta, and suggest how tolerance to placental antigens can be maintained systemically without being detrimental to host defense. PMID:24391885

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

Hsp70 enhances presentation of FMDV antigen to bovine CD4+ T cells in vitro

PubMed Central

McLaughlin, Kerry; Seago, Julian; Robinson, Lucy; Kelly, Charles; Charleston, Bryan

2010-01-01

Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious acute vesicular disease affecting cloven-hoofed animals, including cattle, sheep and pigs. The current vaccine induces a rapid humoral response, but the duration of the protective antibody response is variable, possibly associated with a variable specific CD4+ T cell response. We investigated the use of heat shock protein 70 (Hsp70) as a molecular chaperone to target viral antigen to the Major Histocompatibility Complex (MHC) class II pathway of antigen presenting cells and generate enhanced MHC II-restricted CD4+ T cell responses in cattle. Monocytes and CD4+ T cells from FMDV vaccinated cattle were stimulated in vitro with complexes of Hsp70 and FMDV peptide, or peptide alone. Hsp70 was found to consistently improve the presentation of a 25-mer FMDV peptide to CD4+ T cells, as measured by T cell proliferation. Complex formation was required for the enhanced effects and Hsp70 alone did not stimulate proliferation. This study provides further evidence that Hsp70:peptide complexes can enhance antigen-specific CD4+ T cell responses in vitro for an important pathogen of livestock. PMID:20167197

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

The level of PPD-specific IFN-gamma-producing CD4+ T cells in the blood predicts the in vivo response to PPD.

PubMed

Martins, Marcia Valéria B S; Lima, Mônica Cristina B S; Duppre, Nadia C; Matos, Haroldo J; Spencer, John S; Brennan, Patrick J; Sarno, Euzenir N; Fonseca, Leila; Pereira, Geraldo M B; Pessolani, Maria Cristina V

2007-05-01

There are no reliable means for detecting subclinical mycobacterial infections. The recent sequencing of several mycobacterial genomes has now afforded new opportunities for the development of pathogen-specific diagnostic tests, critical in the context of leprosy and tuberculosis control. In the present study, we applied a multi-parametric flow cytometric analysis that allowed the investigation of T-cell functions in order to define immunological markers that measure previous exposure to mycobacteria. We compared the in vivo response to PPD, the gold standard skin test reagent for measuring previous exposure to Mycobacterium tuberculosis, with in vitro parameters of leukocyte activation in five PPD positive and five PPD negative healthy volunteers. PPD-stimulated peripheral leukocytes expressing CD4, CD69, cutaneous lymphocyte-associated antigen (CLA) and intracellular IFN-gamma were enumerated in whole blood and compared with the size of in vivo PPD-induced induration and IFN-gamma production levels as measured by ELISA in supernatants of PPD-stimulated peripheral blood mononuclear cells. The reactivity to the tuberculin skin test (TST) was associated with markedly increased frequencies of PPD-responsive activated (CD69+) and IFN-gamma-producing CD4+T cells. Detection of PPD-specific IFN-gamma producing leukocytes was restricted to CD4+T cells and a subset of these cells was shown to express the skin homing molecule CLA. Multiple linear regression modeling of responses to PPD showed the highest association between skin test indurations and frequencies of PPD-responsive IFN-gamma-producing CD4+CD69+ T cells. Our data show that the in vitro enumeration of antigen-specific IFN-gamma-producing CD4+ T cells can provide an alternative to the in vivo tuberculin test for the detection of latent Mycobacterium tuberculosis infection. Moreover, the measurement of these immunological parameters can be useful for the screening of new specific antigens defined by the genome

Blocking of CD1d Decreases Trypanosoma cruzi-Induced Activation of CD4-CD8- T Cells and Modulates the Inflammatory Response in Patients With Chagas Heart Disease.

PubMed

Passos, Lívia Silva Araújo; Villani, Fernanda Nobre Amaral; Magalhães, Luísa Mourão Dias; Gollob, Kenneth J; Antonelli, Lis Ribeiro do Vale; Nunes, Maria Carmo Pereira; Dutra, Walderez Ornelas

2016-09-15

The control of inflammatory responses to prevent the deadly cardiac pathology in human Chagas disease is a desirable and currently unattained goal. Double-negative (DN) T cells are important sources of inflammatory and antiinflammatory cytokines in patients with Chagas heart disease and those with the indeterminate clinical form of Chagas disease, respectively. Given the importance of DN T cells in immunoregulatory processes and their potential as targets for controlling inflammation-induced pathology, we studied the involvement of CD1 molecules in the activation and functional profile of Trypanosoma cruzi-specific DN T cells. We observed that parasite stimulation significantly increased the expression of CD1a, CD1b, CD1c, and CD1d by CD14(+) cells from patients with Chagas disease. Importantly, among the analyzed molecules, only CD1d expression showed an association with the activation of DN T cells, as well as with worse ventricular function in patients with Chagas disease. Blocking of CD1d-mediated antigen presentation led to a clear reduction of DN T-cell activation and a decrease in the expression of interferon γ (IFN-γ) by DN T cells. Thus, our results showed that antigen presentation via CD1d is associated with activation of DN T cells in Chagas disease and that CD1d blocking leads to downregulation of IFN-γ by DN T cells from patients with Chagas heart disease, which may be a potential target for preventing progression of inflammation-mediated dilated cardiomyopathy. © 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.

CD137+CD154- Expression As a Regulatory T Cell (Treg)-Specific Activation Signature for Identification and Sorting of Stable Human Tregs from In Vitro Expansion Cultures.

PubMed

Nowak, Anna; Lock, Dominik; Bacher, Petra; Hohnstein, Thordis; Vogt, Katrin; Gottfreund, Judith; Giehr, Pascal; Polansky, Julia K; Sawitzki, Birgit; Kaiser, Andrew; Walter, Jörn; Scheffold, Alexander

2018-01-01

Regulatory T cells (Tregs) are an attractive therapeutic tool for several different immune pathologies. Therapeutic Treg application often requires prolonged in vitro culture to generate sufficient Treg numbers or to optimize their functionality, e.g., via genetic engineering of their antigen receptors. However, purity of clinical Treg expansion cultures is highly variable, and currently, it is impossible to identify and separate stable Tregs from contaminating effector T cells, either ex vivo or after prior expansion. This represents a major obstacle for quality assurance of expanded Tregs and raises significant safety concerns. Here, we describe a Treg activation signature that allows identification and sorting of epigenetically imprinted Tregs even after prolonged in vitro culture. We show that short-term reactivation resulted in expression of CD137 but not CD154 on stable FoxP3+ Tregs that displayed a demethylated Treg-specific demethylated region, high suppressive potential, and lack of inflammatory cytokine expression. We also applied this Treg activation signature for rapid testing of chimeric antigen receptor functionality in human Tregs and identified major differences in the signaling requirements regarding CD137 versus CD28 costimulation. Taken together, CD137+CD154- expression emerges as a universal Treg activation signature ex vivo and upon in vitro expansion allowing the identification and isolation of epigenetically stable antigen-activated Tregs and providing a means for their rapid functional testing in vitro .

Human Macrophages Escape Inhibition of Major Histocompatibility Complex-Dependent Antigen Presentation by Cytomegalovirus and Drive Proliferation and Activation of Memory CD4+ and CD8+ T Cells.

PubMed

Frascaroli, Giada; Lecher, Carina; Varani, Stefania; Setz, Corinna; van der Merwe, Johannes; Brune, Wolfram; Mertens, Thomas

2018-01-01

Human cytomegalovirus (HCMV) persistently infects 40-90% of the human population but in the face of a normal immune system, viral spread and dissemination are efficiently controlled thus preventing clinically signs and disease. HCMV-infected hosts produce a remarkably large amount of HCMV-specific CD4 + and CD8 + T cells that can even reach 20-50% of total T memory cells in the elderly. How HCMV may elicit such large and long-lasting T-cell responses in the absence of detectable viremia has not been elucidated yet. Additionally, HCMV is known to encode several gene products that potently inhibit T-cell recognition of infected cells. The best characterized are the four immune evasive US2, US3, US6, and US11 genes that by different mechanisms account for major histocompatibility complex (MHC) class I and class II degradation and intracellular retention in infected cells. By infecting M1 and M2 human macrophages (Mφ) with the wild-type HCMV strain TB40E or a mutant virus deleted of the four immune evasive genes US2, US3, US6, and US11, we demonstrated that human Mφ counteract the inhibitory potential of the US2-11 genes and remain capable to present peptides via MHC class I and class II molecules. Moreover, by sorting the infected and bystander cells, we provide evidence that both infected and bystander Mφ contribute to antigen presentation to CD4 + and CD8 + T cells. The T cells responding to TB40E-infected Mφ show markers of the T effector memory compartment, produce interferon-γ, and express the lytic granule marker CD107a on the cell surface, thus mirroring the HCMV-specific T cells present in healthy seropositive individuals. All together, our findings reveal that human Mφ escape inhibition of MHC-dependent antigen presentation by HCMV and continue to support T cell proliferation and activation after HCMV infection. Taking into account that Mφ are natural targets of HCMV infection and a site of viral reactivation from latency, our findings support the

In Vitro Pre-Clinical Validation of Suicide Gene Modified Anti-CD33 Redirected Chimeric Antigen Receptor T-Cells for Acute Myeloid Leukemia

PubMed Central

Minagawa, Kentaro; Jamil, Muhammad O.; AL-Obaidi, Mustafa; Pereboeva, Larisa; Salzman, Donna; Erba, Harry P.; Lamb, Lawrence S.; Bhatia, Ravi; Mineishi, Shin

2016-01-01

Background Approximately fifty percent of patients with acute myeloid leukemia can be cured with current therapeutic strategies which include, standard dose chemotherapy for patients at standard risk of relapse as assessed by cytogenetic and molecular analysis, or high-dose chemotherapy with allogeneic hematopoietic stem cell transplant for high-risk patients. Despite allogeneic hematopoietic stem cell transplant about 25% of patients still succumb to disease relapse, therefore, novel strategies are needed to improve the outcome of patients with acute myeloid leukemia. Methods and findings We developed an immunotherapeutic strategy targeting the CD33 myeloid antigen, expressed in ~ 85–90% of patients with acute myeloid leukemia, using chimeric antigen receptor redirected T-cells. Considering that administration of CAR T-cells has been associated with cytokine release syndrome and other potential off-tumor effects in patients, safety measures were here investigated and reported. We genetically modified human activated T-cells from healthy donors or patients with acute myeloid leukemia with retroviral supernatant encoding the inducible Caspase9 suicide gene, a ΔCD19 selectable marker, and a humanized third generation chimeric antigen receptor recognizing human CD33. ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells had a 75±3.8% (average ± standard error of the mean) chimeric antigen receptor expression, were able to specifically lyse CD33+ targets in vitro, including freshly isolated leukemic blasts from patients, produce significant amount of tumor-necrosis-factor-alpha and interferon-gamma, express the CD107a degranulation marker, and proliferate upon antigen specific stimulation. Challenging ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells with programmed-death-ligand-1 enriched leukemia blasts resulted in significant killing like observed for the programmed-death-ligand-1 negative leukemic blasts fraction. Since the administration of 10 nanomolar of a

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

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.

CD1c presentation of synthetic glycolipid antigens with foreign alkyl branching motifs

PubMed Central

de Jong, Annemieke; Arce, Eva Casas; Cheng, Tan-Yun; van Summeren, Ruben P.; Feringa, Ben L.; Dudkin, Vadim; Crich, David; Matsunaga, Isamu; Minnaard, Adriaan J.; Moody, D. Branch

2009-01-01

Summary Human CD1c is a protein that activates αβ T cells by presenting self antigens, synthetic mannosyl phosphodolichols and mycobacterial mannosyl phosphopolyketides. To determine which molecular structures of antigens mediate a T cell response, we measured activation by structurally divergent M. tuberculosis mannosyl-β1-phosphomycoketides as well as by synthetic analogs produced by two methods that yield either stereorandom or stereospecific methyl branching patterns. T cell responses required both a phosphate and a β-linked mannose unit, and showed preference for C30–34 lipid units with methyl branches in the S-configuration. Thus, in all cases T cell responses were strongest for synthetic compounds that mimicked the natural branched lipids produced by mycobacterial polyketide synthase 12. Incorporation of methylmalonate to form branched lipids is a common bacterial lipid synthesis pathway that is absent in vertebrates, so the preferential recognition of branched lipids may represent a new type of lipid-based pathogen associated molecular pattern (PAMP). PMID:18022562

Identification of DRG-1 As a Melanoma-Associated Antigen Recognized by CD4+ Th1 Cells

PubMed Central

Kiniwa, Yukiko; Li, Jiang; Wang, Mingjun; Sun, Chuang; Lee, Jeffrey E.; Wang, Rong-Fu; Wang, Helen Y.

2015-01-01

Immunotherapy has emerged as a promising strategy for the treatment of metastatic melanoma. Clinical studies have demonstrated the feasibility of cancer immunotherapy using tumor antigens recognized by CD8+ T cells. However, the overall immune responses induced by these antigens are too weak and transient to induce tumor regression in the majority of patients who received immunization. A growing body of evidence suggests that CD4+ T helper (Th) cells play an important role in antitumor immunity. Therefore, the identification of MHC class II-restricted tumor antigens capable of stimulating CD4+ T cells may provide opportunities for developing effective cancer vaccines. To this end, we describe the identification of developmentally regulated GTP-binding protein 1 (DRG-1) as a melanoma-associated antigen recognized by HLA-DR11-restricted CD4+ Th1 cells. Epitope mapping analysis showed that the DRG1248-268 epitope of DRG-1 was required for T cell recognition. Reverse transcription-polymerase chain reaction revealed that DRG-1 was highly expressed in melanoma cell lines but not in normal tissues. DRG-1 knockdown by lentiviral-based shRNA suppressed melanoma cell proliferation and soft agar colony formation. Taken together, these data suggest that DRG-1 plays an important role in melanoma cell growth and transformation, indicating that DRG1 may represent a novel target for CD4+ T cell-mediated immunotherapy in melanoma. PMID:25993655

Identification of DRG-1 As a Melanoma-Associated Antigen Recognized by CD4+ Th1 Cells.

PubMed

Kiniwa, Yukiko; Li, Jiang; Wang, Mingjun; Sun, Chuang; Lee, Jeffrey E; Wang, Rong-Fu; Wang, Helen Y

2015-01-01

Immunotherapy has emerged as a promising strategy for the treatment of metastatic melanoma. Clinical studies have demonstrated the feasibility of cancer immunotherapy using tumor antigens recognized by CD8(+) T cells. However, the overall immune responses induced by these antigens are too weak and transient to induce tumor regression in the majority of patients who received immunization. A growing body of evidence suggests that CD4(+) T helper (Th) cells play an important role in antitumor immunity. Therefore, the identification of MHC class II-restricted tumor antigens capable of stimulating CD4(+) T cells may provide opportunities for developing effective cancer vaccines. To this end, we describe the identification of developmentally regulated GTP-binding protein 1 (DRG-1) as a melanoma-associated antigen recognized by HLA-DR11-restricted CD4(+) Th1 cells. Epitope mapping analysis showed that the DRG1248-268 epitope of DRG-1 was required for T cell recognition. Reverse transcription-polymerase chain reaction revealed that DRG-1 was highly expressed in melanoma cell lines but not in normal tissues. DRG-1 knockdown by lentiviral-based shRNA suppressed melanoma cell proliferation and soft agar colony formation. Taken together, these data suggest that DRG-1 plays an important role in melanoma cell growth and transformation, indicating that DRG1 may represent a novel target for CD4(+) T cell-mediated immunotherapy in melanoma.

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

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.

CD137+CD154− Expression As a Regulatory T Cell (Treg)-Specific Activation Signature for Identification and Sorting of Stable Human Tregs from In Vitro Expansion Cultures

PubMed Central

Nowak, Anna; Lock, Dominik; Bacher, Petra; Hohnstein, Thordis; Vogt, Katrin; Gottfreund, Judith; Giehr, Pascal; Polansky, Julia K.; Sawitzki, Birgit; Kaiser, Andrew; Walter, Jörn; Scheffold, Alexander

2018-01-01

Regulatory T cells (Tregs) are an attractive therapeutic tool for several different immune pathologies. Therapeutic Treg application often requires prolonged in vitro culture to generate sufficient Treg numbers or to optimize their functionality, e.g., via genetic engineering of their antigen receptors. However, purity of clinical Treg expansion cultures is highly variable, and currently, it is impossible to identify and separate stable Tregs from contaminating effector T cells, either ex vivo or after prior expansion. This represents a major obstacle for quality assurance of expanded Tregs and raises significant safety concerns. Here, we describe a Treg activation signature that allows identification and sorting of epigenetically imprinted Tregs even after prolonged in vitro culture. We show that short-term reactivation resulted in expression of CD137 but not CD154 on stable FoxP3+ Tregs that displayed a demethylated Treg-specific demethylated region, high suppressive potential, and lack of inflammatory cytokine expression. We also applied this Treg activation signature for rapid testing of chimeric antigen receptor functionality in human Tregs and identified major differences in the signaling requirements regarding CD137 versus CD28 costimulation. Taken together, CD137+CD154− expression emerges as a universal Treg activation signature ex vivo and upon in vitro expansion allowing the identification and isolation of epigenetically stable antigen-activated Tregs and providing a means for their rapid functional testing in vitro. PMID:29467769

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.

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

HIV Envelope gp120 Alters T Cell Receptor Mobilization in the Immunological Synapse of Uninfected CD4 T Cells and Augments T Cell Activation

PubMed Central

Deng, Jing; Mitsuki, Yu-ya; Shen, Guomiao; Ray, Jocelyn C.; Cicala, Claudia; Arthos, James; Dustin, Michael L.

2016-01-01

ABSTRACT HIV is transmitted most efficiently from cell to cell, and productive infection occurs mainly in activated CD4 T cells. It is postulated that HIV exploits immunological synapses formed between CD4 T cells and antigen-presenting cells to facilitate the targeting and infection of activated CD4 T cells. This study sought to evaluate how the presence of the HIV envelope (Env) in the CD4 T cell immunological synapse affects synapse formation and intracellular signaling to impact the downstream T cell activation events. CD4 T cells were applied to supported lipid bilayers that were reconstituted with HIV Env gp120, anti-T cell receptor (anti-TCR) monoclonal antibody, and ICAM-1 to represent the surface of HIV Env-bearing antigen-presenting cells. The results showed that the HIV Env did not disrupt immunological synapse formation. Instead, the HIV Env accumulated with TCR at the center of the synapse, altered the kinetics of TCR recruitment to the synapse and affected synapse morphology over time. The HIV Env also prolonged Lck phosphorylation at the synapse and enhanced TCR-induced CD69 upregulation, interleukin-2 secretion, and proliferation to promote virus infection. These results suggest that HIV uses the immunological synapse as a conduit not only for selective virus transmission to activated CD4 T cells but also for boosting the T cell activation state, thereby increasing its likelihood of undergoing productive replication in targeted CD4 T cells. IMPORTANCE There are about two million new HIV infections every year. A better understanding of how HIV is transmitted to susceptible cells is critical to devise effective strategies to prevent HIV infection. Activated CD4 T cells are preferentially infected by HIV, although how this is accomplished is not fully understood. This study examined whether HIV co-opts the normal T cell activation process through the so-called immunological synapse. We found that the HIV envelope is recruited to the center of the

Enhancing Antitumor Efficacy of Chimeric Antigen Receptor T Cells Through Constitutive CD40L Expression

PubMed Central

Curran, Kevin J; Seinstra, Beatrijs A; Nikhamin, Yan; Yeh, Raymond; Usachenko, Yelena; van Leeuwen, Dayenne G; Purdon, Terence; Pegram, Hollie J; Brentjens, Renier J

2015-01-01

Adoptive cell therapy with genetically modified T cells expressing a chimeric antigen receptor (CAR) is a promising therapy for patients with B-cell acute lymphoblastic leukemia. However, CAR-modified T cells (CAR T cells) have mostly failed in patients with solid tumors or low-grade B-cell malignancies including chronic lymphocytic leukemia with bulky lymph node involvement. Herein, we enhance the antitumor efficacy of CAR T cells through the constitutive expression of CD40 ligand (CD40L, CD154). T cells genetically modified to constitutively express CD40L (CD40L-modified T cells) demonstrated increased proliferation and secretion of proinflammatory TH1 cytokines. Further, CD40L-modified T cells augmented the immunogenicity of CD40+ tumor cells by the upregulated surface expression of costimulatory molecules (CD80 and CD86), adhesion molecules (CD54, CD58, and CD70), human leukocyte antigen (HLA) molecules (Class I and HLA-DR), and the Fas-death receptor (CD95). Additionally, CD40L-modified T cells induced maturation and secretion of the proinflammatory cytokine interleukin-12 by monocyte-derived dendritic cells. Finally, tumor-targeted CD19-specific CAR/CD40L T cells exhibited increased cytotoxicity against CD40+ tumors and extended the survival of tumor-bearing mice in a xenotransplant model of CD19+ systemic lymphoma. This preclinical data supports the clinical application of CAR T cells additionally modified to constitutively express CD40L with anticipated enhanced antitumor efficacy. PMID:25582824

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.

