A novel mechanism by which tissue transglutaminase activates signaling events that promote cell survival.

PubMed

Boroughs, Lindsey K; Antonyak, Marc A; Cerione, Richard A

2014-04-04

Tissue transglutaminase (tTG) functions as a GTPase and an acyl transferase that catalyzes the formation of protein cross-links. tTG expression is frequently up-regulated in human cancer, where it has been implicated in various aspects of cancer progression, including cell survival and chemo-resistance. However, the extent to which tTG cooperates with other proteins within the context of a cancer cell, versus its intrinsic ability to confer transformed characteristics to cells, is poorly understood. To address this question, we asked what effect the ectopic expression of tTG in a non-transformed cellular background would have on the behavior of the cells. Using NIH3T3 fibroblasts stably expressing a Myc-tagged form of tTG, we found that tTG strongly protected these cells from serum starvation-induced apoptosis and triggered the activation of the PI3-kinase/mTOR Complex 1 (mTORC1)/p70 S6-kinase pathway. We determined that tTG forms a complex with the non-receptor tyrosine kinase c-Src and PI3-kinase, and that treating cells with inhibitors to block tTG function (monodansylcadaverine; MDC) or c-Src kinase activity (PP2) disrupted the formation of this complex, and prevented tTG from activating the PI3-kinase pathway. Moreover, treatment of fibroblasts over-expressing tTG with PP2, or with inhibitors that inactivate components of the PI3-kinase pathway, including PI3-kinase (LY294002) and mTORC1 (rapamycin), ablated the tTG-promoted survival of the cells. These findings demonstrate that tTG has an intrinsic capability to stimulate cell survival through a novel mechanism that activates PI3-kinase signaling events, thus highlighting tTG as a potential target for the treatment of human cancer.

The Pathogenesis of Human Cervical Epithelium Cells Induced by Interacting with Trichomonas vaginalis

PubMed Central

Lin, Wei-Chen; Chang, Wei-Ting; Chang, Tsuey-Yu; Shin, Jyh-Wei

2015-01-01

Background Trichomonas vaginalis is a protozoan parasite that occurs in the urogenital-vaginal tract and is the primary causative agent of trichomoniasis, a common sexually transmitted disease in humans. The aggregation of this protozoan tends to destroy epithelial cells and induce pathogenesis. Principal Findings This study cultured T. vaginalis and human cervical epithelial cells (Z172) under the same conditions in the experiments. Following co-culturing for ten hours, the protozoans became attached to Z172, such that the cells presented a round shape and underwent shrinkage. Time-lapse recording and flow cytometry on interacted Z172 revealed that 70% had been disrupted, 18% presented a necrosis-like morphology and 8% showed signs of apoptosis. Gene expression profiling revealed in the seven inflammatory Z172 genes as well as in T. vaginalis genes that code for adhesion proteins 65 and 65-1. Significance These results suggest that cytopathogenic effects progress while Z172 is in contact with T. vaginalis, and the resulting morphological changes can be categorized as disruption. PMID:25901354

Comorbidity between HTLV-1-associated adult T-cell lymphoma/leukemia and verrucous carcinoma: a case report

PubMed Central

Moreno, Luis

2017-01-01

Abstract Background: Adult T-cell Leukemia/Lymphoma (ATLL) is classified as a peripheral CD4+ T-cell neoplasm caused by the human T-cell lymphotropic virus type 1 (HTLV-1). Typical symptoms are associated with leukemic infiltration; however, atypical and exaggerated manifestations of verrucous carcinoma have also been described. Case report: We present here the case of a patient with multiple skin lesions, ischemic necrosis in the hallux and lymphadenopathies. Biopsies were taken, which showed verrucous epidermal carcinoma and cutaneous lymphoma. Splenomegaly and adenopathy in mesentery, retro peritoneum and lymph node chains in the limbs were observed. Bone marrow examination showed findings compatible with T-cell leukemia/lymphoma; and it was ELISA positive for HTLV-1/2. Treatment and outcome: The patient had a good initial response to a CHOP scheme (cyclophosphamide, doxorubicin, vincristine and prednisone) with filgrastim. However, the patient had a relapse and died before the second cycle. Clinical relevance: Comorbidity could lead to the associated risk factors model. According to this model, secondary immunodeficiency caused by HTLV-1 may induce the development of verrucous carcinomas; alternatively, the disease could be due to a correlation between HTLV-1 and the human papillomavirus (HPV). PMID:28559645

Eomesoderminlo CTLA4hi Alloreactive CD8+ Memory T Cells Are Associated With Prolonged Renal Transplant Survival Induced by Regulatory Dendritic Cell Infusion in CTLA4Ig-Treated Non-Human Primates

PubMed Central

Ezzelarab, Mohamed B.; Lu, Lien; Guo, Hao; Zahorchak, Alan F.; Shufesky, William F.; Cooper, David K.C.; Morelli, Adrian E.; Thomson, Angus W.

2015-01-01

Background Memory T cells (Tmem), particularly those resistant to costimulation blockade (CB), are a major barrier to transplant tolerance. The transcription factor Eomesodermin (Eomes) is critical for Tmem development and maintenance, but its expression by alloactivated T cells has not been examined in non-human primates. Methods We evaluated Eomes and co-inhibitory cytotoxic T lymphocyte antigen-4 (CTLA4) expression by alloactivated rhesus monkey T cells in the presence of CTLA4 immunoglobulin (Ig), both in vitro and in renal allograft recipients treated with CTLA4Ig, with or without regulatory dendritic cell (DCreg) infusion. Results In normal monkeys, CD8+ T cells expressed significantly more Eomes than CD4+T cells. By contrast, CD8+T cells displayed minimal CTLA4. Among T cell subsets, central Tmem (Tcm) expressed the highest levels of Eomes. Notably, EomesloCTLA4hi cells displayed higher levels of CD25 and Foxp3 than EomeshiCTLA4lo CD8+ T cells. Following allostimulation, distinct proliferating EomesloCTLA4hi and EomeshiCTLA4lo CD8+ T cell populations were identified, with a high proportion of Tcm being EomesloCTLA4hi. CB with CTLA4Ig during allostimulation of CD8+T cells reduced CTLA4 but not Eomes expression, significantly reducing EomesloCTLA4hi cells. After transplantation with CB and rapamycin, donor-reactive EomesloCTLA4hi CD8+T cells were reduced. However, in monkeys also given DCreg, absolute numbers of these cells were elevated significantly. Conclusions Low Eomes and high CTLA4 expression by donor-reactive CD8+ Tmem is associated with prolonged renal allograft survival induced by DCreg infusion in CTLA4Ig-treated monkeys. Prolonged allograft survival associated with DCreg infusion may be related to maintenance of donor-reactive EomesloCTLA4hi Tcm. PMID:26680373

Human Parainfluenza Virus-3 can be Targeted by Rapidly ex vivo Expanded T-Lymphocytes

PubMed Central

McLaughlin, Lauren P.; Lang, Haili; Williams, Elizabeth; Wright, Kaylor E.; Powell, Allison; Cruz, Conrad R; Colberg-Poley, Anamaris M.; Barese, Cecilia; Hanley, Patrick J.; Bollard, Catherine M.; Keller, Michael D.