Eliciting Epitope-Specific CD8+ T Cell Response by Immunization with Microbial Protein Antigens Formulated with α-Galactosylceramide: Theory, Practice, and Protocols.

PubMed

Gilchuk, Pavlo; Knight, Frances C; Wilson, John T; Joyce, Sebastian

2017-01-01

CD8+ cytotoxic T lymphocytes confer protection against infectious diseases caused by viruses, bacteria, and parasites. Hence, significant efforts have been invested into devising ways to generate CD8+ T cell-targeted vaccines. Generation of microbe-free protein subunit vaccines requires a thorough knowledge of protective target antigens. Such antigens are proteolytically processed peptides presented by MHC class I molecules. To induce a robust antigen-specific CD8+ T cell response through vaccination, it is essential to formulate the antigen with an effective adjuvant. Here, we describe a versatile method for generating high-frequency antigen-specific CD8+ T cells through immunization of mice using the invariant natural killer T cell agonist α-galactosylceramide as the adjuvant.

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

The antigenic complex in HIT binds to B cells via complement and complement receptor 2 (CD21)

PubMed Central

Khandelwal, Sanjay; Lee, Grace M.; Hester, C. Garren; Poncz, Mortimer; McKenzie, Steven E.; Sachais, Bruce S.; Rauova, Lubica; Kelsoe, Garnett; Cines, Douglas B.; Frank, Michael

2016-01-01

Heparin-induced thrombocytopenia is a prothrombotic disorder caused by antibodies to platelet factor 4 (PF4)/heparin complexes. The mechanism that incites such prevalent anti-PF4/heparin antibody production in more than 50% of patients exposed to heparin in some clinical settings is poorly understood. To investigate early events associated with antigen exposure, we first examined the interaction of PF4/heparin complexes with cells circulating in whole blood. In healthy donors, PF4/heparin complexes bind preferentially to B cells (>90% of B cells bind to PF4/heparin in vitro) relative to neutrophils, monocytes, or T cells. Binding of PF4 to B cells is heparin dependent, and PF4/heparin complexes are found on circulating B cells from some, but not all, patients receiving heparin. Given the high proportion of B cells that bind PF4/heparin, we investigated complement as a mechanism for noncognate antigen recognition. Complement is activated by PF4/heparin complexes, co-localizes with antigen on B cells from healthy donors, and is present on antigen-positive B cells in patients receiving heparin. Binding of PF4/heparin complexes to B cells is mediated through the interaction between complement and complement receptor 2 (CR2 [CD21]). To the best of our knowledge, these are the first studies to demonstrate complement activation by PF4/heparin complexes, opsonization of PF4/heparin to B cells via CD21, and the presence of complement activation fragments on circulating B cells in some patients receiving heparin. Given the critical contribution of complement to humoral immunity, our observations provide new mechanistic insights into the immunogenicity of PF4/heparin complexes. PMID:27412887

An extracatalytic function of CD45 in B cells is mediated by CD22

PubMed Central

Coughlin, Sarah; Noviski, Mark; Mueller, James L.; Chuwonpad, Ammarina; Raschke, William C.; Weiss, Arthur; Zikherman, Julie

2015-01-01

The receptor-like tyrosine phosphatase CD45 regulates antigen receptor signaling by dephosphorylating the C-terminal inhibitory tyrosine of the src family kinases. However, despite its abundance, the function of the large, alternatively spliced extracellular domain of CD45 has remained elusive. We used normally spliced CD45 transgenes either incorporating a phosphatase-inactivating point mutation or lacking the cytoplasmic domain to uncouple the enzymatic and noncatalytic functions of CD45 in lymphocytes. Although these transgenes did not alter T-cell signaling or development irrespective of endogenous CD45 expression, both partially rescued the phenotype of CD45-deficient B cells. We identify a noncatalytic role for CD45 in regulating tonic, but not antigen-mediated, B-cell antigen receptor (BCR) signaling through modulation of the function of the inhibitory coreceptor CD22. This finding has important implications for understanding how naïve B cells maintain tonic BCR signaling while restraining inappropriate antigen-dependent activation to preserve clonal “ignorance.” PMID:26561584

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.

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

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

Regulation of cytokine signaling by B cell antigen receptor and CD40-controlled expression of heparan sulfate proteoglycans.

PubMed

van der Voort, R; Keehnen, R M; Beuling, E A; Spaargaren, M; Pals, S T

2000-10-16

Recently, biochemical, cell biological, and genetic studies have converged to reveal that integral membrane heparan sulfate proteoglycans (HSPGs) are critical regulators of growth and differentiation of epithelial and connective tissues. As a large number of cytokines involved in lymphoid tissue homeostasis or inflammation contain potential HS-binding domains, HSPGs presumably also play important roles in the regulation of the immune response. In this report, we explored the expression, regulation, and function of HSPGs on B lymphocytes. We demonstrate that activation of the B cell antigen receptor (BCR) and/or CD40 induces a strong transient expression of HSPGs on human tonsillar B cells. By means of these HSPGs, the activated B cells can bind hepatocyte growth factor (HGF), a cytokine that regulates integrin-mediated B cell adhesion and migration. This interaction with HGF is highly selective since the HSPGs did not bind the chemokine stromal cell-derived factor (SDF)-1 alpha, even though the affinities of HGF and SDF-1alpha for heparin are similar. On the activated B cells, we observed induction of a specific HSPG isoform of CD44 (CD44-HS), but not of other HSPGs such as syndecans or glypican-1. Interestingly, the expression of CD44-HS on B cells strongly promotes HGF-induced signaling, resulting in an HS-dependent enhanced phosphorylation of Met, the receptor tyrosine kinase for HGF, as well as downstream signaling molecules including Grb2-associated binder 1 (Gab1) and Akt/protein kinase B (PKB). Our results demonstrate that the BCR and CD40 control the expression of HSPGs, specifically CD44-HS. These HSPGs act as functional coreceptors that selectively promote cytokine signaling in B cells, suggesting a dynamic role for HSPGs in antigen-specific B cell differentiation.

Clonotype and repertoire changes drive the functional improvement of HIV-specific CD8 T cell populations under conditions of limited antigenic stimulation

PubMed Central

Janbazian, Loury; Price, David A.; Canderan, Glenda; Filali-Mouhim, Abdelali; Asher, Tedi E.; Ambrozak, David R.; Scheinberg, Phillip; Boulassel, Mohamad Rachid; Routy, Jean-Pierre; Koup, Richard A.; Douek, Daniel C.; Sekaly, Rafick-Pierre; Trautmann, Lydie

2011-01-01

Persistent exposure to cognate antigen leads to the functional impairment and exhaustion of HIV-specific CD8 T cells. Antigen withdrawal, due either to antiretroviral treatment or the emergence of epitope escape mutations, causes HIV-specific CD8 T cell responses to wane over time. However, this process does not continue to extinction, and residual CD8 T cells likely play an important role in the control of HIV replication. Here, we conducted a longitudinal analysis of clonality, phenotype and function to define the characteristics of HIV-specific CD8 T cell populations that persist under conditions of limited antigenic stimulation. Antigen decay was associated with dynamic changes in the TCR repertoire, increased expression of CD45RA and CD127, decreased expression of PD-1 and the emergence of poly-functional HIV-specific CD8 T cells. High definition analysis of individual clonotypes revealed that the antigen loss-induced gain of function within HIV-specific CD8 T cell populations could be attributed to two non-exclusive mechanisms: (i) functional improvement of persisting clonotypes; and, (ii) recruitment of particular clonotypes endowed with superior functional capabilities. PMID:22210916

FOXP3, CBLB and ITCH gene expression and cytotoxic T lymphocyte antigen 4 expression on CD4+CD25high T cells in multiple sclerosis

PubMed Central

Sellebjerg, F; Krakauer, M; Khademi, M; Olsson, T; Sørensen, P S

2012-01-01

Expression of the forkhead box protein 3 (FoxP3) transcription factor is regulated by the E3 ubiquitin ligases Itch and Cbl-b and induces regulatory activity CD4+CD25high T cells. Treatment with interferon (IFN)-β enhances regulatory T cell activity in multiple sclerosis (MS). We studied the phenotype of CD4+CD25high T cells in MS by flow cytometry and its relationship with expression of the FOXP3, ITCH and CBLB genes. We found that untreated MS patients had lower cell surface expression of cytotoxic T lymphocyte antigen 4 (CTLA-4) on CD4+CD25high T cells and higher intracellular CTLA-4 expression than healthy controls. Cell surface expression of CTLA-4 on CD4+CD25high T cells correlated with expression of FOXP3 mRNA in untreated patients and increased significantly with time from most recent injection in patients treated with IFN-β. FOXP3 mRNA expression correlated with CBLB and ITCH and T helper type 2 cytokine mRNA expression in MS patients. These data link expression of FOXP3, CBLB and ITCH mRNA and CTLA-4 expression on the surface of CD4+CD25high T cell in MS. We hypothesize that this may reflect alterations in the inhibitory effect of CTLA-4 or in regulatory T cell function. PMID:23039885

Relation of follicular dendritic reticulum cells to Reed-Sternberg cells of Hodgkin's disease with emphasis on the expression of CD21 antigen.

PubMed Central

Delsol, G.; Meggetto, F.; Brousset, P.; Cohen-Knafo, E.; al Saati, T.; Rochaix, P.; Gorguet, B.; Rubin, B.; Voigt, J. J.; Chittal, S.

1993-01-01

Based on observations of 66 cases, in which tissues were specially processed to optimize the simultaneous preservation of cell membrane antigens and morphology, we provide evidence in favor of a relationship between follicular dendritic reticulum cells (FDRC) and Reed-Sternberg (RS) cells of Hodgkin's disease (HD) other than the lymphocyte predominance subtype. RS cells were intimately related to the FDRC network (75% of cases), and the expression of CD21 antigen was frequent (41% of cases). Exclusive expression of CD21 antigen was found in 11 cases of HD, while the expression of other B-cell-associated markers (CD19, CD20, CD22) was both variable and inconsistent. The expression of T-cell antigens (CD3, CD4, CD8) was rare. Null phenotype of RS cells was observed in 27 of 66 cases (41%). Epstein-Barr virus (EBV) nucleic acids were found in 34 of 66 (51.5%) cases. Double labeling techniques showed the presence of EBV-positive RS cells within the FDRC network. A non-B-cell origin of RS cells was supported by the differential expression of EBV latent antigens in HD (latent membrane protein+, EB nuclear antigen 2-), which is unusual in EBV-driven lymphoblastoid cell lines and EBV-positive B-cell lymphomas. FDRC and RS cells are known to share morphological traits (binucleated cells), and both cell types possess Fc receptor for IgG. The hypothesis is further backed by the findings of CD15 antigen expression by occasional RS-like dysplastic FDRC in Castleman's disease (five cases), which is characterized by hyperplasia of FDRC. Whether FDRC might be the only cells involved in the conversion to RS cells by the loss or gain of antigens remains to be determined. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7685151

Children with postsurgical capillary leak syndrome can be distinguished by antigen expression on neutrophils and monocytes

NASA Astrophysics Data System (ADS)

Tarnok, Attila; Pipek, Michal; Valet, Guenter; Richter, Jacqueline; Hambsch, Joerg; Schneider, Peter

1999-04-01

Our initial studies indicate that children who develop post- operative capillary leak syndrome (CLS) following cardiac surgery with cardiopulmonary bypass (CPB) can be distinguished based on their pre-operative level of circulating cytokines an adhesion molecules. We tested flow cytometric analysis of surface antigen expression as a potential assay for risk assessment of CLS. 24th preoperative blood samples were stained with monoclonal antibodies for the adhesion molecules ICAM-1, LFA1, MAC1, (beta) -integrin, activation markers CD25, CD54, CD69, HLA- DR, CD14 or CD4. Cells were measured on a dual-laser flow cytometer calibrated with microbeads. Antigen expression was detected as mean fluorescence intensity. The data indicate, that neutrophils of CLS patients express preoperatively higher levels of LFA1 and monocytes higher levels of HLA-DR and activation markers thus are in a state of activation. This could in combination with surgical trauma and CPB lead to their additional stimulation and migration into sites of inflammation and induce postoperative CLS. It is planned to set up a Flow-Classification program for individual risk assessment. By discriminate analysis over 80 percent of the patients were correctly classified. Our preliminary study indicates that flow cytometry with its low samples requirements and rapid access of the results could be a powerful tool to perform risk assessment prior to pediatric open heart surgery.

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.

Antitumor action of lymphokin-activated cells of patients with soft tissue sarcomas and melanomas in dependence on expression of MHC classes I and II antigenes.

PubMed

Berezhnaya, N M; Vinnichuk, U D; Belova, O B; Baranovich, V V

2006-09-01

To study expression of major histocompatibility complex (MHC) classes and antigens and CD25, CD71, Ki-67, CD54, CD56, CD11b, PCNA on lymphocytes and tumor cells and antitumor action of lymphocytes activated with IL-2. Tumor explants (soft tissue sarcoma, n = 20, melanoma, n = 25) were co-cultivated in diffusion chambers with autologous lymphocytes; antitumor action was evaluated by morphologic patterns of explant's growth. Expression of CD25, CD71, Ki-67, CD54, CD56, CD11b, PCNA was evaluated by the method of indirect fluorescence using respective monoclonal antibodies. The highest antitumor action of lymphocytes toward soft tissue sarcoma and melanoma cells is observed if tumor cells are expressing MHC class I antigens. In the cases of soft tissue sarcoma no correlation between the level of antitumor activity of lymphocytes and expression of CD25, CD71, Ki-67, CD54, CD56, CD11b, PCNA has been found, whilst in the case of melanoma it is associated with the high level of CD11b expression. There is a direct correlation between sensitivity of soft tissue sarcoma and melanoma cells to action of lymphokin-activated killer cells and the level of MHC class I antigens.

Multiplexed BioCD for prostate specific antigen detection

NASA Astrophysics Data System (ADS)

Wang, Xuefeng; Zhao, Ming; Nolte, David D.

2008-02-01

Specific protein concentrations in human body fluid can serve as diagnostic markers for some diseases, and a quantitative and high-throughput technique for multiplexed protein detection would speed up diagnosis and facilitate medical research. For this purpose, our group developed the BioCD, a spinning-disc interferometric biosensor on which antibody is immobilized. The detection system adopts a common-path scheme making it ultra stable. The scaling mass sensitivity is below 10 pg/mm for protein surface density. A 25000-spot antibody BioCD was fabricated to measure the concentration of prostate specific antigen (PSA), a protein indicating prostate cancer if its level is high. Statistical analysis of our immunoassay results projects that the detection limit of PSA would reach 20 pg/ml in a 2 mg/ml background solution. For future prospects, a multiplexed BioCD can be produced for simultaneous diagnosis of diverse diseases. For instance, 100 markers above 200 pg/ml could be measured on a single disc given that the detection limit is inversely proportional to square root of the number of spots.

Disruption of HLA class II antigen presentation in Burkitt lymphoma: implication of a 47 000 MW acid labile protein in CD4+ T-cell recognition

PubMed Central

God, Jason M; Zhao, Dan; Cameron, Christine A; Amria, Shereen; Bethard, Jennifer R; Haque, Azizul

2014-01-01

While Burkitt lymphoma (BL) has a well-known defect in HLA class I-mediated antigen presentation, the exact role of BL-associated HLA class II in generating a poor CD4+ T-cell response remains unresolved. Here, we found that BL cells are deficient in their ability to optimally stimulate CD4+ T cells via the HLA class II pathway. This defect in CD4+ T-cell recognition was not associated with low levels of co-stimulatory molecules on BL cells, as addition of external co-stimulation failed to elicit CD4+ T-cell activation by BL. Further, the defect was not caused by faulty antigen/class II interaction, because antigenic peptides bound with measurable affinity to BL-associated class II molecules. Interestingly, functional class II–peptide complexes were formed at acidic pH 5·5, which restored immune recognition. Acidic buffer (pH 5·5) eluate from BL cells contained molecules that impaired class II-mediated antigen presentation and CD4+ T-cell recognition. Biochemical analysis showed that these molecules were greater than 30 000 molecular weight in size, and proteinaceous in nature. In addition, BL was found to have decreased expression of a 47 000 molecular weight enolase-like molecule that enhances class II-mediated antigen presentation in B cells, macrophages and dendritic cells, but not in BL cells. These findings demonstrate that BL likely has multiple defects in HLA class II-mediated antigen presentation and immune recognition, which may be exploited for future immunotherapies. PMID:24628049

Reactivity of commercially available monoclonal antibodies to human CD antigens with peripheral blood leucocytes of dromedary camels (Camelus dromedarius)

PubMed Central

Hussen, Jamal; Shawaf, Turke; Al-herz, Abdulkareem Imran; Alturaifi, Hussain R.; Alluwaimi, Ahmed M.

2017-01-01

Monoclonal antibodies (mAbs) to cell surface molecules have been proven as a key tool for phenotypic and functional characterization of the cellular immune response. One of the major difficulties in studying camel cellular immunity consists in the lack of mAbs that dtect their leukocyte differentiation antigens. In the present study two-parameter flow cytometry was used to screen existing commercially available mAbs to human leukocyte antigens and major histocompatibility molecules (MHC) for their reactivity with camel leukocytes. The comparison of patterns of reactivity obtained after labelling human and camel leukocytes have shown that mAbs specific to human cluster of differentiation (CD) 18, CD11a, CD11b and CD14 are predicted to be cross-reactive with homologous camel antigens. PMID:28652982

CD3ζ-based chimeric antigen receptors mediate T cell activation via cis- and trans-signalling mechanisms: implications for optimization of receptor structure for adoptive cell therapy

PubMed Central

Bridgeman, J S; Ladell, K; Sheard, V E; Miners, K; Hawkins, R E; Price, D A; Gilham, D E

2014-01-01

Chimeric antigen receptors (CARs) can mediate redirected lysis of tumour cells in a major histocompatibility complex (MHC)-independent manner, thereby enabling autologous adoptive T cell therapy for a variety of malignant neoplasms. Currently, most CARs incorporate the T cell receptor (TCR) CD3ζ signalling chain; however, the precise mechanisms responsible for CAR-mediated T cell activation are unclear. In this study, we used a series of immunoreceptor tyrosine-based activation motif (ITAM)-mutant and transmembrane-modified receptors to demonstrate that CARs activate T cells both directly via the antigen-ligated signalling chain and indirectly via associated chains within the TCR complex. These observations allowed us to generate new receptors capable of eliciting polyfunctional responses in primary human T cells. This work increases our understanding of CAR function and identifies new avenues for the optimization of CAR-based therapeutic interventions. PMID:24116999

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

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

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.

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

Tumor-derived vaccines containing CD200 inhibit immune activation: implications for immunotherapy.

PubMed

Xiong, Zhengming; Ampudia-Mesias, Elisabet; Shaver, Rob; Horbinski, Craig M; Moertel, Christopher L; Olin, Michael R

2016-09-01

There are over 400 ongoing clinical trials using tumor-derived vaccines. This approach is especially attractive for many types of brain tumors, including glioblastoma, yet so far the clinical response is highly variable. One contributor to poor response is CD200, which acts as a checkpoint blockade, inducing immune tolerance. We demonstrate that, in response to vaccination, glioma-derived CD200 suppresses the anti-tumor immune response. In contrast, a CD200 peptide inhibitor that activates antigen-presenting cells overcomes immune tolerance. The addition of the CD200 inhibitor significantly increased leukocyte infiltration into the vaccine site, cytokine and chemokine production, and cytolytic activity. Our data therefore suggest that CD200 suppresses the immune system's response to vaccines, and that blocking CD200 could improve the efficacy of cancer immunotherapy.

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

Use of Engineered Exosomes Expressing HLA and Costimulatory Molecules to Generate Antigen-specific CD8+ T Cells for Adoptive Cell Therapy.

PubMed

Kim, Sueon; Sohn, Hyun-Jung; Lee, Hyun-Joo; Sohn, Dae-Hee; Hyun, Seung-Joo; Cho, Hyun-Il; Kim, Tai-Gyu

2017-04-01

Dendritic cell-derived exosomes (DEX) comprise an efficient stimulator of T cells. However, the production of sufficient DEX remains a barrier to their broad applicability in immunotherapeutic approaches. In previous studies, genetically engineered K562 have been used to generate artificial antigen presenting cells (AAPC). Here, we isolated exosomes from K562 cells (referred to as CoEX-A2s) engineered to express human leukocyte antigen (HLA)-A2 and costimulatory molecules such as CD80, CD83, and 41BBL. CoEX-A2s were capable of stimulating antigen-specific CD8 T cells both directly and indirectly via CoEX-A2 cross-dressed cells. Notably, CoEX-A2s also generated similar levels of HCMV pp65-specific and MART1-specific CD8 T cells as DEX in vitro. The results suggest that these novel exosomes may provide a crucial reagent for generating antigen-specific CD8 T cells for adoptive cell therapies against viral infection and tumors.

Antigen presenting capacity of murine splenic myeloid cells.

PubMed

Hey, Ying-Ying; Quah, Benjamin; O'Neill, Helen C

2017-01-11

The spleen is an important site for hematopoiesis. It supports development of myeloid cells from bone marrow-derived precursors entering from blood. Myeloid subsets in spleen are not well characterised although dendritic cell (DC) subsets are clearly defined in terms of phenotype, development and functional role. Recently a novel dendritic-like cell type in spleen named 'L-DC' was distinguished from other known dendritic and myeloid cells by its distinct phenotype and developmental origin. That study also redefined splenic eosinophils as well as resident and inflammatory monocytes in spleen. L-DC are shown to be distinct from known splenic macrophages and monocyte subsets. Using a new flow cytometric procedure, it has been possible to identify and isolate L-DC in order to assess their functional competence and ability to activate T cells both in vivo and in vitro. L-DC are readily accessible to antigen given intravenously through receptor-mediated endocytosis. They are also capable of CD8 + T cell activation through antigen cross presentation, with subsequent induction of cytotoxic effector T cells. L-DC are MHCII - cells and unable to activate CD4 + T cells, a property which clearly distinguishes them from conventional DC. The myeloid subsets of resident monocytes, inflammatory monocytes, neutrophils and eosinophils, were found to have varying capacities to take up antigen, but were uniformly unable to activate either CD4 + T cells or CD8 + T cells. The results presented here demonstrate that L-DC in spleen are distinct from other myeloid cells in that they can process antigen for CD8 + T cell activation and induction of cytotoxic effector function, while both L-DC and myeloid subsets remain unable to activate CD4 + T cells. The L-DC subset in spleen is therefore distinct as an antigen presenting cell.

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

CD45RO enriches for activated, highly mutated human germinal center B cells

PubMed Central

Jackson, Stephen M.; Harp, Natessa; Patel, Darshna; Zhang, Jeffrey; Willson, Savannah; Kim, Yoon J.; Clanton, Christian

2007-01-01

To date, there is no consensus regarding the influence of different CD45 isoforms during peripheral B-cell development. Examining correlations between surface CD45RO expression and various physiologic processes ongoing during the germinal center (GC) reaction, we hypothesized that GC B cells, like T cells, that up-regulate surface RO should progressively acquire phenotypes commonly associated with activated, differentiating lymphocytes. GC B cells (IgD−CD38+) were subdivided into 3 surface CD45RO fractions: RO−, RO+/−, and RO+. We show here that the average number of mutations per IgVH transcript increased in direct correlation with surface RO levels. Conjunctional use of RO and CD69 further delineated low/moderately and highly mutated fractions. Activation-induced cytidine deaminase (AID) mRNA was slightly reduced among RO+ GC B cells, suggesting that higher mutation averages are unlikely due to elevated somatic mutation activity. Instead, RO+ GC B cells were negative for Annexin V, comprised mostly (93%) of CD77− centrocytes, and were enriched for CD69+ cells. Collectively, RO+ GC B cells occupy what seems to be a specialized niche comprised mostly of centrocytes that may be in transition between activation states. These findings are among the first to sort GC B cells into populations enriched for live mutated cells solely using a single extracellular marker. PMID:17644737

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

Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells.

PubMed

Kochenderfer, James N; Yu, Zhiya; Frasheri, Dorina; Restifo, Nicholas P; Rosenberg, Steven A

2010-11-11

Adoptive T-cell therapy with anti-CD19 chimeric antigen receptor (CAR)-expressing T cells is a new approach for treating advanced B-cell malignancies. To evaluate anti-CD19-CAR-transduced T cells in a murine model of adoptive T-cell therapy, we developed a CAR that specifically recognized murine CD19. We used T cells that were retrovirally transduced with this CAR to treat mice bearing a syngeneic lymphoma that naturally expressed the self-antigen murine CD19. One infusion of anti-CD19-CAR-transduced T cells completely eliminated normal B cells from mice for at least 143 days. Anti-CD19-CAR-transduced T cells eradicated intraperitoneally injected lymphoma cells and large subcutaneous lymphoma masses. The antilymphoma efficacy of anti-CD19-CAR-transduced T cells was critically dependent on irradiation of mice before anti-CD19-CAR-transduced T-cell infusion. Anti-CD19-CAR-transduced T cells had superior antilymphoma efficacy compared with the anti-CD19 monoclonal antibody from which the anti-CD19 CAR was derived. Our results demonstrated impressive antilymphoma activity and profound destruction of normal B cells caused by anti-CD19-CAR-transduced T cells in a clinically relevant murine model.

Combining a CD20 chimeric antigen receptor and an inducible caspase 9 suicide switch to improve the efficacy and safety of T cell adoptive immunotherapy for lymphoma.

PubMed

Budde, Lihua E; Berger, Carolina; Lin, Yukang; Wang, Jinjuan; Lin, Xubin; Frayo, Shani E; Brouns, Shaunda A; Spencer, David M; Till, Brian G; Jensen, Michael C; Riddell, Stanley R; Press, Oliver W

2013-01-01

Modification of T cells with chimeric antigen receptors (CAR) has emerged as a promising treatment modality for human malignancies. Integration of co-stimulatory domains into CARs can augment the activation and function of genetically targeted T cells against tumors. However, the potential for insertional mutagenesis and toxicities due to the infused cells have made development of safe methods for removing transferred cells an important consideration. We have genetically modified human T cells with a lentiviral vector to express a CD20-CAR containing both CD28 and CD137 co-stimulatory domains, a "suicide gene" relying on inducible activation of caspase 9 (iC9), and a truncated CD19 selectable marker. Rapid expansion (2000 fold) of the transduced T cells was achieved in 28 days after stimulation with artificial antigen presenting cells. Transduced T cells exhibited effective CD20-specific cytotoxic activity in vitro and in a mouse xenograft tumor model. Activation of the iC9 suicide switch resulted in efficient removal of transduced T cells both in vitro and in vivo. Our work demonstrates the feasibility and promise of this approach for treating CD20(+) malignancies in a safe and more efficient manner. A phase I clinical trial using this approach in patients with relapsed indolent B-NHL is planned.

Intimate association of Thy-1 and the T-cell antigen receptor with the CD45 tyrosine phosphatase

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

Volarevic, S.; Burns, C.M.; Sussman, J.J.

1990-09-01

Immunoprecipitation of Thy-1 from Triton X-100 detergent lysates of surface-iodinated and chemically cross-linked T cells precipitated at least first major and discrete bands. Four of these bands were identified as Thy-1, CD45 (a trasmembrane tyrosine phosphatase), a major histocompatibility complex-encoded class I molecule, and {beta}{sub 2}-microglobulin. Similar analyses revealed that CD45 was coprecipitated from lysates of cross-linker-treated cells by antibodies to the T-cell antigen receptor (TCR). The same pattern of coprecipitated bands was observed when digitonin was used to lyse untreated cells. Immunoprecipitation of Thy-1 or the TCR from lysates of cross-linked T cells precipitated CD45 tyrosine phosphatase activity. Calculationsmore » based upon the amounts of coprecipitated enzymatic activity or TCR {zeta} chain indicate that a substantial fraction of Thy-1 and TCR complexes can be cross-linked to CD45. These data support a model in which the dependence of Thy-1 signaling on TCR coexpression is due to their common interaction with a tyrosine phosphatase and provide a possible structural basis for the influence of CD45 on TCR-mediated signaling.« less

Enumeration of antigen-specific CD8+ T lymphocytes by single-platform, HLA tetramer-based flow cytometry: a European multicenter evaluation.

PubMed

Heijnen, Ingmar A F M; Barnett, David; Arroz, Maria J; Barry, Simon M; Bonneville, Marc; Brando, Bruno; D'hautcourt, Jean-Luc; Kern, Florian; Tötterman, Thomas H; Marijt, Erik W A; Bossy, David; Preijers, Frank W M B; Rothe, Gregor; Gratama, Jan W

2004-11-01

HLA class I peptide tetramers represent powerful diagnostic tools for detection and monitoring of antigen-specific CD8(+) T cells. The impetus for the current multicenter study is the critical need to standardize tetramer flow cytometry if it is to be implemented as a routine diagnostic assay. Hence, the European Working Group on Clinical Cell Analysis set out to develop and evaluate a single-platform tetramer-based method that used cytomegalovirus (CMV) as the antigenic model. Absolute numbers of CMV-specific CD8(+) T cells were obtained by combining the percentage of tetramer-binding cells with the absolute CD8(+) T-cell count. Six send-outs of stabilized blood from healthy individuals or CMV-carrying donors with CMV-specific CD8(+) T-cell counts of 3 to 10 cells/microl were distributed to 7 to 16 clinical sites. These sites were requested to enumerate CD8(+) T cells and, in the case of CMV-positive donors, CMV-specific subsets on three separate occasions using the standard method. Between-site coefficients of variation of less than 10% (absolute CD8(+) T-cell counts) and approximately 30% (percentage and absolute numbers of CMV-specific CD8(+) T cells) were achieved. Within-site coefficients of variation were approximately 5% (absolute CD8(+) T-cell counts), approximately 9% (percentage CMV-specific CD8(+) T cells), and approximately 17% (absolute CMV-specific CD8(+) T-cell counts). The degree of variation tended to correlate inversely with the proportion of CMV-specific CD8(+) T-cell subsets. The single-platform MHC tetramer-based method for antigen-specific CD8(+) T-cell counting has been evaluated by a European group of laboratories and can be considered a reproducible assay for routine enumeration of antigen-specific CD8(+) T cells. (c) 2004 Wiley-Liss, Inc.

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

CD Nomenclature 2015: Human Leukocyte Differentiation Antigen Workshops as a Driving Force in Immunology.

PubMed

Engel, Pablo; Boumsell, Laurence; Balderas, Robert; Bensussan, Armand; Gattei, Valter; Horejsi, Vaclav; Jin, Bo-Quan; Malavasi, Fabio; Mortari, Frank; Schwartz-Albiez, Reinhard; Stockinger, Hannes; van Zelm, Menno C; Zola, Heddy; Clark, Georgina

2015-11-15

CD (cluster of differentiation) Ags are cell surface molecules expressed on leukocytes and other cells relevant for the immune system. CD nomenclature has been universally adopted by the scientific community and is officially approved by the International Union of Immunological Societies and sanctioned by the World Health Organization. It provides a unified designation system for mAbs, as well as for the cell surface molecules that they recognize. This nomenclature was established by the Human Leukocyte Differentiation Antigens Workshops. In addition to defining the CD nomenclature, these workshops have been instrumental in identifying and determining the expression and function of cell surface molecules. Over the past 30 y, the data generated by the 10 Human Leukocyte Differentiation Antigens Workshops have led to the characterization and formal designation of more than 400 molecules. CD molecules are commonly used as cell markers, allowing the identification and isolation of leukocyte populations, subsets, and differentiation stages. mAbs against these molecules have proven to be essential for biomedical research and diagnosis, as well as in biotechnology. More recently, they have been recognized as invaluable tools for the treatment of several malignancies and autoimmune diseases. In this article, we describe how the CD nomenclature was established, present the official updated list of CD molecules, and provide a rationale for their usefulness in the 21st century. Copyright © 2015 by The American Association of Immunologists, Inc.

Partial CD25 Antagonism Enables Dominance of Antigen-Inducible CD25high FOXP3+ Regulatory T Cells As a Basis for a Regulatory T Cell-Based Adoptive Immunotherapy

PubMed Central

Wilkinson, Daniel S.; Ghosh, Debjani; Nickle, Rebecca A.; Moorman, Cody D.; Mannie, Mark D.

2017-01-01

FOXP3+ regulatory T cells (Tregs) represent a promising platform for effective adoptive immunotherapy of chronic inflammatory disease, including autoimmune diseases such as multiple sclerosis. Successful Treg immunotherapy however requires new technologies to enable long-term expansion of stable, antigen-specific FOXP3+ Tregs in cell culture. Antigen-specific activation of naïve T cells in the presence of TGF-β elicits the initial differentiation of the FOXP3+ lineage, but these Treg lines lack phenotypic stability and rapidly transition to a conventional T cell (Tcon) phenotype during in vitro propagation. Because Tregs and Tcons differentially express CD25, we hypothesized that anti-CD25 monoclonal antibodies (mAbs) would only partially block IL-2 signaling in CD25high FOXP3+ Tregs while completely blocking IL-2 responses of CD25low-intermediate Tcons to enable preferential outgrowth of Tregs during in vitro propagation. Indeed, murine TGF-β-induced MOG-specific Treg lines from 2D2 transgenic mice that were maintained in IL-2 with the anti-CD25 PC61 mAb rapidly acquired and indefinitely maintained a FOXP3high phenotype during long-term in vitro propagation (>90% FOXP3+ Tregs), whereas parallel cultures lacking PC61 rapidly lost FOXP3. These results pertained to TGF-β-inducible “iTregs” because Tregs from 2D2-FIG Rag1−/− mice, which lack thymic or natural Tregs, were stabilized by continuous culture in IL-2 and PC61. MOG-specific and polyclonal Tregs upregulated the Treg-associated markers Neuropilin-1 (NRP1) and Helios (IKZF2). Just as PC61 stabilized FOXP3+ Tregs during expansion in IL-2, TGF-β fully stabilized FOXP3+ Tregs during cellular activation in the presence of dendritic cells and antigen/mitogen. Adoptive transfer of blastogenic CD25high FOXP3+ Tregs from MOG35-55-specific 2D2 TCR transgenic mice suppressed experimental autoimmune encephalomyelitis in pretreatment and therapeutic protocols. In conclusion, low IL-2 concentrations coupled

CD25 and CD69 induction by α4β1 outside-in signalling requires TCR early signalling complex proteins

PubMed Central

Cimo, Ann-Marie; Ahmed, Zamal; McIntyre, Bradley W.; Lewis, Dorothy E.; Ladbury, John E.

2013-01-01

Distinct signalling pathways producing diverse cellular outcomes can utilize similar subsets of proteins. For example, proteins from the TCR (T-cell receptor) ESC (early signalling complex) are also involved in interferon-α receptor signalling. Defining the mechanism for how these proteins function within a given pathway is important in understanding the integration and communication of signalling networks with one another. We investigated the contributions of the TCR ESC proteins Lck (lymphocyte-specific kinase), ZAP-70 (ζ-chain-associated protein of 70 kDa), Vav1, SLP-76 [SH2 (Src homology 2)-domain-containing leukocyte protein of 76 kDa] and LAT (linker for activation of T-cells) to integrin outside-in signalling in human T-cells. Lck, ZAP-70, SLP-76, Vav1 and LAT were activated by α4β1 outside-in signalling, but in a manner different from TCR signalling. TCR stimulation recruits ESC proteins to activate the mitogen-activated protein kinase ERK (extracellular-signal-regulated kinase). α4β1 outside-in-mediated ERK activation did not require TCR ESC proteins. However, α4β1 outside-in signalling induced CD25 and co-stimulated CD69 and this was dependent on TCR ESC proteins. TCR and α4β1 outside-in signalling are integrated through the common use of TCR ESC proteins; however, these proteins display functionally distinct roles in these pathways. These novel insights into the cross-talk between integrin outside-in and TCR signalling pathways are highly relevant to the development of therapeutic strategies to overcome disease associated with T-cell deregulation. PMID:23758320

Isolation of tumor antigen-specific single-chain variable fragments using a chimeric antigen receptor bicistronic retroviral vector in a Mammalian screening protocol.

PubMed

Lipowska-Bhalla, Grazyna; Gilham, David E; Hawkins, Robert E; Rothwell, Dominic G

2013-12-01

The clinical potential of chimeric antigen receptors in adoptive cellular therapy is beginning to be realized with several recent clinical trials targeting CD19 showing promising results in advanced B cell malignancies. This increased efficacy corresponds with improved engineering of the chimeric receptors with the latest-generation receptors eliciting greater signaling and proliferation potential. However, the antigen-binding single-chain variable fragment (scFv) domain of the receptors is critical in determining the activity of the chimeric receptor-expressing T cells, as this determines specificity and affinity to the tumor antigen. In this study, we describe a mammalian T cell line screening protocol employing a 2A-based bicistronic retroviral vector to isolate functional scFvs. This approach involves expression of the scFv library in a chimeric antigen receptor, and is based on selection of clones capable of stimulating CD69 upregulation in a T cell line and has a number of advantages over previously described methods in that the use of a 2A cassette ensures the exclusion of nonexpressing scFvs and the screening using a chimeric receptor in a mammalian T cell line ensures selection in the optimum context for therapeutic use. Proof-of-principle experiments show that the protocol was capable of a 10(5)-fold enrichment of positive clones after three rounds of selection. Furthermore, an antigen-specific clone was successfully isolated from a partially enriched scFv library, confirming the strength of the protocol. This approach has the potential to identify novel scFvs of use in adoptive T cell therapy and, potentially, wider antibody-based applications.

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

Engagement of Cytotoxic T Lymphocyte–associated Antigen 4 (CTLA-4) Induces Transforming Growth Factor β (TGF-β) Production by Murine CD4+ T Cells

PubMed Central

Chen, Wanjun; Jin, Wenwen; Wahl, Sharon M.

1998-01-01

Evidence indicates that cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) may negatively regulate T cell activation, but the basis for the inhibitory effect remains unknown. We report here that cross-linking of CTLA-4 induces transforming growth factor β (TGF-β) production by murine CD4+ T cells. CD4+ T helper type 1 (Th1), Th2, and Th0 clones all secrete TGF-β after antibody cross-linking of CTLA-4, indicating that induction of TGF-β by CTLA-4 signaling represents a ubiquitous feature of murine CD4+ T cells. Stimulation of the CD3–T cell antigen receptor complex does not independently induce TGF-β, but is required for optimal CTLA-4–mediated TGF-β production. The consequences of cross-linking of CTLA-4, together with CD3 and CD28, include inhibition of T cell proliferation and interleukin (IL)-2 secretion, as well as suppression of both interferon γ (Th1) and IL-4 (Th2). Moreover, addition of anti–TGF-β partially reverses this T cell suppression. When CTLA-4 was cross-linked in T cell populations from TGF-β1 gene–deleted (TGF-β1−/−) mice, the T cell responses were only suppressed 38% compared with 95% in wild-type mice. Our data demonstrate that engagement of CTLA-4 leads to CD4+ T cell production of TGF-β, which, in part, contributes to the downregulation of T cell activation. CTLA-4, through TGF-β, may serve as a counterbalance for CD28 costimulation of IL-2 and CD4+ T cell activation. PMID:9815262

Commensal oral bacteria antigens prime human dendritic cells to induce Th1, Th2 or Treg differentiation.

PubMed

Kopitar, A N; Ihan Hren, N; Ihan, A

2006-02-01

In various immunopathologic conditions, bacterial flora induce an immune response which results in inflammatory manifestations, e.g. periapical granuloma. Dendritic cells provide the main orchestration of specific immune responses. The aim of our study was to test the capacity of distinct oral bacterial antigens (prepared from Streptococcus mitis, Propionibacterium acnes, and Bacteroides spp.) to prime human dendritic cells for stimulation of the T-lymphocyte response. To assess the T-lymphocyte response, the expression of CD25, CD69, intracellular interferon gamma (cIFN-gamma), and intracellular interleukin 4 (cIL-4) was determined. Dendritic cells were prepared from leukocyte buffy coat from healthy blood donors. Monocytes were stimulated with IL-4 and GM-CSF and dendritic cells activated with bacterial lysates. Cell suspensions contained up to 90% dendritic cells, which represented 2-12% of the initial number of mononuclear cells. Lymphocyte subsets that developed in lymphocyte cultures after 1 week of stimulation were analyzed by flow cytometry. Dendritic cells, primed with antigens of Bacteroides fragilis have shown significantly higher activation and expression of intercellular IFN-gamma by T lymphocytes compared to negative controls. The dendritic cells primed with antigens of P. acnes had no effect on T-lymphocyte activation or cytokine production; instead they induced differentiation of T lymphocytes into CD25bright cells (regulatory T cells) with a potentially inhibitory effect on immune response. Dendritic cells primed with antigens of S. mitis induced increased expression of cIL-4. We conclude that commensal oral bacteria antigens prepared from B. fragilis, S. mitis, and P. acnes prime human dendritic cells to induce Th1, Th2, and T(reg) differentiation, respectively. This may advance our understanding of immunopathologic manifestations in the oral cavity and offer new possibilities for redirecting immune responses in mucosal vaccination.