2016-01-01

Background Human Parainfluenza virus-3 (HPIV) is a common cause of respiratory infection in immunocompromised patients, and presently has no effective therapies. Virus-specific T-cell therapy has been successful for the treatment or prevention of viral infections in immunocompromised patients, but requires determination of T-cell antigens on targeted viruses. Methods HPIV3-specific T cells were expanded from peripheral blood of healthy donors using a rapid generation protocol targeting four HPIV3 proteins. Immunophenotyping was performed by flow cytometry. Viral specificity was determined by IFN-γ ELISpot, intracellular cytokine staining, and cytokine measurements from culture supernatants by Luminex assay. Cytotoxic activity was tested by 51Cr release and CD107a mobilization assays. Virus-specific T-cells targeting 6 viruses were then produced by rapid protocol, and the phenotype of HPIV3-specific T-cells was determined by immunomagnetic sorting for IFN-γ producing cells. Results HPIV3-specific T cells were expanded from 13 healthy donors. HPIV3-specific T-cells showed a CD4+ predominance (mean CD4:CD8 ratio 2.89), and demonstrated specificity for multiple HPIV3 antigens. The expanded T-cells were polyfunctional based on cytokine production, but only had a minor cytotoxic component. T cells targeting six viruses in a single product similarly showed HPIV3 specificity, with a predominant effector memory phenotype (CD3+/CD45RA-/CCR7-) in responder cells. Discussion HPIV3-specific T cells can be produced using a rapid ex vivo protocol from healthy donors and are predominantly CD4+ T-cells with Th1 activity. HPIV3 epitopes can also be successfully targeted alongside multiple other viral epitopes in production of 6-virus T-cells, without loss of HPIV3 specificity. These products may be clinically beneficial to combat HPIV3 infections by adoptive T-cell therapy in immune compromised patients. PMID:27692559

The Tuberculosis Vaccine Candidate Bacillus Calmette-Guérin ΔureC::hly Coexpressing Human Interleukin-7 or -18 Enhances Antigen-Specific T Cell Responses in Mice

PubMed Central

Rao, Martin; Vogelzang, Alexis; Kaiser, Peggy; Schuerer, Stefanie; Kaufmann, Stefan H. E.; Gengenbacher, Martin

2013-01-01

Bacillus Calmette–Guérin (BCG), the only approved tuberculosis vaccine, provides only limited protection. Previously, we generated a recombinant derivative (BCG ΔureC::hly), which secretes the pore-forming toxin listeriolysin O (LLO) of Listeria monocytogenes. This vaccine shows superior protection against tuberculosis in preclinical models and is safe in humans. Here we describe two new vaccine strains which express human interleukin-7 (hIL)-7 or hIL-18 in the genetic background of BCG ΔureC::hly to modulate specific T cell immunity. Both strains exhibited an uncompromised in vitro growth pattern, while inducing a proinflammatory cytokine profile in human dendritic cells (DCs). Human DCs harbouring either strain efficiently promoted secretion of IL-2 by autologous T cells in a coculture system, suggesting superior immunogenicity. BALB/c mice vaccinated with BCG ΔureC::hly, BCG ΔureC::hly_hIL7 or BCG ΔureC::hly_hIL18 developed a more robust Th1 response than after vaccination with parental BCG. Both strains provided significantly better protection than BCG in a murine Mycobacterium tuberculosis challenge model but efficacy remained comparable to that afforded by BCG ΔureC::hly. We conclude that expression of hIL-7 or hIL-18 enhanced specific T cell responses but failed to improve protection over BCG ΔureC::hly in mice. PMID:24236077

Co-Introduced Functional CCR2 Potentiates In Vivo Anti-Lung Cancer Functionality Mediated by T Cells Double Gene-Modified to Express WT1-Specific T-Cell Receptor

PubMed Central

Asai, Hiroaki; Fujiwara, Hiroshi; An, Jun; Ochi, Toshiki; Miyazaki, Yukihiro; Nagai, Kozo; Okamoto, Sachiko; Mineno, Junichi; Kuzushima, Kiyotaka; Shiku, Hiroshi; Inoue, Hirofumi; Yasukawa, Masaki