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.

Production of anti-CD14 monoclonal antibody using synthetic peptide of human CD14 as immunizing antigen.

PubMed

Maleki, Leili Aghebati; Shanehbandi, Dariush; Majidi, Jafar; Yusefi, Mehdi; Abdolalizadeh, Jalal; Orangi, Mona; Baradaran, Behzad

2013-01-01

CD14 is a myeloid differentiation antigen expressed primarily on peripheral blood monocytes, dendritic cells and macrophages. It is a key regulator of inflammatory responses to gram-negative bacteria, oxidative burst and septic shock. The aim of this study was to produce and characterize monoclonal antibody against CD14 for use in detection and diagnosis of monocytes. To produce MAb against CD14 protein, mice were immunized with two KLH-conjugated CD14 peptides. The spleen cells of the immunized mice were then fused with SP2/0 by hybridoma technique. Fused cells were grown in selective medium and cloned by limiting dilution method. The desired clones were selected and supernatants of hybridoma cells were screened by ELISA for antibody. Monoclonal antibody was purified by chromatography and confirmed by SDS-PAGE. Finally, immunoblotting and flowcytometry were recruited to explore the specificity of the MAb. Our results showed successful production and characterization of anti CD14 monoclonal antibody. The MAb was IgG2a with Kappa light chain and immunobloting and flowcytometry results demonstrated specific reactivity of this MAb with CD14. The results show that, the produced anti- CD14 MAb is highly specific and functional in biomedical applications such as flow cytometry and western blotting and could be utilized for identification of monocytes.

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.

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

The T-cell antigen CD5 acts as a receptor and substrate for the protein-tyrosine kinase p56lck.

PubMed Central

Raab, M; Yamamoto, M; Rudd, C E

1994-01-01

CD5 is a T-cell-specific antigen which binds to the B-cell antigen CD72 and acts as a coreceptor in the stimulation of T-cell growth. CD5 associates with the T-cell receptor zeta chain (TcR zeta)/CD3 complex and is rapidly phosphosphorylated on tyrosine residues as a result of TcR zeta/CD3 ligation. However, despite this, the mechanism by which CD5 generates intracellular signals is unclear. In this study, we demonstrate that CD5 is coupled to the protein-tyrosine kinase p56lck and can act as a substrate for p56lck. Coexpression of CD5 with p56lck in the baculovirus expression system resulted in the phosphorylation of CD5 on tyrosine residues. Further, anti-CD5 and anti-p56lck coprecipitated each other in a variety of detergents, including Nonidet P-40 and Triton X-100. Anti-CD5 also precipitated the kinase from various T cells irrespective of the expression of TcR zeta/CD3 or CD4. No binding between p59fyn(T) and CD5 was detected in T cells. The binding of p56lck to CD5 induced a 10- to 15-fold increase in p56lck catalytic activity, as measured by in vitro kinase analysis. In vivo labelling with 32P(i) also showed a four- to fivefold increase in Y-394 occupancy in p56lck when associated with CD5. The use of glutathione S-transferase-Lck fusion proteins in precipitation analysis showed that the SH2 domain of p56lck could recognize CD5 as expressed in the baculovirus expression system. CD5 interaction with p56lck represents a novel variant of a receptor-kinase complex in which receptor can also serve as substrate. The CD5-p56lck interaction is likely to play roles in T-cell signalling and T-B collaboration. Images PMID:7513045

The leukocyte common antigen (CD45): a putative receptor-linked protein tyrosine phosphatase.

PubMed Central

Charbonneau, H; Tonks, N K; Walsh, K A; Fischer, E H

1988-01-01

A major protein tyrosine phosphatase (PTPase 1B) has been isolated in essentially homogeneous form from the soluble and particulate fractions of human placenta. Unexpectedly, partial amino acid sequences displayed no homology with the primary structures of the protein Ser/Thr phosphatases deduced from cDNA clones. However, the sequence is strikingly similar to the tandem C-terminal homologous domains of the leukocyte common antigen (CD45). A 157-residue segment of PTPase 1B displayed 40% and 33% sequence identity with corresponding regions from cytoplasmic domains I and II of human CD45. Similar degrees of identity have been observed among the catalytic domains of families of regulatory proteins such as protein kinases and cyclic nucleotide phosphodiesterases. On this basis, it is proposed that the CD45 family has protein tyrosine phosphatase activity and may represent a set of cell-surface receptors involved in signal transduction. This suggests that the repertoire of signal transduction mechanisms may include the direct control of an intracellular protein tyrosine phosphatase, offering the possibility of a regulatory balance with those protein tyrosine kinases that act at the internal surface of the membrane. Images PMID:2845400

Hyper-reactive cloned mice generated by direct nuclear transfer of antigen-specific CD4+ T cells.

PubMed

Kaminuma, Osamu; Katayama, Kazufumi; Inoue, Kimiko; Saeki, Mayumi; Nishimura, Tomoe; Kitamura, Noriko; Shimo, Yusuke; Tofukuji, Soichi; Ishida, Satoru; Ogonuki, Narumi; Kamimura, Satoshi; Oikawa, Mami; Katoh, Shigeki; Mori, Akio; Shichijo, Michitaka; Hiroi, Takachika; Ogura, Atsuo

2017-06-01

T-cell receptor (TCR)-transgenic mice have been employed for evaluating antigen-response mechanisms, but their non-endogenous TCR might induce immune response differently than the physiologically expressed TCR Nuclear transfer cloning produces animals that retain the donor genotype in all tissues including germline and immune systems. Taking advantage of this feature, we generated cloned mice that carry endogenously rearranged TCR genes from antigen-specific CD4 + T cells. We show that T cells of the cloned mice display distinct developmental pattern and antigen reactivity because of their endogenously pre-rearranged TCRα (rTα) and TCRβ (rTβ) alleles. These alleles were transmitted to the offspring, allowing us to establish a set of mouse lines that show chronic-type allergic phenotypes, that is, bronchial and nasal inflammation, upon local administrations of the corresponding antigens. Intriguingly, the existence of either rTα or rTβ is sufficient to induce in vivo hypersensitivity. These cloned mice expressing intrinsic promoter-regulated antigen-specific TCR are a unique animal model with allergic predisposition for investigating CD4 + T-cell-mediated pathogenesis and cellular commitment in immune diseases. © 2017 The Authors.

Combining a CD20 Chimeric Antigen Receptor and an Inducible Caspase 9 Suicide Switch to Improve the Efficacy and Safety of T Cell Adoptive Immunotherapy for Lymphoma

PubMed Central

Budde, Lihua E.; Berger, Carolina; Lin, Yukang; Wang, Jinjuan; Lin, Xubin; Frayo, Shani E.; Brouns, Shaunda A.; Spencer, David M.; Till, Brian G.; Jensen, Michael C.; Riddell, Stanley R.; Press, Oliver W.

2013-01-01

Modification of T cells with chimeric antigen receptors (CAR) has emerged as a promising treatment modality for human malignancies. Integration of co-stimulatory domains into CARs can augment the activation and function of genetically targeted T cells against tumors. However, the potential for insertional mutagenesis and toxicities due to the infused cells have made development of safe methods for removing transferred cells an important consideration. We have genetically modified human T cells with a lentiviral vector to express a CD20-CAR containing both CD28 and CD137 co-stimulatory domains, a “suicide gene” relying on inducible activation of caspase 9 (iC9), and a truncated CD19 selectable marker. Rapid expansion (2000 fold) of the transduced T cells was achieved in 28 days after stimulation with artificial antigen presenting cells. Transduced T cells exhibited effective CD20-specific cytotoxic activity in vitro and in a mouse xenograft tumor model. Activation of the iC9 suicide switch resulted in efficient removal of transduced T cells both in vitro and in vivo. Our work demonstrates the feasibility and promise of this approach for treating CD20+ malignancies in a safe and more efficient manner. A phase I clinical trial using this approach in patients with relapsed indolent B-NHL is planned. PMID:24358223

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

Co-stimulatory function in primary germinal center responses: CD40 and B7 are required on distinct antigen-presenting cells.

PubMed

Watanabe, Masashi; Fujihara, Chiharu; Radtke, Andrea J; Chiang, Y Jeffrey; Bhatia, Sumeena; Germain, Ronald N; Hodes, Richard J

2017-09-04

T cell-dependent germinal center (GC) responses require coordinated interactions of T cells with two antigen-presenting cell (APC) populations, B cells and dendritic cells (DCs), in the presence of B7- and CD40-dependent co-stimulatory pathways. Contrary to the prevailing paradigm, we found unique cellular requirements for B7 and CD40 expression in primary GC responses to vaccine immunization with protein antigen and adjuvant: B7 was required on DCs but was not required on B cells, whereas CD40 was required on B cells but not on DCs in the generation of antigen-specific follicular helper T cells, antigen-specific GC B cells, and high-affinity class-switched antibody production. There was, in fact, no requirement for coexpression of B7 and CD40 on the same cell in these responses. Our findings support a substantially revised model for co-stimulatory function in the primary GC response, with crucial and distinct contributions of B7- and CD40-dependent pathways expressed by different APC populations and with important implications for understanding how to optimize vaccine responses or limit autoimmunity. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

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

Increased frequency of human leukocyte antigen-E inhibitory receptor CD94/NKG2A-expressing peritoneal natural killer cells in patients with endometriosis.

PubMed

Galandrini, Ricciarda; Porpora, Maria Grazia; Stoppacciaro, Antonella; Micucci, Federica; Capuano, Cristina; Tassi, Ilaria; Di Felice, Alessia; Benedetti-Panici, Pierluigi; Santoni, Angela

2008-05-01

To analyze the frequency of peritoneal natural killer (NK) cells expressing the human leukocyte antigen (HLA)-E receptor CD94/NKG2A in patients with endometriosis. Case-control study. University hospital. Stage III and stage IV endometriosis, according to the revised American Society for Reproductive Medicine classification, was laparoscopically and histologically confirmed in 11 and 9 patients, respectively; 13 subjects without endometriosis were selected for the control group. Collection of peripheral venous blood, peritoneal fluid, endometriotic tissue, and normal endometrium in subjects undergoing laparoscopy. Surface expression levels of CD94/NKG2A and CD94/NKG2C were detected by three-color cytofluorometric analysis. Semiquantitative HLA-E messenger RNA expression analysis was performed in endometriotic lesions and in eutopic endometrium. NK cell-mediated cytotoxic activity toward HLA-E positive target, DT360 cell line, was also determined. In women with endometriosis, the percentage of CD94/NKG2A-positive peritoneal NK cells was significantly higher than in the control group. The CD94/NKG2A ligand, HLA-E, was detected at high levels in endometriotic tissue as messenger RNA transcript. Target cells bearing HLA-E were resistant to NK cell-mediated lysis in a CD94/NKG2A-dependent manner. Increased expression of CD94/NKG2A in peritoneal NK cells may mediate the resistance of endometriotic tissue to NK cell-mediated lysis, thus contributing to the progression of the disease.

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

Microfluidic squeezing for intracellular antigen loading in polyclonal B-cells as cellular vaccines

NASA Astrophysics Data System (ADS)

Lee Szeto, Gregory; van Egeren, Debra; Worku, Hermoon; Sharei, Armon; Alejandro, Brian; Park, Clara; Frew, Kirubel; Brefo, Mavis; Mao, Shirley; Heimann, Megan; Langer, Robert; Jensen, Klavs; Irvine, Darrell J.

2015-05-01

B-cells are promising candidate autologous antigen-presenting cells (APCs) to prime antigen-specific T-cells both in vitro and in vivo. However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to “professional” APCs such as dendritic cells. Here we utilize a microfluidic device that employs many parallel channels to pass single cells through narrow constrictions in high throughput. This microscale “cell squeezing” process creates transient pores in the plasma membrane, enabling intracellular delivery of whole proteins from the surrounding medium into B-cells via mechano-poration. We demonstrate that both resting and activated B-cells process and present antigens delivered via mechano-poration exclusively to antigen-specific CD8+T-cells, and not CD4+T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8+T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ. Finally, antigen-loaded B-cells were also able to prime antigen-specific CD8+T-cells in vivo when adoptively transferred into mice. Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8+T-cells, and decoupling of antigen uptake from B-cell activation.

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

The diabetes type 1 locus Idd6 modulates activity of CD4+CD25+ regulatory T-cells.

PubMed

Rogner, Ute Christine; Lepault, Françoise; Gagnerault, Marie-Claude; Vallois, David; Morin, Joëlle; Avner, Philip; Boitard, Christian

2006-01-01

The genetic locus Idd6 confers susceptibility to the spontaneous development of type 1 diabetes in the NOD mouse. Our studies on disease resistance of the congenic mouse strain NOD.C3H 6.VIII showed that Idd6 influences T-cell activities in the peripheral immune system and suggest that a major mechanism by which the Idd6 locus modifies diabetes development is via modulation of regulatory T-cell activities. Our transfer experiments using total splenocytes and purified T-cells demonstrated that the locus specifically controls the efficiency of disease protection mediated by the regulatory CD4(+)CD25(+) T-cell subset. Our data also implicate the Idd6 locus in controlling the balance between infiltrating lymphocytes and antigen-presenting cells within the pancreatic islet.

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

Activated CD4+ and CD8+ cells in the colonic mucosa of ulcerative colitis patients: their relationship to HLA-DR antigen expression on the colonic epithelium and serum soluble CD25 levels.

PubMed

Sasakawa, T; Takizawa, H; Bannai, H; Narisawa, R; Asakura, H

1995-01-01

This study was performed to clarify the relationship between activated (HLA-DR-expressing) CD4+ and CD8+ cells in the colonic lamina propria of ulcerative colitis and other immunological factors, i.e., epithelial DR expression, serum soluble CD25 levels, and colonic mucosal CD25+ cells. The frequency of epithelial DR expression was positively correlated with the numbers of CD4+ and CD8+ cells. The percentages activated CD4+/CD4+ cells were higher in mucosae with DR- epithelium than in mucosae with DR+ epithelium. The serum soluble CD25 levels were increased in ulcerative colitis, and there was an inverse correlation between these levels and the relative number of activated CD4+ cells in untreated active disease. These results suggest that interactions among mucosal CD4+ cells, colonic epithelium, and serum soluble CD25 might play an important role in the pathogenesis of ulcerative colitis.

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

Manufacture of Clinical-Grade CD19-Specific T Cells Stably Expressing Chimeric Antigen Receptor Using Sleeping Beauty System and Artificial Antigen Presenting Cells

PubMed Central

Singh, Harjeet; Figliola, Matthew J.; Dawson, Margaret J.; Olivares, Simon; Zhang, Ling; Yang, Ge; Maiti, Sourindra; Manuri, Pallavi; Senyukov, Vladimir; Jena, Bipulendu; Kebriaei, Partow; Champlin, Richard E.; Huls, Helen; Cooper, Laurence J. N.

2013-01-01

Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28) that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB) transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC) in the presence of interleukin (IL)-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT). We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼1010 T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion. PMID:23741305

Naïve and memory CD8 T cell pool homeostasis in advanced aging: impact of age and of antigen-specific responses to cytomegalovirus.

PubMed

Vescovini, Rosanna; Fagnoni, Francesco Fausto; Telera, Anna Rita; Bucci, Laura; Pedrazzoni, Mario; Magalini, Francesca; Stella, Adriano; Pasin, Federico; Medici, Maria Cristina; Calderaro, Adriana; Volpi, Riccardo; Monti, Daniela; Franceschi, Claudio; Nikolich-Žugich, Janko; Sansoni, Paolo

2014-04-01

Alterations in the circulating CD8+ T cell pool, with a loss of naïve and accumulation of effector/effector memory cells, are pronounced in older adults. However, homeostatic forces that dictate such changes remain incompletely understood. This observational cross-sectional study explored the basis for variability of CD8+ T cell number and composition of its main subsets: naïve, central memory and effector memory T cells, in 131 cytomegalovirus (CMV) seropositive subjects aged over 60 years. We found great heterogeneity of CD8+ T cell numbers, which was mainly due to variability of the CD8 + CD28- T cell subset regardless of age. Analysis, by multiple regression, of distinct factors revealed that age was a predictor for the loss in absolute number of naïve T cells, but was not associated with changes in central or effector memory CD8+ T cell subsets. By contrast, the size of CD8+ T cells specific to pp65 and IE-1 antigens of CMV, predicted CD28 - CD8+ T cell, antigen-experienced CD8+ T cell, and even total CD8+ T cell numbers, but not naïve CD8+ T cell loss. These results indicate a clear dichotomy between the homeostasis of naïve and antigen-experienced subsets of CD8+ T cells which are independently affected, in human later life, by age and antigen-specific responses to CMV, respectively.

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

CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy.

PubMed

Fry, Terry J; Shah, Nirali N; Orentas, Rimas J; Stetler-Stevenson, Maryalice; Yuan, Constance M; Ramakrishna, Sneha; Wolters, Pamela; Martin, Staci; Delbrook, Cindy; Yates, Bonnie; Shalabi, Haneen; Fountaine, Thomas J; Shern, Jack F; Majzner, Robbie G; Stroncek, David F; Sabatino, Marianna; Feng, Yang; Dimitrov, Dimiter S; Zhang, Ling; Nguyen, Sang; Qin, Haiying; Dropulic, Boro; Lee, Daniel W; Mackall, Crystal L

2018-01-01

Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent effects in relapsed and/or refractory pre-B cell acute lymphoblastic leukemia (B-ALL), but antigen loss is a frequent cause of resistance to CD19-targeted immunotherapy. CD22 is also expressed in most cases of B-ALL and is usually retained following CD19 loss. We report results from a phase 1 trial testing a new CD22-targeted CAR (CD22-CAR) in 21 children and adults, including 17 who were previously treated with CD19-directed immunotherapy. Dose-dependent antileukemic activity was observed, with complete remission obtained in 73% (11/15) of patients receiving ≥1 × 10 6 CD22-CAR T cells per kg body weight, including 5 of 5 patients with CD19 dim or CD19 - B-ALL. Median remission duration was 6 months. Relapses were associated with diminished CD22 site density that likely permitted CD22 + cell escape from killing by CD22-CAR T cells. These results are the first to establish the clinical activity of a CD22-CAR in B-ALL, including leukemia resistant to anti-CD19 immunotherapy, demonstrating potency against B-ALL comparable to that of CD19-CAR at biologically active doses. Our results also highlight the critical role played by antigen density in regulating CAR function.

MHC Class II and CD9 in human eosinophils localize to detergent-resistant membrane microdomains.

PubMed

Akuthota, Praveen; Melo, Rossana C N; Spencer, Lisa A; Weller, Peter F

2012-02-01

Eosinophils function in murine allergic airways inflammation as professional antigen-presenting cells (APCs). In murine professional APC cell types, optimal functioning of MHC Class II depends on its lateral association in plasma membranes and colocalization with the tetraspanin CD9 into detergent-resistant membrane microdomains (DRMs). With human eosinophils, we evaluated the localization of MHC Class II (HLA-DR) to DRMs and the functional significance of such localization. In granulocyte-macrophage colony-stimulating factor-stimulated human eosinophils, antibody cross-linked HLA-DR colocalized by immunofluorescence microscopy focally on plasma membranes with CD9 and the DRM marker ganglioside GM1. In addition, HLA-DR coimmunoprecipitates with CD9 after chemical cross-linking of CD9. HLA-DR and CD9 were localized by Western blotting in eosinophil DRM subcellular fractions. DRM disruption with the cholesterol-depleting agent methyl-β-cyclodextrin decreased eosinophil surface expression of HLA-DR and CD9. We show that CD9 is abundant on the surface of eosinophils, presenting the first electron microscopy data of the ultrastructural immunolocalization of CD9 in human eosinophils. Disruption of HLA-DR-containing DRMs decreased the ability of superantigen-loaded human eosinophils to stimulate CD4(+) T-cell activation (CD69 expression), proliferation, and cytokine production. Our results, which demonstrate that eosinophil MHC Class II localizes to DRMs in association with CD9 in a functionally significant manner, represent a novel insight into the organization of the antigen presentation complex of human eosinophils.