2013-01-01

Background and Purpose Although gene-modification of T cells to express tumor-related antigen-specific T-cell receptor (TCR) or chimeric antigen receptor (CAR) has clinically proved promise, there still remains room to improve the clinical efficacy of re-directed T-cell based antitumor adoptive therapy. In order to achieve more objective clinical responses using ex vivo-expanded tumor-responsive T cells, the infused T cells need to show adequate localized infiltration into the tumor. Methodology/Principal Findings Human lung cancer cells variously express a tumor antigen, Wilms' Tumor gene product 1 (WT1), and an inflammatory chemokine, CCL2. However, CCR2, the relevant receptor for CCL2, is rarely expressed on activated T-lymphocytes. A HLA-A2402+ human lung cancer cell line, LK79, which expresses high amounts of both CCL2 and WT1 mRNA, was employed as a target. Normal CD8+ T cells were retrovirally gene-modified to express both CCR2 and HLA-A*2402-restricted and WT1235–243 nonapeptide-specific TCR as an effector. Anti-tumor functionality mediated by these effector cells against LK79 cells was assessed both in vitro and in vivo. Finally the impact of CCL2 on WT1 epitope-responsive TCR signaling mediated by the effector cells was studied. Introduced CCR2 was functionally validated using gene-modified Jurkat cells and human CD3+ T cells both in vitro and in vivo. Double gene-modified CD3+ T cells successfully demonstrated both CCL2-tropic tumor trafficking and cytocidal reactivity against LK79 cells in vitro and in vivo. CCL2 augmented the WT1 epitope-responsive TCR signaling shown by relevant luciferase production in double gene-modified Jurkat/MA cells to express luciferase and WT1-specific TCR, and CCL2 also dose-dependently augmented WT1 epitope-responsive IFN-γ production and CD107a expression mediated by these double gene-modifiedCD3+ T cells. Conclusion/Significance Introduction of the CCL2/CCR2 axis successfully potentiated in vivo anti-lung cancer reactivity mediated by CD8+ T cells double gene-modified to express WT1-specific TCR and CCR2 not only via CCL2-tropic tumor trafficking, but also CCL2-enhanced WT1-responsiveness. PMID:23441216

Invariant NKT Cell Response to Dengue Virus Infection in Human

PubMed Central

Matangkasombut, Ponpan; Chan-in, Wilawan; Opasawaschai, Anunya; Pongchaikul, Pisut; Tangthawornchaikul, Nattaya; Vasanawathana, Sirijitt; Limpitikul, Wannee; Malasit, Prida; Duangchinda, Thaneeya; Screaton, Gavin; Mongkolsapaya, Juthathip

2014-01-01

Background Dengue viral infection is a global health threat without vaccine or specific treatment. The clinical outcome varies from asymptomatic, mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF). While adaptive immune responses were found to be detrimental in the dengue pathogenesis, the roles of earlier innate events remain largely uninvestigated. Invariant natural killer T (iNKT) cells represent innate-like T cells that could dictate subsequent adaptive response but their role in human dengue virus infection is not known. We hypothesized that iNKT cells play a role in human dengue infection. Methods Blood samples from a well-characterized cohort of children with DF, DHF, in comparison to non-dengue febrile illness (OFI) and healthy controls at various time points were studied. iNKT cells activation were analyzed by the expression of CD69 by flow cytometry. Their cytokine production was then analyzed after α-GalCer stimulation. Further, the CD1d expression on monocytes, and CD69 expression on conventional T cells were measured. Results iNKT cells were activated during acute dengue infection. The level of iNKT cell activation associates with the disease severity. Furthermore, these iNKT cells had altered functional response to subsequent ex vivo stimulation with α-GalCer. Moreover, during acute dengue infection, monocytic CD1d expression was also upregulated and conventional T cells also became activated. Conclusion iNKT cells might play an early and critical role in the pathogenesis of severe dengue viral infection in human. Targeting iNKT cells and CD1d serve as a potential therapeutic strategy for severe dengue infection in the future. PMID:24945350

Immunoregulatory effects of human dental pulp-derived stem cells on T cells: comparison of transwell co-culture and mixed lymphocyte reaction systems.