MHC Class II and CD9 in Human Eosinophils Localize to Detergent-Resistant Membrane Microdomains

PubMed Central

Akuthota, Praveen; Melo, Rossana C. N.; Spencer, Lisa A.

2012-01-01

Eosinophils function in murine allergic airways inflammation as professional antigen-presenting cells (APCs). In murine professional APC cell types, optimal functioning of MHC Class II depends on its lateral association in plasma membranes and colocalization with the tetraspanin CD9 into detergent-resistant membrane microdomains (DRMs). With human eosinophils, we evaluated the localization of MHC Class II (HLA-DR) to DRMs and the functional significance of such localization. In granulocyte-macrophage colony-stimulating factor–stimulated human eosinophils, antibody cross-linked HLA-DR colocalized by immunofluorescence microscopy focally on plasma membranes with CD9 and the DRM marker ganglioside GM1. In addition, HLA-DR coimmunoprecipitates with CD9 after chemical cross-linking of CD9. HLA-DR and CD9 were localized by Western blotting in eosinophil DRM subcellular fractions. DRM disruption with the cholesterol-depleting agent methyl-β-cyclodextrin decreased eosinophil surface expression of HLA-DR and CD9. We show that CD9 is abundant on the surface of eosinophils, presenting the first electron microscopy data of the ultrastructural immunolocalization of CD9 in human eosinophils. Disruption of HLA-DR–containing DRMs decreased the ability of superantigen-loaded human eosinophils to stimulate CD4+ T-cell activation (CD69 expression), proliferation, and cytokine production. Our results, which demonstrate that eosinophil MHC Class II localizes to DRMs in association with CD9 in a functionally significant manner, represent a novel insight into the organization of the antigen presentation complex of human eosinophils. PMID:21885678

Reduced CD5(+) CD24(hi) CD38(hi) and interleukin-10(+) regulatory B cells in active anti-neutrophil cytoplasmic autoantibody-associated vasculitis permit increased circulating autoantibodies.

PubMed

Aybar, L T; McGregor, J G; Hogan, S L; Hu, Y; Mendoza, C E; Brant, E J; Poulton, C J; Henderson, C D; Falk, R J; Bunch, D O

2015-05-01

Pathogenesis of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is B cell-dependent, although how particular B cell subsets modulate immunopathogenesis remains unknown. Although their phenotype remains controversial, regulatory B cells (Bregs ), play a role in immunological tolerance via interleukin (IL)-10. Putative CD19(+) CD24(hi) CD38(hi) and CD19(+) CD24(hi) CD27(+) Bregs were evaluated in addition to their CD5(+) subsets in 69 patients with ANCA-associated vasculitis (AAV). B cell IL-10 was verified by flow cytometry following culture with CD40 ligand and cytosine-phosphate-guanosine (CpG) DNA. Patients with active disease had decreased levels of CD5(+) CD24(hi) CD38(hi) B cells and IL-10(+) B cells compared to patients in remission and healthy controls (HCs). As IL-10(+) and CD5(+) CD24(hi) CD38(hi) B cells normalized in remission within an individual, ANCA titres decreased. The CD5(+) subset of CD24(hi) CD38(hi) B cells decreases in active disease and rebounds during remission similarly to IL-10-producing B cells. Moreover, CD5(+) B cells are enriched in the ability to produce IL-10 compared to CD5(neg) B cells. Together these results suggest that CD5 may identify functional IL-10-producing Bregs . The malfunction of Bregs during active disease due to reduced IL-10 expression may thus permit ANCA production. © 2014 British Society for Immunology.

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

T cells targeting a neuronal paraneoplastic antigen mediate tumor rejection and trigger CNS autoimmunity with humoral activation.

PubMed

Blachère, Nathalie E; Orange, Dana E; Santomasso, Bianca D; Doerner, Jessica; Foo, Patricia K; Herre, Margaret; Fak, John; Monette, Sébastien; Gantman, Emily C; Frank, Mayu O; Darnell, Robert B

2014-11-01

Paraneoplastic neurologic diseases (PND) involving immune responses directed toward intracellular antigens are poorly understood. Here, we examine immunity to the PND antigen Nova2, which is expressed exclusively in central nervous system (CNS) neurons. We hypothesized that ectopic expression of neuronal antigen in the periphery could incite PND. In our C57BL/6 mouse model, CNS antigen expression limits antigen-specific CD4+ and CD8+ T-cell expansion. Chimera experiments demonstrate that this tolerance is mediated by antigen expression in nonhematopoietic cells. CNS antigen expression does not limit tumor rejection by adoptively transferred transgenic T cells but does limit the generation of a memory population that can be expanded upon secondary challenge in vivo. Despite mediating cancer rejection, adoptively transferred transgenic T cells do not lead to paraneoplastic neuronal targeting. Preliminary experiments suggest an additional requirement for humoral activation to induce CNS autoimmunity. This work provides evidence that the requirements for cancer immunity and neuronal autoimmunity are uncoupled. Since humoral immunity was not required for tumor rejection, B-cell targeting therapy, such as rituximab, may be a rational treatment option for PND that does not hamper tumor immunity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Prolonged neutropenia due to antihuman neutrophil antigen 2 (CD177) antibody after bone marrow transplantation.

PubMed

Wada, Taizo; Miyamoto, Satoshi; Okamoto, Hiroyuki; Matsuda, Yusuke; Toma, Tomoko; Imai, Kohsuke; Takagi, Masatoshi; Morio, Tomohiro; Yachie, Akihiro

2017-07-01

We describe a patient who presented with prolonged neutropenia due to anti-human neutrophil antigen (HNA)-2 (CD177) antibody after allogeneic bone marrow transplantation. A granulocyte immunofluorescence test showed bimodal expression of antineutrophil antibody that resulted from specific binding of anti-HNA-2 to CD177 + neutrophils from healthy donors. The patient did not respond to granulocyte colony stimulating factor, which is able to upregulate CD177 expression on neutrophils. The low percentage of CD177 + cells in the few remaining neutrophils supports the possibility of elimination of CD177-upregulated neutrophils. These findings might explain an inferior response to neutrophil growth factors in neutropenia secondary to anti-HNA-2 antibody. © 2016 Wiley Periodicals, Inc.

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

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.

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

Increase of Alternatively Activated Antigen Presenting Cells in Active Experimental Autoimmune Encephalomyelitis.

PubMed

Wasser, Beatrice; Pramanik, Gautam; Hess, Moritz; Klein, Matthias; Luessi, Felix; Dornmair, Klaus; Bopp, Tobias; Zipp, Frauke; Witsch, Esther

2016-12-01

The importance of CD11c + antigen-presenting cells (APCs) in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) is well accepted and the gate keeper function of perivascular CD11c + APCs has been demonstrated. CD11c can be expressed by APCs from external sources or by central nervous system (CNS) resident APCs such as microglia. Yet, changes in the gene expression pattern of CNS CD11c + APCs during disease are still unclear and differentially expressed genes might play a decisive role in EAE progression. Due to their low numbers in the diseased brain and due to the absence of considerable numbers in the healthy CNS, analysis of CNS CD11c + cells is technically difficult. To ask whether the CD11c + APC population contributes to remission of EAE disease, we used Illumina deep mRNA sequencing (RNA-Seq) and quantitative real time polymerase chain reaction (qRT-PCR) analyses to identify the transcriptome of CD11c + APCs during disease course. We identified a battery of genes that were significantly regulated during the exacerbation of the disease compared to remission and relapse. Three of these genes, Arginase-1, Chi3l3 and Ms4a8a, showed a higher expression at the exacerbation than at later time points during the disease, both in SJL/J and in C57BL/6 mice, and could be attributed to alternatively activated APCs. Expression of Arginase-1, Chi3l3 and Ms4a8a genes was linked to the disease phase of EAE rather than to disease score. Expression of these genes suggested that APCs resembling alternatively activated macrophages are involved during the first wave of neuroinflammation and can be directly associated with the disease progress.

Feasibility and Safety of RNA-transfected CD20-specific Chimeric Antigen Receptor T Cells in Dogs with Spontaneous B Cell Lymphoma.

PubMed

Panjwani, M Kazim; Smith, Jenessa B; Schutsky, Keith; Gnanandarajah, Josephine; O'Connor, Colleen M; Powell, Daniel J; Mason, Nicola J

2016-09-01

Preclinical murine models of chimeric antigen receptor (CAR) T cell therapy are widely applied, but are greatly limited by their inability to model the complex human tumor microenvironment and adequately predict safety and efficacy in patients. We therefore sought to develop a system that would enable us to evaluate CAR T cell therapies in dogs with spontaneous cancers. We developed an expansion methodology that yields large numbers of canine T cells from normal or lymphoma-diseased dogs. mRNA electroporation was utilized to express a first-generation canine CD20-specific CAR in expanded T cells. The canine CD20 (cCD20) CAR expression was efficient and transient, and electroporated T cells exhibited antigen-specific interferon-gamma (IFN-γ) secretion and lysed cCD20+ targets. In a first-in-canine study, autologous cCD20-ζ CAR T cells were administered to a dog with relapsed B cell lymphoma. Treatment was well tolerated and led to a modest, but transient, antitumor activity, suggesting that stable CAR expression will be necessary for durable clinical remissions. Our study establishes the methodologies necessary to evaluate CAR T cell therapy in dogs with spontaneous malignancies and lays the foundation for use of outbred canine cancer patients to evaluate the safety and efficacy of next-generation CAR therapies and their optimization prior to translation into humans.

Feasibility and Safety of RNA-transfected CD20-specific Chimeric Antigen Receptor T Cells in Dogs with Spontaneous B Cell Lymphoma

PubMed Central

Panjwani, M Kazim; Smith, Jenessa B; Schutsky, Keith; Gnanandarajah, Josephine; O'Connor, Colleen M; Powell, Daniel J; Mason, Nicola J

2016-01-01

Preclinical murine models of chimeric antigen receptor (CAR) T cell therapy are widely applied, but are greatly limited by their inability to model the complex human tumor microenvironment and adequately predict safety and efficacy in patients. We therefore sought to develop a system that would enable us to evaluate CAR T cell therapies in dogs with spontaneous cancers. We developed an expansion methodology that yields large numbers of canine T cells from normal or lymphoma-diseased dogs. mRNA electroporation was utilized to express a first-generation canine CD20-specific CAR in expanded T cells. The canine CD20 (cCD20) CAR expression was efficient and transient, and electroporated T cells exhibited antigen-specific interferon-gamma (IFN-γ) secretion and lysed cCD20+ targets. In a first-in-canine study, autologous cCD20-ζ CAR T cells were administered to a dog with relapsed B cell lymphoma. Treatment was well tolerated and led to a modest, but transient, antitumor activity, suggesting that stable CAR expression will be necessary for durable clinical remissions. Our study establishes the methodologies necessary to evaluate CAR T cell therapy in dogs with spontaneous malignancies and lays the foundation for use of outbred canine cancer patients to evaluate the safety and efficacy of next-generation CAR therapies and their optimization prior to translation into humans. PMID:27401141

Crosslinking CD4 by human immunodeficiency virus gp120 primes T cells for activation-induced apoptosis

PubMed Central

1992-01-01

During human immunodeficiency virus (HIV) infection there is a profound and selective decrease in the CD4+ population of T lymphocytes. The mechanism of this depletion is not understood, as only a small fraction of all CD4+ cells appear to be productively infected with HIV-1 in seropositive individuals. In the present study, crosslinking of bound gp120 on human CD4+ T cells followed by signaling through the T cell receptor for antigen was found to result in activation-dependent cell death by a form of cell suicide termed apoptosis, or programmed cell death. The data indicate that even picomolar concentrations of gp120 prime T cells for activation-induced cell death, suggesting a mechanism for CD4+ T cell depletion in acquired immune deficiency syndrome (AIDS), particularly in the face of concurrent infection and antigenic challenge with other organisms. These results also provide an explanation for the enhancement of infection by certain antibodies against HIV, and for the paradox that HIV appears to cause AIDS after the onset of antiviral immunity. PMID:1402655

First characterization of fish CD22: An inhibitory role in the activation of peripheral blood leukocytes.

PubMed

Li, Yi-Qun; Zhang, Jian; Li, Jun; Sun, Li

2017-08-01

In mammals, CD22 is a member of the Ig superfamily that serves as an inhibitor during B cell responses to foreign antigens. In this study, we characterized for the first time a fish CD22 from tongue sole Cynoglossus semilaevis (CsCD22). CsCD22 possesses the conserved structural features of CD22 and shares 35%-54% sequence identities with other fish CD22. mRNA expression of CsCD22 was most abundant in head kidney and heart. CsCD22 protein was detected on the surface of peripheral blood leukocytes (PBL). In the presence of rCsCD22 antibody, the proliferation, phagocytosis, and antibacterial activity of PBL were significantly increased. These results indicate for the first time that fish CD22 plays an inhibitory role in PBL activation. Copyright © 2017 Elsevier B.V. All rights reserved.

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

Rapid and preferential distribution of blood-borne αCD3εAb to the liver is followed by local stimulation of T cells and natural killer T cells

PubMed Central

Wingender, Gerhard; Schumak, Beatrix; Schurich, Anna; Gessner, J Engelbert; Endl, Elmar; Limmer, Andreas; Knolle, Percy A

2006-01-01

Dissemination of soluble molecules or antigens via the blood stream is considered to lead to a uniform distribution in the various organs of the body, but organ-specific microarchitecture and vascularization may influence this. Following intravenous injection of αCD3ε antibody (αCD3εAb) we observed clear differences in antibody binding to Fcγ receptor (FcγR)+ antigen-presenting cells (APCs) or T lymphocytes in different organs. Significant binding of blood-borne αCD3εAb was only detected in the spleen and liver and not in the thymus or lymph node. In the spleen, only 10% of dendritic cells/macrophages and 40% of T-cell receptor (TCR)-β+ cells were positive for αCD3εAb, and, dependent on FcγR-mediated cross-linking of αCD3εAb, a similar percentage of splenic TCR-β+ cells were stimulated and became CD69+. Stimulation of TCR-β+ cells in the liver was at least as efficient as in the spleen, but almost all T cells and all scavenger liver sinusoidal endothelial cells bound αCD3εAb. In contrast to CD69 up-regulation, only CD4+ natural killer T (NKT) cells and CD11ahigh CD8+ T cells were activated by αCD3εAb and expressed interferon (IFN)-γ. Again, IFN-γ release from NKT/T cells was at least as efficient in the liver as in the spleen. Taken together, our results support the notion that the combination of extensive hepatic vascularization and very high scavenger activity allows the liver to fulfill its metabolic tasks and to promote stimulation of the large but widely distributed hepatic population of NKT/T cells. PMID:16423047

CD22 and autoimmune disease.

PubMed

Dörner, Thomas; Shock, Anthony; Smith, Kenneth G C

2012-10-01

CD22 is a 140-kDa member of the Siglec family of cell surface proteins that is expressed by most mature B-cell lineages. As a co-receptor of the B-cell receptor (BCR), it is known to contribute to the sensitive control of the B-cell response to antigen. Cross-linking of CD22 and the BCR by antigen triggers the phosphorylation of CD22, which leads to activation of signaling molecules such as phosphatases. Signal transduction pathways involving CD22 have been explored in a number of mouse models, some of which have provided evidence that in the absence of functional CD22, B cells have a "hyperactivated" phenotype, and suggest that loss of CD22 function could contribute to the pathogenesis of autoimmune diseases. Modulating CD22 activity has therefore been suggested as a possible therapeutic approach to such diseases. For example, the novel CD22-targeting monoclonal antibody epratuzumab is currently under investigation as a treatment for the connective tissue disorder systemic lupus erythematosus (SLE).

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.

Effective antigen presentation to helper T cells by human eosinophils.

PubMed

Farhan, Ruhaifah K; Vickers, Mark A; Ghaemmaghami, Amir M; Hall, Andrew M; Barker, Robert N; Walsh, Garry M

2016-12-01

Although eosinophils are inflammatory cells, there is increasing attention on their immunomodulatory roles. For example, murine eosinophils can present antigen to CD4 + T helper (Th) cells, but it remains unclear whether human eosinophils also have this ability. This study determined whether human eosinophils present a range of antigens, including allergens, to activate Th cells, and characterized their expression of MHC class II and co-stimulatory molecules required for effective presentation. Human peripheral blood eosinophils purified from non-allergic donors were pulsed with the antigens house dust mite extract (HDM), Timothy Grass extract (TG) or Mycobacterium tuberculosis purified protein derivative (PPD), before co-culture with autologous CD4 + Th cells. Proliferative and cytokine responses were measured, with eosinophil expression of HLA-DR/DP/DQ and the co-stimulatory molecules CD40, CD80 and CD86 determined by flow cytometry. Eosinophils pulsed with HDM, TG or PPD drove Th cell proliferation, with the response strength dependent on antigen concentration. The cytokine responses varied with donor and antigen, and were not biased towards any particular Th subset, often including combinations of pro- and anti-inflammatory cytokines. Eosinophils up-regulated surface expression of HLA-DR/DP/DQ, CD80, CD86 and CD40 in culture, increases that were sustained over 5 days when incubated with antigens, including HDM, or the major allergens it contains, Der p I or Der p II. Human eosinophils can, therefore, act as effective antigen-presenting cells to stimulate varied Th cell responses against a panel of antigens including HDM, TG or PPD, an ability that may help to determine the development of allergic disease. © 2016 John Wiley & Sons Ltd.

Intraperitoneal Administration of a Tumor-Associated Antigen SART3, CD40L, and GM-CSF Gene-Loaded Polyplex Micelle Elicits a Vaccine Effect in Mouse Tumor Models

PubMed Central

Furugaki, Kouichi; Cui, Lin; Kunisawa, Yumi; Osada, Kensuke; Shinkai, Kentaro; Tanaka, Masao; Kataoka, Kazunori; Nakano, Kenji

2014-01-01

Polyplex micelles have demonstrated biocompatibility and achieve efficient gene transfection in vivo. Here, we investigated a polyplex micelle encapsulating genes encoding the tumor-associated antigen squamous cell carcinoma antigen recognized by T cells-3 (SART3), adjuvant CD40L, and granulocyte macrophage colony-stimulating factor (GM-CSF) as a DNA vaccine platform in mouse tumor models with different types of major histocompatibility antigen complex (MHC). Intraperitoneally administrated polyplex micelles were predominantly found in the lymph nodes, spleen, and liver. Compared with mock controls, the triple gene vaccine significantly prolonged the survival of mice harboring peritoneal dissemination of CT26 colorectal cancer cells, of which long-term surviving mice showed complete rejection when re-challenged with CT26 tumors. Moreover, the DNA vaccine inhibited the growth and metastasis of subcutaneous CT26 and Lewis lung tumors in BALB/c and C57BL/6 mice, respectively, which represent different MHC haplotypes. The DNA vaccine highly stimulated both cytotoxic T lymphocyte and natural killer cell activities, and increased the infiltration of CD11c+ DCs and CD4+/CD8a+ T cells into tumors. Depletion of CD4+ or CD8a+ T cells by neutralizing antibodies deteriorated the anti-tumor efficacy of the DNA vaccine. In conclusion, a SART3/CD40L+GM-CSF gene-loaded polyplex micelle can be applied as a novel vaccine platform to elicit tumor rejection immunity regardless of the recipient MHC haplotype. PMID:25013909

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.

Shared target antigens on cancer cells and tissue stem cells: go or no-go for CAR T cells?

PubMed

Hombach, Andreas A; Abken, Hinrich

2017-02-01

Adoptive therapy with chimeric antigen receptor (CAR) T cells redirected towards CD19 produces remissions of B cell malignancies, however, it also eradicates healthy B cells sharing the target antigen. Such 'on-target off-tumor' toxicity raises serious safety concerns when the target antigen is also expressed by tissue stem cells, with the risk of lasting tissue destruction. Areas covered: We discuss CAR T cell targeting of activation antigens versus lineage associated antigens on the basis of recent experimental and animal data and the literature in the field. Expert commentary: Targeting an activation associated antigen which is transiently expressed by stem cells seems to be safe, like CAR T cells targeting CD30 spare CD30 + hematopoietic stem and progenitor cells while eliminating CD30 + lymphoma cells, whereas targeting lineage associated antigens which increase in expression during cell maturation, like folate receptor-β and CD123, is of risk to destruct tissue stem cells.