PubMed

Demircan, Pinar Cetinalp; Sariboyaci, Ayla Eker; Unal, Zehra Seda; Gacar, Gulcin; Subasi, Cansu; Karaoz, Erdal

2011-11-01

BACKGROUND AIMS. Studies performed using human and animal models have indicated the immunoregulatory capability of mesenchymal stromal cells in several lineages. We investigated whether human dental pulp-derived stem cells (hDP-SC) have regulatory effects on phytohemagglutinin (PHA)-activated CD3(+) T cells. We aimed to define the regulatory mechanisms associated with hDP-SC that occur in mixed lymphocyte reaction (MLR) and transwell systems with PHA-CD3(+) T cells and hDP-SC at a ratio of 1:1. METHODS. Proliferation, apoptosis and pro- and anti-inflammatory cytokines of PHA-CD3(+)T cells, the expression of Regulatory T cells (Treg) markers and some regulatory factors related to hDP-SC, were studied in Both transwell and MLR are co-cultures systems. RESULTS. Anti-proliferative and apoptotic effects of hDP-SC were determined in co-culture systems. Elevated expression levels of human leukocyte antigen (HLA)-G, hepatocyte growth factor (HGF)-β1, intracellular adhesion molecule (ICAM-1)-1, interleukin (IL)-6, IL-10, transforming growth factor (TGF)-β1, vascular adhesion molecule (VCAM)-1 and vascular endothelial growth factor (VEGF) by hDP-SC were detected in the co-culture systems. We observed decreased expression levels of pro-inflammatory cytokines [interferon (IFN)-γ, IL-2, IL-6 receptor (R), IL-12, Interleukin-17A (IL-17A), tumor necrosis factor (TNF)-α] and increased expression levels of anti-inflammatory cytokine [inducible protein (IP)-10] from PHA-CD3(+) T cells in the transwell system. Expression of Treg (CD4(+) CD25(+) Foxp3(+)) markers was significantly induced by hDP-SC in both co-culture systems. We observed apoptosis of PHA-CD3(+) T cells with 24 h using time-lapse camera photographs and active caspase labeling; it is likely that paracrine soluble factors and molecular signals secreted by hDP-SC led this apoptosis. CONCLUSIONS. We suggest that hDP-SC have potent immunoregulatory functions because of their soluble factors and cytokines via paracrine mechanisms associated with PHA-CD3(+) T cells, which could contribute to clinical therapies.

γ/δ T cell subsets in human aging using the classical α/β T cell model.

PubMed

Vasudev, Anusha; Ying, Crystal Tan Tze; Ayyadhury, Shamini; Puan, Kia Joo; Andiappan, Anand Kumar; Nyunt, Ma Shwe Zin; Shadan, Nurhidaya Binte; Mustafa, Seri; Low, Ivy; Rotzschke, Olaf; Fulop, Tamas; Ng, Tze Pin; Larbi, Anis

2014-10-01

Aging is associated with an increased susceptibility to infections and diseases. It has also been associated with reduced functionality and altered distribution of immune cells, especially T cells. Whereas classical α/β T cells, especially CD8(+) T cells, were shown to be highly susceptible to aging, the effects of viral persistent stimulations on the fate of γ/δ T cells are much less documented. Healthy, elderly individuals of Chinese ethnical background were recruited under the aegis of SLAS-II. In this observational study, γ/δ T cell populations were characterized by flow cytometry and compared with the α/β CD4(+) and CD8(+) T cells in elderly and young controls. In our study, we identified a reduced frequency of γ/δ T cells but not α/β T cells with aging. The classical markers of α/β T cell aging, including CD28, CD27, and CD57, did not prove significant for γ/δ T cells. The extreme range of expression of these markers in γ/δ T cells was responsible for the lack of relationship between γ/δ T cell subsets, CD4/CD8 ratio, and anti-CMV titers that was significant for α/β T cells and, especially, CD8(+) T cells. Although markers of aging for γ/δ T cells are not clearly identified, our data collectively suggest that the presence of CD27 γ/δ T cells is associated with markers of α/β T cell aging. © 2014 Society for Leukocyte Biology.