Nuclear location of an endogenously expressed antigen, EBNA1, restricts access to macroautophagy and the range of CD4 epitope display.

PubMed

Leung, Carol S; Haigh, Tracey A; Mackay, Laura K; Rickinson, Alan B; Taylor, Graham S

2010-02-02

Whereas exogenously acquired proteins are the major source of antigens feeding the MHC class II pathway in antigen-presenting cells, some endogenously expressed antigens also access that pathway but the rules governing such access are poorly understood. Here we address this using Epstein-Barr virus (EBV)-coded nuclear antigen EBNA1, a protein naturally expressed in EBV-infected B lymphoblastoid cell lines (LCLs) and a source of multiple CD4(+) T cell epitopes. Using CD4(+) T cell clones against three indicator epitopes, we find that two epitopes are weakly displayed on the LCL surface whereas the third is undetectable, a pattern of limited epitope presentation that is maintained even when nuclear expression of EBNA1 is induced to high supraphysiological levels. Inhibitor and siRNA studies show that, of the two epitopes weakly presented under these conditions, one involves macroautophagy, and the second involves antigen delivery to the MHC II pathway by another endogenous route. In contrast, when EBNA1 is expressed as a cytoplasmic protein, all three CD4 epitopes are processed and presented much more efficiently, and all involve macroautophagy. We conclude that EBNA1's nuclear location limits its accessibility to the macroautophagy pathway and, in consequence, limits the level and range of EBNA1 CD4 epitopes naturally displayed on the infected cell surface.

Expression patterns of lectin-like natural killer receptors, inhibitory CD94/NKG2A, and activating CD94/NKG2C on decidual CD56bright natural killer cells differ from those on peripheral CD56dim natural killer cells.

PubMed

Kusumi, Maki; Yamashita, Takahiro; Fujii, Tomoyuki; Nagamatsu, Takeshi; Kozuma, Shiro; Taketani, Yuji

2006-06-01

The balance of inhibitory and activating natural killer (NK) receptors on maternal decidual NK cells, most of which are CD56bright, is thought to be crucial for the proper growth of trophoblasts in placenta. A lectin-like NK receptor, CD94/NKG2, is the receptor for human leukocyte antigen (HLA)-E, which is expressed on trophoblasts. To clarify the mechanism regulating the activity of decidual NK cells during pregnancy, we investigated the expression patterns of inhibitory NK receptor, CD94/NKG2A, and activating receptor, CD94/NKG2C, on decidual NK cells in an early stage of normal pregnancy and compared them with those on peripheral NK cells, most of which are CD56dim. The rate of NKG2A-positive cells was significantly higher for decidual CD56bright NK cells than for peripheral CD56dim NK cells, but the rates of NKG2C-positive cells were comparable between the two cell types. Interestingly, peripheral CD56dim NK cells reciprocally expressed inhibitory NKG2A and activating NKG2C, but decidual CD56bright NK cells that expressed activating NKG2C simultaneously expressed inhibitory NKG2A. The co-expression of inhibitory and activating NKG2 receptors may fine-tune the immunoregulatory functions of the decidual NK cells to control the trophoblast invasion in constructing placenta.

Anti-CD22 and anti-CD79B antibody drug conjugates are active in different molecular diffuse large B-cell lymphoma subtypes.

PubMed

Pfeifer, M; Zheng, B; Erdmann, T; Koeppen, H; McCord, R; Grau, M; Staiger, A; Chai, A; Sandmann, T; Madle, H; Dörken, B; Chu, Y-W; Chen, A I; Lebovic, D; Salles, G A; Czuczman, M S; Palanca-Wessels, M C; Press, O W; Advani, R; Morschhauser, F; Cheson, B D; Lenz, P; Ott, G; Polson, A G; Mundt, K E; Lenz, G

2015-07-01

Antibody drug conjugates (ADCs), in which cytotoxic drugs are linked to antibodies targeting antigens on tumor cells, represent promising novel agents for the treatment of malignant lymphomas. Pinatuzumab vedotin is an anti-CD22 ADC and polatuzumab vedotin an anti-CD79B ADC that are both linked to the microtubule-disrupting agent monomethyl auristatin E (MMAE). In the present study, we analyzed the activity of these agents in different molecular subtypes of diffuse large B-cell lymphoma (DLBCL) both in vitro and in early clinical trials. Both anti-CD22-MMAE and anti-CD79B-MMAE were highly active and induced cell death in the vast majority of activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL cell lines. Similarly, both agents induced cytotoxicity in models with and without mutations in the signaling molecule CD79B. In line with these observations, relapsed and refractory DLBCL patients of both subtypes responded to these agents. Importantly, a strong correlation between CD22 and CD79B expression in vitro and in vivo was not detectable, indicating that patients should not be excluded from anti-CD22-MMAE or anti-CD79B-MMAE treatment because of low target expression. In summary, these studies suggest that pinatuzumab vedotin and polatuzumab vedotin are active agents for the treatment of patients with different subtypes of DLBCL.

Characterisation of an epigenetically altered CD4+ CD28+ Kir+ T cell subset in autoimmune rheumatic diseases by multiparameter flow cytometry

PubMed Central

Strickland, Faith M; Patel, Dipak; Somers, Emily; Robida, Aaron M; Pihalja, Michael; Swartz, Richard; Marder, Wendy; Richardson, Bruce

2016-01-01

Objectives Antigen-specific CD4+ T cells epigenetically modified with DNA methylation inhibitors overexpress genes normally suppressed by this mechanism, including CD11a, CD70, CD40L and the KIR gene family. The altered cells become autoreactive, losing restriction for nominal antigen and responding to self-class II major histocompatibility complex (MHC) molecules without added antigen, and are sufficient to cause a lupus-like disease in syngeneic mice. T cells overexpressing the same genes are found in patients with active lupus. Whether these genes are co-overexpressed on the same or different cells is unknown. The goal of this study was to determine whether these genes are overexpressed on the same or different T cells and whether this subset of CD4+ T cells is also present in patients with lupus and other rheumatic diseases. Methods Multicolour flow cytometry was used to compare CD11a, CD70, CD40L and KIR expression on CD3+CD4+CD28+ T cells to their expression on experimentally demethylated CD3+CD4+CD28+ T cells and CD3+CD4+CD28+ T cells from patients with active lupus and other autoimmune diseases. Results Experimentally demethylated CD4+ T cells and T cells from patients with active lupus have a CD3+CD4+CD28+CD11ahiCD70+CD40LhiKIR+ subset, and the subset size is proportional to lupus flare severity. A similar subset is found in patients with other rheumatic diseases including rheumatoid arthritis, systemic sclerosis and Sjögren's syndrome but not retroperitoneal fibrosis. Conclusions Patients with active autoimmune rheumatic diseases have a previously undescribed CD3+CD4+CD28+CD11ahiCD70+CD40LhiKIR+ T cell subset. This subset may play an important role in flares of lupus and related autoimmune rheumatic diseases, provide a biomarker for disease activity and serve as a novel therapeutic target for the treatment of lupus flares. PMID:27099767

Molecular recognition of microbial lipid-based antigens by T cells.

PubMed

Gras, Stephanie; Van Rhijn, Ildiko; Shahine, Adam; Le Nours, Jérôme

2018-05-01

The immune system has evolved to protect hosts from pathogens. T cells represent a critical component of the immune system by their engagement in host defence mechanisms against microbial infections. Our knowledge of the molecular recognition by T cells of pathogen-derived peptidic antigens that are presented by the major histocompatibility complex glycoproteins is now well established. However, lipids represent an additional, distinct chemical class of molecules that when presented by the family of CD1 antigen-presenting molecules can serve as antigens, and be recognized by specialized subsets of T cells leading to antigen-specific activation. Over the past decades, numerous CD1-presented self- and bacterial lipid-based antigens have been isolated and characterized. However, our understanding at the molecular level of T cell immunity to CD1 molecules presenting microbial lipid-based antigens is still largely unexplored. Here, we review the insights and the molecular basis underpinning the recognition of microbial lipid-based antigens by T cells.

RBP4 activates antigen-presenting cells leading to adipose tissue inflammation and systemic insulin resistance

PubMed Central

Moraes-Vieira, Pedro M.; Yore, Mark M.; Dwyer, Peter M.; Syed, Ismail; Aryal, Pratik; Kahn, Barbara B.

2014-01-01

Insulin resistance is a major cause of diabetes and is highly associated with adipose tissue (AT) inflammation in obesity. RBP4, a retinol-transporter, is elevated in insulin resistance and contributes to increased diabetes risk. We aimed to determine the mechanisms for RBP4-induced insulin resistance. Here we show that RBP4 elevation causes AT inflammation by activating innate immunity which elicits an adaptive immune-response. RBP4-overexpressing mice (RBP4-Ox) are insulin-resistant and glucose-intolerant and have increased AT macrophage and CD4 T-cell infiltration. In RBP4-Ox, AT CD206+ macrophages express pro-inflammatory markers and activate CD4 T-cells while maintaining alternatively-activated macrophage markers. These effects result from direct activation of AT antigen-presenting cells (APCs) by RBP4 through a JNK-dependent pathway. Transfer of RBP4-activated APCs into normal mice is sufficient to induce AT inflammation, insulin resistance and glucose intolerance. Thus, RBP4 causes insulin resistance, at least partly, by activating AT APCs which induce CD4 T-cell Th1 polarization and AT inflammation. PMID:24606904

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

Chimeric Antigen Receptors to CD276 for Treating Cancer | NCI Technology Transfer Center | TTC

Cancer.gov

This licensing opportunity from the National Cancer Institute concerns the development of CARs comprising an antigen-binding fragment derived from the MGA271 antibody. The resulting CARs can be used in adoptive cell therapy treatment for neuroblastoma and other tumors that express CD276.

Correlation between CD16a binding and immuno effector functionality of an antigen specific immunoglobulin Fc fragment (Fcab).

PubMed

Kainer, Manuela; Antes, Bernhard; Wiederkum, Susanne; Wozniak-Knopp, Gordana; Bauer, Anton; Rüker, Florian; Woisetschläger, Max

2012-10-15

Antigen binding immunoglobulin Fc fragments (Fcab) are generated by engineering loop regions in the CH3 domain of human IgG1 Fc. Variants of an Fcab specific for Her-2 were designed to display either enhanced (S239D:A330L:I332E) or diminished (L234A:L235A) binding affinities to the Fc receptor CD16a based on mutations described previously. The two mutant Fcab proteins demonstrated the expected modulation of CD16a binding. Interaction with recombinant or cell surface expressed Her-2 was unaffected in both mutants compared to the parental Fcab. Binding affinities for CD16a correlated with the ADCC-potencies of the Fcab variants. Additional studies indicated that the L234A:L235A variant Fcab had equivalent structural features as the unmodified Fcab since their DSC profiles were similar and antigen binding after re-folding upon partial heat denaturation had not changed. Introduction of the S239D:A330L:I332E mutations resulted in a significant reduction of the CH2 domain melting temperature, a moderate decrease of the thermal transition of the CH3 domain and lower antigen binding after thermal stress compared to the parental Fcab. We conclude that the known correlation between CD16a binding affinity and ADCC potency is also valid in Fcab proteins and that antigen specific Fcab molecules can be further engineered for fine tuning of immuno effector functions. Copyright © 2012 Elsevier Inc. All rights reserved.

Live Imaging of Influenza Infection of the Trachea Reveals Dynamic Regulation of CD8+ T Cell Motility by Antigen.

PubMed

Lambert Emo, Kris; Hyun, Young-Min; Reilly, Emma; Barilla, Christopher; Gerber, Scott; Fowell, Deborah; Kim, Minsoo; Topham, David J

2016-09-01

During a primary influenza infection, cytotoxic CD8+ T cells need to infiltrate the infected airways and engage virus-infected epithelial cells. The factors that regulate T cell motility in the infected airway tissue are not well known. To more precisely study T cell infiltration of the airways, we developed an experimental model system using the trachea as a site where live imaging can be performed. CD8+ T cell motility was dynamic with marked changes in motility on different days of the infection. In particular, significant changes in average cell velocity and confinement were evident on days 8-10 during which the T cells abruptly but transiently increase velocity on day 9. Experiments to distinguish whether infection itself or antigen affect motility revealed that it is antigen, not active infection per se that likely affects these changes as blockade of peptide/MHC resulted in increased velocity. These observations demonstrate that influenza tracheitis provides a robust experimental foundation to study molecular regulation of T cell motility during acute virus infection.

Live Imaging of Influenza Infection of the Trachea Reveals Dynamic Regulation of CD8+ T Cell Motility by Antigen

PubMed Central

Lambert Emo, Kris; Hyun, Young-min; Barilla, Christopher; Gerber, Scott; Fowell, Deborah; Kim, Minsoo

2016-01-01

During a primary influenza infection, cytotoxic CD8+ T cells need to infiltrate the infected airways and engage virus-infected epithelial cells. The factors that regulate T cell motility in the infected airway tissue are not well known. To more precisely study T cell infiltration of the airways, we developed an experimental model system using the trachea as a site where live imaging can be performed. CD8+ T cell motility was dynamic with marked changes in motility on different days of the infection. In particular, significant changes in average cell velocity and confinement were evident on days 8–10 during which the T cells abruptly but transiently increase velocity on day 9. Experiments to distinguish whether infection itself or antigen affect motility revealed that it is antigen, not active infection per se that likely affects these changes as blockade of peptide/MHC resulted in increased velocity. These observations demonstrate that influenza tracheitis provides a robust experimental foundation to study molecular regulation of T cell motility during acute virus infection. PMID:27644089

Transiently Reduced PI3K/Akt Activity Drives the Development of Regulatory Function in Antigen-Stimulated Naïve T-Cells

PubMed Central

Hasenberg, Mike; Reichardt, Peter; Gunzer, Matthias

2013-01-01

Regulatory T-cells (Tregs) are central for immune homeostasis and divided in thymus-derived natural Tregs and peripherally induced iTreg. However, while phenotype and function of iTregs are well known, a remarkable lack exists in knowledge about signaling mechanisms leading to their generation from naïve precursors in peripheral tissues. Using antigen specific naïve T-cells from mice, we investigated CD4+ CD25+ FoxP3- iTreg induction during antigen-specific T-cell receptor (TCR) stimulation with weak antigen presenting cells (APC). We show that early signaling pathways such as ADAM-17-activation appeared similar in developing iTreg and effector cells (Teff) and both initially shedded CD62-L. But iTreg started reexpressing CD62-L after 24 h while Teff permanently downmodulated it. Furthermore, between 24 and 72 hours iTreg presented with significantly lower phosphorylation levels of Akt-S473 suggesting lower activity of the PI3K/Akt-axis. This was associated with a higher expression of the Akt hydrophobic motif-specific phosphatase PHLPP1 in iTreg. Importantly, the lack of costimulatory signals via CD28 from weak APC was central for the development of regulatory function in iTreg but not for the reappearance of CD62-L. Thus, T-cells display a window of sensitivity after onset of TCR triggering within which the intensity of the PI3K/Akt signal controls entry into either effector or regulatory pathways. PMID:23874604

Attrition of memory CD8 T cells during sepsis requires LFA-1.

PubMed

Serbanescu, Mara A; Ramonell, Kimberly M; Hadley, Annette; Margoles, Lindsay M; Mittal, Rohit; Lyons, John D; Liang, Zhe; Coopersmith, Craig M; Ford, Mandy L; McConnell, Kevin W

2016-11-01

CD8 T cell loss and dysfunction have been implicated in the increased susceptibility to opportunistic infections during the later immunosuppressive phase of sepsis, but CD8 T cell activation and attrition in early sepsis remain incompletely understood. With the use of a CLP model, we assessed CD8 T cell activation at 5 consecutive time points and found that activation after sepsis results in a distinct phenotype (CD69 + CD25 int CD62L HI ) independent of cognate antigen recognition and TCR engagement and likely through bystander-mediated cytokine effects. Additionally, we observed that sepsis concurrently results in the preferential depletion of a subset of memory-phenotype CD8 T cells that remain "unactivated" (i.e., fail to up-regulate activation markers) by apoptosis. Unactivated CD44 HI OT-I cells were spared from sepsis-induced attrition, as were memory-phenotype CD8 T cells of mice treated with anti-LFA-1 mAb, 1 h after CLP. Perhaps most importantly, we demonstrate that attrition of memory phenotype cells may have a pathologic significance, as elevated IL-6 levels were associated with decreased numbers of memory-phenotype CD8 T cells in septic mice, and preservation of this subset after administration of anti-LFA-1 mAb conferred improved survival at 7 d. Taken together, these data identify potentially modifiable responses of memory-phenotype CD8 T cells in early sepsis and may be particularly important in the application of immunomodulatory therapies in sepsis. © Society for Leukocyte Biology.

Attrition of memory CD8 T cells during sepsis requires LFA-1

PubMed Central

Serbanescu, Mara A.; Ramonell, Kimberly M.; Hadley, Annette; Margoles, Lindsay M.; Mittal, Rohit; Lyons, John D.; Liang, Zhe; Coopersmith, Craig M.; Ford, Mandy L.; McConnell, Kevin W.

2016-01-01

CD8 T cell loss and dysfunction have been implicated in the increased susceptibility to opportunistic infections during the later immunosuppressive phase of sepsis, but CD8 T cell activation and attrition in early sepsis remain incompletely understood. With the use of a CLP model, we assessed CD8 T cell activation at 5 consecutive time points and found that activation after sepsis results in a distinct phenotype (CD69+CD25intCD62LHI) independent of cognate antigen recognition and TCR engagement and likely through bystander-mediated cytokine effects. Additionally, we observed that sepsis concurrently results in the preferential depletion of a subset of memory-phenotype CD8 T cells that remain “unactivated” (i.e., fail to up-regulate activation markers) by apoptosis. Unactivated CD44HI OT-I cells were spared from sepsis-induced attrition, as were memory-phenotype CD8 T cells of mice treated with anti-LFA-1 mAb, 1 h after CLP. Perhaps most importantly, we demonstrate that attrition of memory phenotype cells may have a pathologic significance, as elevated IL-6 levels were associated with decreased numbers of memory-phenotype CD8 T cells in septic mice, and preservation of this subset after administration of anti-LFA-1 mAb conferred improved survival at 7 d. Taken together, these data identify potentially modifiable responses of memory-phenotype CD8 T cells in early sepsis and may be particularly important in the application of immunomodulatory therapies in sepsis. PMID:27286793

CD107a Expression and IFN-γ Production as Markers for Evaluation of Cytotoxic CD3+ CD8+ T Cell Response to CMV Antigen in Women with Recurrent Spontaneous Abortion

PubMed Central

Tarokhian, Batoul; Sherkat, Roya; Nasr Esfahani, Mohamma Hossein; Adib, Minoo; Kiani Esfahani, Abbas; Ataei, Behrooz

2014-01-01

Background: Some evidence has shown a relationship between primary human cytomegalovirus (CMV) infection and pregnancy loss. The impact of CMV infection reactivation during pregnancy on adverse pregnancy outcomes is not completely understood. It is proposed that altered immune response, and therefore, recurrence or reactivation of latent CMV infection may relate to recurrent spontaneous abortion (RSA); however, few data are available in this regard. To find out about any cell mediated defect and reactivation of latent CMV infection in women with RPL, cellular immunity to the virus has been evaluated by specific cytotoxic T lymphocyte (CTL) response to CMV. Materials and Methods: In a case control study, CTL CD107a expression and in- tercellular IFN-γ production in response to CMV pp65 antigen and staphylococcus enterotoxin B (SEB) in women with RSA were assessed by flow cytometric analysis. Forty-four cases with history of recurrent pregnancy and forty-four controls with history of successful pregnancies were included. The FACSCaliber flow cytometer were used for analysis. Results: No significant difference was observed between CD107a expression and IFN-γ production in response to CMV PP65 antigen in RPL patients and control group. How- ever, the cytotoxic response to SEB antigen in patients with RPL was significantly lower than control group (p=0.042). Conclusion: The results of this study show that impaired CD107a expression and IFN-γ production as CTL response to CMV does not appear to be a major contrib- uting and immune incompetence factor in patients with RPL, but cytotoxic T cell response defect to other antigens requires to be assessed further in these patients. PMID:24520502

CD107a Expression and IFN-γ Production as Markers for Evaluation of Cytotoxic CD3+ CD8+ T Cell Response to CMV Antigen in Women with Recurrent Spontaneous Abortion.