New strategies for allergen T cell epitope identification: going beyond IgE

PubMed Central

Schulten, Véronique; Peters, Bjoern; Sette, Alessandro

2014-01-01

Background Type I allergy and allergic asthma are common diseases in the developed world associated with IgE antibodies and Th2 cell reactivity. To date, the only causative treatment for allergic disease is specific immunotherapy (SIT). Method Here, we review recent works from our laboratory focused on identifying human T cell epitopes associated with allergic disease and their potential use as biomarkers or therapeutic targets for SIT. In previous studies, we have mapped T cell epitopes associated with the major ten Timothy grass (Tg) allergens, defined on the basis of human IgE reactivity by ELISPOT. Results Interestingly, in about 33% of allergic donors no T cell epitopes from overlapping peptides spanning the entire sequences of these allergens were identified, despite vigorous T cell responses to the Tg extract. Using a bioinformatics-proteomic approach, we identified a set of 93 novel Tg proteins, many of which were found to elicit IL-5 production in T cells from allergic donors despite lacking IgE reactivity. Next, we assessed T cell responses to the novel Tg proteins in donors who had been treated with subcutaneous specific immunotherapy (SCIT). A subset of these proteins showed a strong reduction of IL-5 responses in donors who had received SCIT compared to allergic donors, which correlated with patient's self-reported improvement of allergic symptoms. Conclusion A bioinformatics-proteomic approach has successfully identified additional Tg-derived T cell targets independent of IgE reactivity. This method can be applied to other allergies potentially leading to the discovery of promising therapeutic targets for allergen-specific immunotherapy. PMID:25402674

The effect of convection on infrared detection by antennal warm cells in the bloodsucking bug Rhodnius prolixus

PubMed Central

Tichy, Harald

2015-01-01

Previous work revealed that bloodsucking bugs can discriminate between oscillating changes in infrared (IR) radiation and air temperature (T) using two types of warm cells located in peg-in-pit sensilla and tapered hairs (Zopf LM, Lazzari CR, Tichy H. J Neurophysiol 111: 1341–1349, 2014). These two stimuli are encoded and discriminated by the response quotient of the two warm cell types. IR radiation stimulates the warm cell in the peg-in-pit sensillum more strongly than that in the tapered hair. T stimuli evoke the reverse responses; they stimulate the latter more strongly than the former. In nature, IR and T cues are always present with certain radiation intensities and air temperatures, here referred to as background IR radiation and background T. In this article, we found that the response quotient permits the discrimination of IR and T oscillations even in the presence of different backgrounds. We show that the two warm cells respond well to IR oscillations if the background T operates by natural convection but poorly at forced convection, even if the background T is higher than at natural convection. Background IR radiation strongly affects the responses to T oscillations: the discharge rates of both warm cells are higher the higher the power of the IR background. We compared the warm cell responses with the T measured inside small model objects shaped like a cylinder, a cone, or a disc. The experiments indicate that passive thermal effects of the sense organs rather than intrinsic properties of the sensory cells are responsible for the observed results. PMID:25609113

Prevalence of human T-cell lymphotropic virus types 1 and 2 in blood donors of the Caruaru Blood Center (Hemope)

PubMed Central

de Lima, Waleska Mayara Gomes; Esteves, Fabrício Andrade Martins; Torres, Maria do Carmo Morais Rodrigues; Pires, Edna Suely Feitosa

2013-01-01

Background There is difficulty in gathering data on the prevalence of human T-cell lymphotropic virus in blood donors as confirmatory testing is not mandatory in Brazil. This suggests there may be an underreporting of the prevalence. Objective To estimate the prevalence of human T-cell lymphotropic virus types 1 and 2 in donors of a blood bank in Caruaru, Brazil. Methods This was an observational, epidemiological, descriptive, longitudinal and retrospective study with information about the serology of donors of the Caruaru Blood Center, Fundação de Hematologia e Hemoterapia de Pernambuco (Hemope) from May 2006 to December 2010. The data were analyzed using the Excel 2010 computer program (Microsoft Office®). Results Of 61,881 donors, 60 (0.096%) individuals were identified as potential carriers of human T-cell lymphotropic virus types 1 and 2. Of these, 28 (0.045%) were positive and 32 (0.051%) had inconclusive results in the serological screening. Forty-five (0.072%) were retested; 17 were positive (0.027%) and 3 inconclusive (0.005%). After confirmatory tests, 8 were positive (0.013%). Six (75%) of the confirmed cases were women. Conclusion Epidemiological surveys like this are very important in order to create campaigns to attract donors and reduce the costs of laboratory tests. PMID:24106445

A single point mutation in cyclin T1 eliminates binding to Hexim1, Cdk9 and RNA but not to AFF4 and enforces repression of HIV transcription

PubMed Central

2014-01-01

Background Human immunodeficiency virus (HIV) gene expression is primarily regulated at the step of transcription elongation. The viral Tat protein recruits the Positive Transcription Elongation Factor b (P-TEFb) and the Super Elongation Complex (SEC) to the HIV promoter and enhances transcription by host RNA polymerase II. Results To map residues in the cyclin box of cyclin T1 that mediate the binding of P-TEFb to its interacting host partners and support HIV transcription, a pool of N-terminal cyclin T1 mutants was generated. Binding and functional assays in cells identified specific positions in cyclin T1 that are important for (i) association of P-TEFb with Hexim1, Cdk9 and SEC/AFF4 (ii) supporting Tat-transactivation in murine cells and (iii) inhibition of basal and Tat-dependent HIV transcription in human cells. Significantly, a unique cyclin T1 mutant where a Valine residue at position 107 was mutated to Glutamate (CycT1-V107E) was identified. CycT1-V107E did not bind to Hexim1 or Cdk9, and also could not assemble on HIV TAR or 7SK-snRNA. However, it bound strongly to AFF4 and its association with HIV Tat was slightly impaired. CycT1-V107E efficiently inhibited HIV replication in human T cell lines and in CD4(+) primary cells, and enforced HIV transcription repression in T cell lines that harbor a transcriptionally silenced integrated provirus. Conclusions This study outlines the mechanism by which CycT1-V107E mutant inhibits HIV transcription and enforces viral latency. It defines the importance of N-terminal residues of cyclin T1 in mediating contacts of P-TEFb with its transcription partners, and signifies the requirement of a functional P-TEFb and SEC in mediating HIV transcription. PMID:24985467

Proof-of-Principle for Immune Control of Global HIV-1 Reactivation In Vivo

PubMed Central

Smith, Nicola M. G.; Mlcochova, Petra; Watters, Sarah A.; Aasa-Chapman, Marlene M. I.; Rabin, Neil; Moore, Sally; Edwards, Simon G.; Garson, Jeremy A.; Grant, Paul R.; Ferns, R. Bridget; Kashuba, Angela; Mayor, Neema P.; Schellekens, Jennifer; Marsh, Steven G. E.; McMichael, Andrew J.; Perelson, Alan S.; Pillay, Deenan; Goonetilleke, Nilu; Gupta, Ravindra K.

2015-01-01

Background. Emerging data relating to human immunodeficiency virus type 1 (HIV-1) cure suggest that vaccination to stimulate the host immune response, particularly cytotoxic cells, may be critical to clearing of reactivated HIV-1–infected cells. However, evidence for this approach in humans is lacking, and parameters required for a vaccine are unknown because opportunities to study HIV-1 reactivation are rare. Methods. We present observations from a HIV-1 elite controller, not treated with combination antiretroviral therapy, who experienced viral reactivation following treatment for myeloma with melphalan and autologous stem cell transplantation. Mathematical modeling was performed using a standard viral dynamic model. Enzyme-linked immunospot, intracellular cytokine staining, and tetramer staining were performed on peripheral blood mononuclear cells; in vitro CD8 T-cell–mediated control of virion production by autologous CD4 T cells was quantified; and neutralizing antibody titers were measured. Results. Viral rebound was measured at 28 000 copies/mL on day 13 post-transplant before rapid decay to <50 copies/mL in 2 distinct phases with t1/2 of 0.71 days and 4.1 days. These kinetics were consistent with an expansion of cytotoxic effector cells and killing of productively infected CD4 T cells. Following transplantation, innate immune cells, including natural killer cells, recovered with virus rebound. However, most striking was the expansion of highly functional HIV-1–specific cytotoxic CD8 T cells, at numbers consistent with those applied in modeling, as virus control was regained. Conclusions. These observations provide evidence that the human immune response is capable of controlling coordinated global HIV-1 reactivation, remarkably with potency equivalent to combination antiretroviral therapy. These data will inform design of vaccines for use in HIV-1 curative interventions. PMID:25778749