PubMed

Tarokhian, Batoul; Sherkat, Roya; Nasr Esfahani, Mohamma Hossein; Adib, Minoo; Kiani Esfahani, Abbas; Ataei, Behrooz

2014-01-01

Some evidence has shown a relationship between primary human cytomegalovirus (CMV) infection and pregnancy loss. The impact of CMV infection reactivation during pregnancy on adverse pregnancy outcomes is not completely understood. It is proposed that altered immune response, and therefore, recurrence or reactivation of latent CMV infection may relate to recurrent spontaneous abortion (RSA); however, few data are available in this regard. To find out about any cell mediated defect and reactivation of latent CMV infection in women with RPL, cellular immunity to the virus has been evaluated by specific cytotoxic T lymphocyte (CTL) response to CMV. In a case control study, CTL CD107a expression and in- tercellular IFN-γ production in response to CMV pp65 antigen and staphylococcus enterotoxin B (SEB) in women with RSA were assessed by flow cytometric analysis. Forty-four cases with history of recurrent pregnancy and forty-four controls with history of successful pregnancies were included. The FACSCaliber flow cytometer were used for analysis. No significant difference was observed between CD107a expression and IFN-γ production in response to CMV PP65 antigen in RPL patients and control group. How- ever, the cytotoxic response to SEB antigen in patients with RPL was significantly lower than control group (p=0.042). The results of this study show that impaired CD107a expression and IFN-γ production as CTL response to CMV does not appear to be a major contrib- uting and immune incompetence factor in patients with RPL, but cytotoxic T cell response defect to other antigens requires to be assessed further in these patients.

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

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.

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

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.

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

Food antigen-induced immune responses in Crohn's disease patients and experimental colitis mice.

PubMed

Kawaguchi, Takaaki; Mori, Maiko; Saito, Keiko; Suga, Yasuyo; Hashimoto, Masaki; Sako, Minako; Yoshimura, Naoki; Uo, Michihide; Danjo, Keiko; Ikenoue, Yuka; Oomura, Kaori; Shinozaki, Junko; Mitsui, Akira; Kajiura, Takayuki; Suzuki, Manabu; Takazoe, Masakazu

2015-04-01

In Crohn's disease (CD), the involvement of food antigens in immune responses remains unclear. The objective of this study was to detect immune responses against food antigens in CD patients and examine the mechanism in a mouse model of colitis. We enrolled 98 CD patients, 50 ulcerative colitis patients, and 52 healthy controls (HCs) to compare the levels of serum immunoglobulin (Ig)Gs against 88 foods. The presence of serum IgGs against foods was also examined in interleukin (IL)-10 knockout (KO) mice in which CD4(+) T cell activation by antigenic food protein was assessed. Mice transferred with IL-10 KO cells received diets with or without food antigens, and the development of colitis was evaluated. The prevalence of IgGs against various foods, especially vegetables, grains, and nuts, was significantly higher in CD patients than in HCs. Similarly, the prevalence of IgGs against food proteins was higher in IL-10 KO mice than in BALB/c mice. Beta-conglycinin, identified as an antigenic food proteins in IL-10 KO mice, induced CD4(+) T cell production of interferon-γ and IL-17 through dendritic cell antigen presentation. Elimination of the food antigens ameliorated the development of colitis in mice without altering the composition of their intestinal microbiota. In CD colitis mice, intestinal inflammation via CD4(+) T cell hyperactivation was induced by food antigens associated with high serum IgG levels and was ameliorated by the elimination of food antigens. This disrupted immunological tolerance to food antigen, which might act as an exacerbating factor, remains to be elucidated in CD patients.

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

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.

Reduced response to Epstein–Barr virus antigens by T-cells in systemic lupus erythematosus patients

PubMed Central

Draborg, Anette Holck; Jacobsen, Søren; Westergaard, Marie; Mortensen, Shila; Larsen, Janni Lisander; Houen, Gunnar; Duus, Karen

2014-01-01

Objective Epstein–Barr virus (EBV) has for long been associated with systemic lupus erythematosus (SLE). In this study, we investigated the levels of latent and lytic antigen EBV-specific T-cells and antibodies in SLE patients. Methods T cells were analyzed by flow cytometry and antibodies were analyzed by enzyme-linked immunosorbent assay. Results SLE patients showed a significantly reduced number of activated (CD69) T-cells upon ex vivo stimulation with EBV nuclear antigen (EBNA) 1 or EBV early antigen diffuse (EBV-EA/D) in whole blood samples compared with healthy controls. Also, a reduced number of T-cells from SLE patients were found to produce interferon-γ upon stimulation with these antigens. Importantly, responses to a superantigen were normal in SLE patients. Compared with healthy controls, SLE patients had fewer EBV-specific T-cells but higher titres of antibodies against EBV. Furthermore, an inverse correlation was revealed between the number of lytic antigen EBV-specific T-cells and disease activity of the SLE patients, with high-activity SLE patients having fewer T-cells than low-activity SLE patients. Conclusions These results indicate a limited or a defective EBV-specific T-cell response in SLE patients, which may suggest poor control of EBV infection in SLE with an immune reaction shift towards a humoral response in an attempt to control viral reactivation. A role for decreased control of EBV as a contributing agent in the development or exacerbation of SLE is proposed. PMID:25396062

Human Cytomegalovirus (HCMV) US2 Protein Interacts with Human CD1d (hCD1d) and Down-Regulates Invariant NKT (iNKT) Cell Activity

PubMed Central

Han, Jihye; Rho, Seung Bae; Lee, Jae Yeon; Bae, Joonbeom; Park, Se Ho; Lee, Suk Jun; Lee, Sang Yeol; Ahn, Curie; Kim, Jae Young; Chun, Taehoon

2013-01-01

To avoid host immune surveillance, human cytomegalovirus (HCMV) encoded endoplasmic reticulum (ER)-membrane glycoprotein US2, which interferes with antigen presenting mechanism of major histocompatibility complex (MHC) class Ia and class II molecules. However, not many attempts have been made to study the effect of HCMV US2 on the expression of MHC class Ib molecules. In this study, we examined the effect of HCMV US2 on the expression and function of human CD1d (hCD1d), which presents glycolipid antigens to invariant NKT (iNKT) cells. Our results clearly showed that the physiological interaction between ER lumenal domain of HCMV US2 and α3 domain of hCD1d was observed within ER. Compared with mature form of hCD1d, immature form of hCD1d is more susceptible to ubiquitin-dependent proteasomal degradation mediated by HCMV US2. Moreover, the ectopic expression of HCMV US2 leads to the down-modulation of iNKT cell activity without significant change of hCD1d expression. These results will advance our understanding of the function of HCMV US2 in immune evasive mechanisms against anti-viral immunity of iNKT cells. PMID:24213674

CD34 Antigen: Determination of Specific Sites of Phosphorylation In Vitro and In Vivo¶

PubMed Central

Deterding, Leesa J.; Williams, Jason G.; Humble, Margaret M.; Petrovich, Robert M.; Wei, Sung-Jen; Trempus, Carol S.; Gates, Matthew B.; Zhu, Feng; Smart, Robert C.; Tennant, Raymond W.; Tomer, Kenneth B.

2010-01-01

CD34, a type I transmembrane glycoprotein, is a surface antigen which is expressed on several cell types, including hematopoietic progenitors, endothelial cells, as well as mast cells. Recently, CD34 has been described as a marker for epidermal stem cells in mouse hair follicles, and is expressed in outer root sheath cells of the human hair follicle. Although the biological function and regulation of CD34 is not well understood, it is thought to be involved in cell adhesion as well as possibly having a role in signal transduction. In addition, CD34 was shown to be critical for skin tumor development in mice, although the exact mechanism remains unknown. Many proteins' functions and biological activities are regulated through post-translational modifications. The extracellular domain of CD34 is heavily glycosylated but the role of these glycans in CD34 function is unknown. Additionally, two sites of tyrosine phosphorylation have been reported on human CD34 and it is known that CD34 is phosphorylated, at least in part, by protein kinase C; however, the precise location of the sites of phosphorylation has not been reported. In an effort to identify specific phosphorylation sites in CD34 and delineate the possible role of protein kinase C, we undertook the identification of the in vitro sites of phosphorylation on the intracellular domain of mouse CD34 (aa 309–382) following PKC treatment. For this work, we are using a combination of enzymatic proteolysis and peptide sequencing by mass spectrometry. After which the in vivo sites of phosphorylation of full-length mouse CD34 expressed from HEK293F cells were determined. The observed in vivo sites of phosphorylation, however, are not consensus PKC sites, but our data indicate that one of these sites may possibly be phosphorylated by AKT2. These results suggest that other kinases, as well as PKC, may have important signaling functions in CD34. PMID:21499536

Cross-talk between Tetraspanin CD9 and Transmembrane Adaptor Protein Non-T Cell Activation Linker (NTAL) in Mast Cell Activation and Chemotaxis*

PubMed Central

Hálová, Ivana; Dráberová, Lubica; Bambousková, Monika; Machyna, Martin; Stegurová, Lucie; Smrž, Daniel; Dráber, Petr

2013-01-01

Chemotaxis, a process leading to movement of cells toward increasing concentrations of chemoattractants, is essential, among others, for recruitment of mast cells within target tissues where they play an important role in innate and adaptive immunity. Chemotaxis is driven by chemoattractants, produced by various cell types, as well as by intrinsic cellular regulators, which are poorly understood. In this study we prepared a new mAb specific for the tetraspanin CD9. Binding of the antibody to bone marrow-derived mast cells triggered activation events that included cell degranulation, Ca2+ response, dephosphorylation of ezrin/radixin/moesin (ERM) family proteins, and potent tyrosine phosphorylation of the non-T cell activation linker (NTAL) but only weak phosphorylation of the linker for activation of T cells (LAT). Phosphorylation of the NTAL was observed with whole antibody but not with its F(ab)2 or Fab fragments. This indicated involvement of the Fcγ receptors. As documented by electron microscopy of isolated plasma membrane sheets, CD9 colocalized with the high-affinity IgE receptor (FcϵRI) and NTAL but not with LAT. Further tests showed that both anti-CD9 antibody and its F(ab)2 fragment inhibited mast cell chemotaxis toward antigen. Experiments with bone marrow-derived mast cells deficient in NTAL and/or LAT revealed different roles of these two adaptors in antigen-driven chemotaxis. The combined data indicate that chemotaxis toward antigen is controlled in mast cells by a cross-talk among FcϵRI, tetraspanin CD9, transmembrane adaptor proteins NTAL and LAT, and cytoskeleton-regulatory proteins of the ERM family. PMID:23443658

CD40 signaling synergizes with TLR-2 in the BCR independent activation of resting B cells.

PubMed

Jain, Shweta; Chodisetti, Sathi Babu; Agrewala, Javed N

2011-01-01

Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB) cells, by involving TLR-2 and CD40--molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID), signifying class switch recombination (CSR). Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.

Dominant role of antigen dose in CD4+Foxp3+ regulatory T cell induction and expansion1

PubMed Central

Turner, Michael S.; Kane, Lawrence P.; Morel, Penelope A.

2009-01-01

The definitions of tolerogenic vs. immunogenic dendritic cells (DC) remain controversial. Immature DC have been shown to induce T regulatory cells (Treg) specific for foreign and allo-antigens. However, we have previously reported that mature DC (G4DC) prevented the onset of autoimmune diabetes whereas immature DC (GMDC) were therapeutically ineffective. In this study, islet-specific CD4+ T cells from BDC2.5 TCR Tg mice were stimulated, in the absence of exogenous cytokine, with GMDC or G4DC pulsed with high- or low-affinity antigenic peptides and examined for Treg induction. Both GMDC and G4DC presenting low peptide doses induced weak TCR signaling via the Akt/mTOR pathway, resulting in significant expansion of Foxp3+ Treg. Furthermore, unpulsed G4DC, but not GMDC, also induced Treg. High peptide doses induced strong Akt/mTOR signaling and favored the expansion of Foxp3neg Th cells. The inverse correlation of Foxp3 and Akt/mTOR signaling was also observed in DO11.10 and OT-II TCR-Tg T cells and was recapitulated with anti-CD3/CD28 stimulation in the absence of DC. IL-6 production in these cultures correlated positively with antigen dose and inversely with Treg expansion. Studies with T cells or DC from IL-6−/− mice revealed that IL-6 production by T cells was more important in the inhibition of Treg induction at low antigen doses. These studies indicate that strength of Akt/mTOR signaling, a critical T cell intrinsic determinant for Treg vs Th induction, can be controlled by adjusting the dose of antigenic peptide. Furthermore, this operates in a dominant fashion over DC phenotype and cytokine production. PMID:19801514

Marek's disease is a natural model for lymphomas overexpressing Hodgkin's disease antigen (CD30)

PubMed Central

Burgess, S. C.; Young, J. R.; Baaten, B. J. G.; Hunt, L.; Ross, L. N. J.; Parcells, M. S.; Kumar, P. M.; Tregaskes, C. A.; Lee, L. F.; Davison, T. F.

2004-01-01

Animal models are essential for elucidating the molecular mechanisms of carcinogenesis. Hodgkin's and many diverse non-Hodgkin's lymphomas overexpress the Hodgkin's disease antigen CD30 (CD30hi), a tumor necrosis factor receptor II family member. Here we show that chicken Marek's disease (MD) lymphoma cells are also CD30hi and are a unique natural model for CD30hi lymphoma. Chicken CD30 resembles an ancestral form, and we identify a previously undescribed potential cytoplasmic signaling domain conserved in chicken, human, and mouse CD30. Our phylogeneic analysis defines a relationship between the structures of human and mouse CD30 and confirms that mouse CD30 represents the ancestral mammalian gene structure. CD30 expression by MD virus (MDV)-transformed lymphocytes correlates with expression of the MDV Meq putative oncogene (a c-Jun homologue) in vivo. The chicken CD30 promoter has 15 predicted high-stringency Meq-binding transcription factor recognition motifs, and Meq enhances transcription from the CD30 promoter in vitro. Plasma proteomics identified a soluble form of CD30. CD30 overexpression is evolutionarily conserved and defines one class of neoplastic transformation events, regardless of etiology. We propose that CD30 is a component of a critical intracellular signaling pathway perturbed in neoplastic transformation. Specific anti-CD30 Igs occurred after infection of genetically MD-resistant chickens with oncogenic MDV, suggesting immunity to CD30 could play a role in MD lymphoma regression. PMID:15356338

Chemokine programming dendritic cell antigen response: part II - programming antigen presentation to T lymphocytes by partially maintaining immature dendritic cell phenotype.

PubMed

Park, Jaehyung; Bryers, James D

2013-05-01

In a companion article to this study,(1) the successful programming of a JAWSII dendritic cell (DC) line's antigen uptake and processing was demonstrated based on pre-treatment of DCs with a specific 'cocktail' of select chemokines. Chemokine pre-treatment modulated cytokine production before and after DC maturation [by lipopolysaccharide (LPS)]. After DC maturation, it induced an antigen uptake and processing capacity at levels 36% and 82% higher than in immature DCs, respectively. Such programming proffers a potential new approach to enhance vaccine efficiency. Unfortunately, simply enhancing antigen uptake does not guarantee the desired activation and proliferation of lymphocytes, e.g. CD4(+) T cells. In this study, phenotype changes and antigen presentation capacity of chemokine pre-treated murine bone marrow-derived DCs were examined in long-term co-culture with antigen-specific CD4(+) T cells to quantify how chemokine pre-treatment may impact the adaptive immune response. When a model antigen, ovalbumin (OVA), was added after intentional LPS maturation of chemokine-treated DCs, OVA-biased CD4(+) T-cell proliferation was initiated from ~ 100% more undivided naive T cells as compared to DCs treated only with LPS. Secretion of the cytokines interferon-γ, interleukin-1β, interleukin-2 and interleukin-10 in the CD4(+) T cell : DC co-culture (with or without chemokine pre-treatment) were essentially the same. Chemokine programming of DCs with a 7 : 3 ratio of CCL3 : CCL19 followed by LPS treatment maintained partial immature phenotypes of DCs, as indicated by surface marker (CD80 and CD86) expression over time. Results here and in our companion paper suggest that chemokine programming of DCs may provide a novel immunotherapy strategy to obviate the natural endocytosis limit of DC antigen uptake, thus potentially increasing DC-based vaccine efficiency. © 2012 Blackwell Publishing Ltd.

Immunosuppressive Effects of Bryoria sp. (Lichen-Forming Fungus) Extracts via Inhibition of CD8+ T-Cell Proliferation and IL-2 Production in CD4+ T Cells.

PubMed

Hwang, Yun-Ho; Lee, Sung-Ju; Kang, Kyung-Yun; Hur, Jae-Seoun; Yee, Sung-Tae

2017-06-28

Lichen-forming fungi are known to have various biological activities, such as antioxidant, antimicrobial, antitumor, antiviral, anti-inflammation, and anti proliferative effects. However, the immunosuppressive effects of Bryoria sp. extract (BSE) have not previously been investigated. In this study, the inhibitory activity of BSE on the proliferation of CD8 + T cells and the mixed lymphocytes reaction (MLR) was evaluated in vitro. BSE was non-toxic in spleen cells and suppressed the growth of splenocytes induced by anti-CD3. The suppressed cell population in spleen cells consisted of CD8 + T cells and their proliferation was inhibited by the treatment with BSE. This extract significantly suppressed the IL-2 associated with T cell growth and IFN-γ as the CD8 + T cell marker. Furthermore, BSE reduced the expression of the IL-2 receptor alpha chain (IL-2Rα) on CD8 + T cells and CD86 on dendritic cells by acting as antigen-presenting cells. Finally, the MLR produced by the co-culture of C57BL/6 and MMC-treated BALB/c was suppressed by BSE. IL-2, IFN-γ, and CD69 on CD8 + T cells in MLR condition were inhibited by BSE. These results indicate that BSE inhibits the MLR via the suppression of IL-2Rα expression in CD8 + T cells. BSE has the potential to be developed as an anti-immunosuppression agent for organ transplants.

EFFECT OF LIPOSOMAL CLODRONATE-DEPENDENT DEPLETION OF PROFESSIONAL ANTIGEN PRESENTING CELLS ON NUMBERS AND PHENOTYPE OF CANINE CD4+CD25+FOXP3+ REGULATORY T CELLS

PubMed Central

Weaver, Kriston F.; Stokes, John V.; Gunnoe, Sagen A.; Follows, Joyce S.; Shafer, Lydia; Ammari, Mais G.; Archer, Todd M.; Thomason, John M.; Mackin, Andrew J.; Pinchuk, Lesya M.

2015-01-01

Regulatory T cells (Tregs) are known to control autoreactivity during and subsequent to the development of the peripheral immune system. Professional antigen presenting cells (APCs), dendritic cells (DCs) and monocytes, have an important role in inducing Tregs. For the first time, this study evaluated proportions and phenotypes of Tregs in canine peripheral blood depleted of professional APCs, utilizing liposomal clodronate (LC) and multicolor flow cytometry analysis. Our results demonstrate that LC exposure promoted short term decreases followed by significant increases in the proportions or absolute numbers of CD4+CD25+FOXP3+ Tregs in dogs. In general, the LC-dependent Treg fluctuations were similar to the changes in the levels of CD14+ monocytes in Walker hounds. However, the proportions of monocytes showed more dramatic changes compared to the proportions of Tregs that were visually unchanged after LC treatment over the study period. At the same time, absolute Treg numbers showed, similarly to the levels of CD14+ monocytes, significant compensatory gains as well as the recovery during the normalization period. We confirm the previous data that CD4+ T cells with the highest CD25 expression were highly enriched for FOXP3. Furthermore, for the first time, we report that CD4+CD25lowFOXP3+ is the major regulatory T cell subset affected by LC exposure. The increases within the lowest CD25 expressers of CD4+FOXP3+ cells together with compensatory gains in the proportion of CD14+ monocytes during compensatory and normalization periods suggest the possible direct or indirect roles of monocytes in active recruitment and generation of Tregs from naïve CD4+ T cells. PMID:25950023

Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy

PubMed Central

Hollyman, Daniel; Stefanski, Jolanta; Przybylowski, Mark; Bartido, Shirley; Borquez-Ojeda, Oriana; Taylor, Clare; Yeh, Raymond; Capacio, Vanessa; Olszewska, Malgorzata; Hosey, James; Sadelain, Michel; Brentjens, Renier J.; Rivière, Isabelle

2009-01-01

Summary Based on promising pre-clinical data demonstrating the eradication of systemic B cell malignancies by CD19-targeted T lymphocytes in vivo in SCID beige mouse models, we are launching Phase 1 clinical trials in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). We present here the validation of the bioprocess we developed for the production and expansion of clinical grade autologous T cells derived from patients with CLL. We demonstrate that T cells genetically modified with a replication-defective gammaretroviral vector derived from the Moloney murine leukemia virus encoding a chimeric antigen receptor (CAR) targeted to CD19 (1928z) can be expanded with Dynabeads® CD3/CD28. This bioprocess allows us to generate clinical doses of 1928z+ T cells in approximately 2 to 3 weeks in a large-scale semi-closed culture system using the Wave bioreactor. These 1928z+ T cells remain biologically functional not only in vitro but also in SCID beige mice bearing disseminated tumors. The validation requirements in terms of T cell expansion, T cell transduction with the 1928z CAR, biological activity, quality control testing and release criteria were met for all four validation runs using apheresis products from patients with CLL. Additionally, following expansion of the T cells, the diversity of the skewed Vβ T cell receptor repertoire was significantly restored. This validated process will be used in phase I clinical trials in patients with chemo-refractory CLL and in patients with relapsed ALL. It can also be adapted for other clinical trials involving the expansion and transduction of patient or donor T cells using any chimeric antigen receptor or T cell receptor. PMID:19238016

CD20-specific adoptive immunotherapy for lymphoma using a chimeric antigen receptor with both CD28 and 4-1BB domains: pilot clinical trial results.

PubMed

Till, Brian G; Jensen, Michael C; Wang, Jinjuan; Qian, Xiaojun; Gopal, Ajay K; Maloney, David G; Lindgren, Catherine G; Lin, Yukang; Pagel, John M; Budde, Lihua E; Raubitschek, Andrew; Forman, Stephen J; Greenberg, Philip D; Riddell, Stanley R; Press, Oliver W

2012-04-26

Cellular immune responses have the potential to elicit dramatic and sustained clinical remissions in lymphoma patients. Recent clinical trial data demonstrate that modification of T cells with chimeric antigen receptors (CARs) is a promising strategy. T cells containing CARs with costimulatory domains exhibit improved activity against tumors. We conducted a pilot clinical trial testing a "third-generation" CD20-specific CAR with CD28 and 4-1BB costimulatory domains in patients with relapsed indolent B-cell and mantle cell lymphomas. Four patients were enrolled, and 3 received T-cell infusions after cyclophosphamide lymphodepletion. Treatment was well tolerated, although one patient developed transient infusional symptoms. Two patients without evaluable disease remained progression-free for 12 and 24 months. The third patient had an objective partial remission and relapsed at 12 months after infusions. Modified T cells were detected by quantitative PCR at tumor sites and up to 1 year in peripheral blood, albeit at low levels. No evidence of host immune responses against infused cells was detected. In conclusion, adoptive immunotherapy with CD20-specific T cells was well tolerated and was associated with antitumor activity. We will pursue alternative gene transfer technologies and culture conditions in future studies to improve CAR expression and cell production efficiency.

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

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.

Lack of Original Antigenic Sin in Recall CD8+ T Cell Responses

PubMed Central

Zehn, Dietmar; Turner, Michael J.; Lefrançois, Leo; Bevan, Michael J.

2010-01-01

In the real world, mice and men are not immunologically naive, having been exposed to numerous antigenic challenges. Prior infections sometimes negatively impact the response to a subsequent infection. This can occur in serial infections with pathogens sharing cross-reactive Ags. At the T cell level it has been proposed that preformed memory T cells, which cross-react with low avidity to epitopes presented in subsequent infections, dampen the response of high-avidity T cells. We investigated this with a series of related MHC class-I restricted Ags expressed by bacterial and viral pathogens. In all cases, we find that high-avidity CD8+ T cell precursors, either naive or memory, massively expand in secondary cross-reactive infections to dominate the response over low-avidity memory T cells. This holds true even when >10% of the CD8+ T cell compartment consists of memory T cells that cross-react weakly with the rechallenge ligand. Occasionally, memory cells generated by low-avidity stimulation in a primary infection recognize a cross-reactive epitope with high avidity and contribute positively to the response to a second infection. Taken together, our data show that the phenomenon of original antigenic sin does not occur in all heterologous infections. PMID:20439913

Upregulation of Immunoproteasome Subunits in Myositis Indicates Active Inflammation with Involvement of Antigen Presenting Cells, CD8 T-Cells and IFNγ

PubMed Central

Ghannam, Khetam; Martinez-Gamboa, Lorena; Spengler, Lydia; Krause, Sabine; Smiljanovic, Biljana; Bonin, Marc; Bhattarai, Salyan; Grützkau, Andreas; Burmester, Gerd-R.

2014-01-01

Objective In idiopathic inflammatory myopathies (IIM) infiltration of immune cells into muscle and upregulation of MHC-I expression implies increased antigen presentation and involvement of the proteasome system. To decipher the role of immunoproteasomes in myositis, we investigated individual cell types and muscle tissues and focused on possible immune triggers. Methods Expression of constitutive (PSMB5, -6, -7) and corresponding immunoproteasomal subunits (PSMB8, -9, -10) was analyzed by real-time RT-PCR in muscle biopsies and sorted peripheral blood cells of patients with IIM, non-inflammatory myopathies (NIM) and healthy donors (HD). Protein analysis in muscle biopsies was performed by western blot. Affymetrix HG-U133 platform derived transcriptome data from biopsies of different muscle diseases and from immune cell types as well as monocyte stimulation experiments were used for validation, coregulation and coexpression analyses. Results Real-time RT-PCR revealed significantly increased expression of immunoproteasomal subunits (PSMB8/-9/-10) in DC, monocytes and CD8+ T-cells in IIM. In muscle biopsies, the immunosubunits were elevated in IIM compared to NIM and exceeded levels of matched blood samples. Proteins of PSMB8 and -9 were found only in IIM but not NIM muscle biopsies. Reanalysis of 78 myositis and 20 healthy muscle transcriptomes confirmed these results and revealed involvement of the antigen processing and presentation pathway. Comparison with reference profiles of sorted immune cells and healthy muscle confirmed upregulation of PSMB8 and -9 in myositis biopsies beyond infiltration related changes. This upregulation correlated highest with STAT1, IRF1 and IFNγ expression. Elevation of T-cell specific transcripts in active IIM muscles was accompanied by increased expression of DC and monocyte marker genes and thus reflects the cell type specific involvement observed in peripheral blood. Conclusions Immunoproteasomes seem to indicate IIM activity and

Upregulation of immunoproteasome subunits in myositis indicates active inflammation with involvement of antigen presenting cells, CD8 T-cells and IFNΓ.

PubMed

Ghannam, Khetam; Martinez-Gamboa, Lorena; Spengler, Lydia; Krause, Sabine; Smiljanovic, Biljana; Bonin, Marc; Bhattarai, Salyan; Grützkau, Andreas; Burmester, Gerd-R; Häupl, Thomas; Feist, Eugen

2014-01-01

In idiopathic inflammatory myopathies (IIM) infiltration of immune cells into muscle and upregulation of MHC-I expression implies increased antigen presentation and involvement of the proteasome system. To decipher the role of immunoproteasomes in myositis, we investigated individual cell types and muscle tissues and focused on possible immune triggers. Expression of constitutive (PSMB5, -6, -7) and corresponding immunoproteasomal subunits (PSMB8, -9, -10) was analyzed by real-time RT-PCR in muscle biopsies and sorted peripheral blood cells of patients with IIM, non-inflammatory myopathies (NIM) and healthy donors (HD). Protein analysis in muscle biopsies was performed by western blot. Affymetrix HG-U133 platform derived transcriptome data from biopsies of different muscle diseases and from immune cell types as well as monocyte stimulation experiments were used for validation, coregulation and coexpression analyses. Real-time RT-PCR revealed significantly increased expression of immunoproteasomal subunits (PSMB8/-9/-10) in DC, monocytes and CD8+ T-cells in IIM. In muscle biopsies, the immunosubunits were elevated in IIM compared to NIM and exceeded levels of matched blood samples. Proteins of PSMB8 and -9 were found only in IIM but not NIM muscle biopsies. Reanalysis of 78 myositis and 20 healthy muscle transcriptomes confirmed these results and revealed involvement of the antigen processing and presentation pathway. Comparison with reference profiles of sorted immune cells and healthy muscle confirmed upregulation of PSMB8 and -9 in myositis biopsies beyond infiltration related changes. This upregulation correlated highest with STAT1, IRF1 and IFNγ expression. Elevation of T-cell specific transcripts in active IIM muscles was accompanied by increased expression of DC and monocyte marker genes and thus reflects the cell type specific involvement observed in peripheral blood. Immunoproteasomes seem to indicate IIM activity and suggest that dominant involvement of

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.

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.

Ndfip1 mediates peripheral tolerance to self and exogenous antigen by inducing cell cycle exit in responding CD4+ T cells

PubMed Central

Altin, John A.; Daley, Stephen R.; Howitt, Jason; Rickards, Helen J.; Batkin, Alison K.; Horikawa, Keisuke; Prasad, Simon J.; Nelms, Keats A.; Kumar, Sharad; Wu, Lawren C.; Tan, Seong-Seng; Cook, Matthew C.; Goodnow, Christopher C.

2014-01-01

The NDFIP1 (neural precursor cell expressed, developmentally down-regulated protein 4 family-interacting protein 1) adapter for the ubiquitin ligase ITCH is genetically linked to human allergic and autoimmune disease, but the cellular mechanism by which these proteins enable foreign and self-antigens to be tolerated is unresolved. Here, we use two unique mouse strains—an Ndfip1-YFP reporter and an Ndfip1-deficient strain—to show that Ndfip1 is progressively induced during T-cell differentiation and activation in vivo and that its deficiency causes a cell-autonomous, Forkhead box P3-independent failure of peripheral CD4+ T-cell tolerance to self and exogenous antigen. In small cohorts of antigen-specific CD4+ cells responding in vivo, Ndfip1 was necessary for tolerogen-reactive T cells to exit cell cycle after one to five divisions and to abort Th2 effector differentiation, defining a step in peripheral tolerance that provides insights into the phenomenon of T-cell anergy in vivo and is distinct from the better understood process of Bcl2-interacting mediator of cell death-mediated apoptosis. Ndfip1 deficiency precipitated autoimmune pancreatic destruction and diabetes; however, this depended on a further accumulation of nontolerant anti-self T cells from strong stimulation by exogenous tolerogen. These findings illuminate a peripheral tolerance checkpoint that aborts T-cell clonal expansion against allergens and autoantigens and demonstrate how hypersensitive responses to environmental antigens may trigger autoimmunity. PMID:24520172

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.

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

Glioblastoma-targeted CD4+ CAR T cells mediate superior antitumor activity.

PubMed

Wang, Dongrui; Aguilar, Brenda; Starr, Renate; Alizadeh, Darya; Brito, Alfonso; Sarkissian, Aniee; Ostberg, Julie R; Forman, Stephen J; Brown, Christine E

2018-05-17

Chimeric antigen receptor-modified (CAR-modified) T cells have shown promising therapeutic effects for hematological malignancies, yet limited and inconsistent efficacy against solid tumors. The refinement of CAR therapy requires an understanding of the optimal characteristics of the cellular products, including the appropriate composition of CD4+ and CD8+ subsets. Here, we investigated the differential antitumor effect of CD4+ and CD8+ CAR T cells targeting glioblastoma-associated (GBM-associated) antigen IL-13 receptor α2 (IL13Rα2). Upon stimulation with IL13Rα2+ GBM cells, the CD8+ CAR T cells exhibited robust short-term effector function but became rapidly exhausted. By comparison, the CD4+ CAR T cells persisted after tumor challenge and sustained their effector potency. Mixing with CD4+ CAR T cells failed to ameliorate the effector dysfunction of CD8+ CAR T cells, while surprisingly, CD4+ CAR T cell effector potency was impaired when coapplied with CD8+ T cells. In orthotopic GBM models, CD4+ outperformed CD8+ CAR T cells, especially for long-term antitumor response. Further, maintenance of the CD4+ subset was positively correlated with the recursive killing ability of CAR T cell products derived from GBM patients. These findings identify CD4+ CAR T cells as a highly potent and clinically important T cell subset for effective CAR therapy.

Glioblastoma-targeted CD4+ CAR T cells mediate superior antitumor activity

PubMed Central

Wang, Dongrui; Starr, Renate; Alizadeh, Darya; Brito, Alfonso; Sarkissian, Aniee; Ostberg, Julie R.; Forman, Stephen J.; Brown, Christine E.

2018-01-01

Chimeric antigen receptor–modified (CAR-modified) T cells have shown promising therapeutic effects for hematological malignancies, yet limited and inconsistent efficacy against solid tumors. The refinement of CAR therapy requires an understanding of the optimal characteristics of the cellular products, including the appropriate composition of CD4+ and CD8+ subsets. Here, we investigated the differential antitumor effect of CD4+ and CD8+ CAR T cells targeting glioblastoma-associated (GBM-associated) antigen IL-13 receptor α2 (IL13Rα2). Upon stimulation with IL13Rα2+ GBM cells, the CD8+ CAR T cells exhibited robust short-term effector function but became rapidly exhausted. By comparison, the CD4+ CAR T cells persisted after tumor challenge and sustained their effector potency. Mixing with CD4+ CAR T cells failed to ameliorate the effector dysfunction of CD8+ CAR T cells, while surprisingly, CD4+ CAR T cell effector potency was impaired when coapplied with CD8+ T cells. In orthotopic GBM models, CD4+ outperformed CD8+ CAR T cells, especially for long-term antitumor response. Further, maintenance of the CD4+ subset was positively correlated with the recursive killing ability of CAR T cell products derived from GBM patients. These findings identify CD4+ CAR T cells as a highly potent and clinically important T cell subset for effective CAR therapy. PMID:29769444

Inhibiting DNA methylation activates cancer testis antigens and expression of the antigen processing and presentation machinery in colon and ovarian cancer cells.

PubMed

Siebenkäs, Cornelia; Chiappinelli, Katherine B; Guzzetta, Angela A; Sharma, Anup; Jeschke, Jana; Vatapalli, Rajita; Baylin, Stephen B; Ahuja, Nita

2017-01-01

Innovative therapies for solid tumors are urgently needed. Recently, therapies that harness the host immune system to fight cancer cells have successfully treated a subset of patients with solid tumors. These responses have been strong and durable but observed in subsets of patients. Work from our group and others has shown that epigenetic therapy, specifically inhibiting the silencing DNA methylation mark, activates immune signaling in tumor cells and can sensitize to immune therapy in murine models. Here we show that colon and ovarian cancer cell lines exhibit lower expression of transcripts involved in antigen processing and presentation to immune cells compared to normal tissues. In addition, treatment with clinically relevant low doses of DNMT inhibitors (that remove DNA methylation) increases expression of both antigen processing and presentation and Cancer Testis Antigens in these cell lines. We confirm that treatment with DNMT inhibitors upregulates expression of the antigen processing and presentation molecules B2M, CALR, CD58, PSMB8, PSMB9 at the RNA and protein level in a wider range of colon and ovarian cancer cell lines and treatment time points than had been described previously. In addition, we show that DNMTi treatment upregulates many Cancer Testis Antigens common to both colon and ovarian cancer. This increase of both antigens and antigen presentation by epigenetic therapy may be one mechanism to sensitize patients to immune therapies.

[Activation of peripheral T lymphocytes in children with epilepsy and production of cytokines].

PubMed

Yang, Jie; Hu, Chongkang; Jiang, Xun

2016-09-01

Objective To study the state of peripheral T lymphocytes and cytokine levels in children with epilepsy. Methods Twenty children with epilepsy and 20 healthy age-matched children were recruited and their peripheral blood was collected. The activation of T lymphocytes was evaluated by detecting the expressions of CD25, CD69 and cytotoxic T lymphocyte-assicated antigen 4 (CTLA4). The function of T lymphocytes was evaluated by detecting the expressions of interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), IL-17A and IL-6. The activation of regulatory T cells (Tregs) was evaluated by detecting the expression of IL-10. Results Children with epilepsy had higher expressions of CD25, CD69 and CTLA-4 in T lymphocytes than the controls did. The expressions of IFN-γ, TNF-α, IL-17A and IL-6 in T lymphocytes of children with epilepsy were higher than those of the controls. Frequency of Tregs producing IL-10 was higher in children with epilepsy as compared with the controls. Conclusion Peripheral T lymphocytes of children with epilepsy are activated and produce cytokines.

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

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…

Antigen smuggling in tuberculosis.

PubMed

Hudrisier, Denis; Neyrolles, Olivier

2014-06-11

The importance of CD4 T lymphocytes in immunity to M. tuberculosis is well established; however, how dendritic cells activate T cells in vivo remains obscure. In this issue of Cell Host & Microbe, Srivastava and Ernst (2014) report a mechanism of antigen transfer for efficient activation of antimycobacterial T cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Expression of CD 68, CD 45 and human leukocyte antigen-DR in central and peripheral giant cell granuloma, giant cell tumor of long bones, and tuberculous granuloma: An immunohistochemical study.

PubMed

Kumar, Anoop; Sherlin, Herald J; Ramani, Pratibha; Natesan, Anuja; Premkumar, Priya

2015-01-01

Multinucleated giant cells (MNCs) form an integral part of numerous bone and soft tissue tumors, tumor-like lesions and are often associated with granulomas of immunological and nonimmunological origin. The presence of various types of giant cells depends on the lesions in which they are present which are difficult to be diagnosed under routine histological techniques. Immunohistochemistry can be used for a better diagnosis and understanding of the origin of various giant cells using various markers of immune response like human leukocyte antigen-DR (HLA-DR) and those expressed on monocytes and macrophages like CD 68 and leukocyte common antigen (LCA). The study group consisted of 10 cases of giant cell tumor (GCT) of long bones, tuberculous granuloma, and giant cell granuloma to evaluate and analyze the expression pattern of LCA, CD 68, and HLA-DR in various giant cell lesions. Strong expression of CD 68 was observed in 80% of the lesions, strong and moderate expression of CD 45 observed in 70% of the lesions among and within the groups. In contrast, HLA-DR demonstrated negative expression in 80% of cases except for tuberculous granuloma where all the 10 cases showed moderate to strong immunoreactivity. CD 68 and CD 45 expression was found in central giant cell granuloma, peripheral giant cell granuloma and GCT, suggesting the origin from mononuclear phagocyte system and considering their clinical behavior of osteoclast type. High expressivity of HLA-DR in tuberculous granulomas which is an essential factor for presentation of the microbial antigen to CD 4 helper cells thus reassuring the fact that they are up-regulated in response to infection.

Comprehensive Approach for Identifying the T Cell Subset Origin of CD3 and CD28 Antibody-Activated Chimeric Antigen Receptor-Modified T Cells.

PubMed

Schmueck-Henneresse, Michael; Omer, Bilal; Shum, Thomas; Tashiro, Haruko; Mamonkin, Maksim; Lapteva, Natalia; Sharma, Sandhya; Rollins, Lisa; Dotti, Gianpietro; Reinke, Petra; Volk, Hans-Dieter; Rooney, Cliona M

2017-07-01

The outcome of therapy with chimeric Ag receptor (CAR)-modified T cells is strongly influenced by the subset origin of the infused T cells. However, because polyclonally activated T cells acquire a largely CD45RO + CCR7 - effector memory phenotype after expansion, regardless of subset origin, it is impossible to know which subsets contribute to the final T cell product. To determine the contribution of naive T cell, memory stem T cell, central memory T cell, effector memory T cell, and terminally differentiated effector T cell populations to the CD3 and CD28-activated CAR-modified T cells that we use for therapy, we followed the fate and function of individually sorted CAR-modified T cell subsets after activation with CD3 and CD28 Abs (CD3/28), transduction and culture alone, or after reconstitution into the relevant subset-depleted population. We show that all subsets are sensitive to CAR transduction, and each developed a distinct T cell functional profile during culture. Naive-derived T cells showed the greatest rate of proliferation but had more limited effector functions and reduced killing compared with memory-derived populations. When cultured in the presence of memory T cells, naive-derived T cells show increased differentiation, reduced effector cytokine production, and a reduced reproliferative response to CAR stimulation. CD3/28-activated T cells expanded in IL-7 and IL-15 produced greater expansion of memory stem T cells and central memory T cell-derived T cells compared with IL-2. Our strategy provides a powerful tool to elucidate the characteristics of CAR-modified T cells, regardless of the protocol used for expansion, reveals the functional properties of each expanded T cell subset, and paves the way for a more detailed evaluation of the effects of manufacturing changes on the subset contribution to in vitro-expanded T cells. Copyright © 2017 by The American Association of Immunologists, Inc.