CD1b-autoreactive T cells contribute to hyperlipidemia-induced skin inflammation in mice

PubMed Central

Bagchi, Sreya; He, Ying; Zhang, Hong; Cao, Liang; Van Rhijn, Ildiko; Moody, D. Branch; Gudjonsson, Johann E.

2017-01-01

A large proportion of human T cells are autoreactive to group 1 CD1 proteins, which include CD1a, CD1b, and CD1c. However, the physiological role of the CD1 proteins remains poorly defined. Here, we have generated a double-transgenic mouse model that expresses human CD1b and CD1c molecules (hCD1Tg) as well as a CD1b-autoreactive TCR (HJ1Tg) in the ApoE-deficient background (hCD1Tg HJ1Tg Apoe–/– mice) to determine the role of CD1-autoreactive T cells in hyperlipidemia-associated inflammatory diseases. We found that hCD1Tg HJ1Tg Apoe–/– mice spontaneously developed psoriasiform skin inflammation characterized by T cell and neutrophil infiltration and a Th17-biased cytokine response. Anti–IL-17A treatment ameliorated skin inflammation in vivo. Additionally, phospholipids and cholesterol preferentially accumulated in diseased skin and these autoantigens directly activated CD1b-autoreactive HJ1 T cells. Furthermore, hyperlipidemic serum enhanced IL-6 secretion by CD1b+ DCs and increased IL-17A production by HJ1 T cells. In psoriatic patients, the frequency of CD1b-autoreactive T cells was increased compared with that in healthy controls. Thus, this study has demonstrated the pathogenic role of CD1b-autoreactive T cells under hyperlipidemic conditions in a mouse model of spontaneous skin inflammation. As a large proportion of psoriatic patients are dyslipidemic, this finding is of clinical significance and indicates that self-lipid–reactive T cells might serve as a possible link between hyperlipidemia and psoriasis. PMID:28463230

Telomere length dynamics in human memory T cells specific for viruses causing acute or latent infections

PubMed Central

2013-01-01

Background Declining telomere length (TL) is associated with T cell senescence. While TL in naïve and memory T cells declines with increasing age, there is limited data on TL dynamics in virus-specific memory CD4+ T cells in healthy adults. We combined BrdU-labeling of virus-stimulated T cells followed with flow cytometry-fluorescent in situ hybridization for TL determination. We analyzed TL in T cells specific for several virus infections: non-recurring acute (vaccinia virus, VACV), recurring-acute (influenza A virus, IAV), and reactivating viruses (varicella-zoster virus, VZV, and cytomegalovirus, CMV) in 10 healthy subjects. Additionally, five subjects provided multiple blood samples separated by up to 10 years. Results VACV- and CMV-specific T cells had longer average TL than IAV-specific CD4+ T cells. Although most virus-specific cells were CD45RA-, we observed a minor population of BrdU+ CD45RA+ T cells characterized by long telomeres. Longitudinal analysis demonstrated a slow decline in average TL in virus-specific T cells. However, in one subject, VZV reactivation led to an increase in average TL in VZV-specific memory T cells, suggesting a conversion of longer TL cells from the naïve T cell repertoire. Conclusions TLs in memory CD4+ T cells in otherwise healthy adults are heterogeneous and follow distinct virus-specific kinetics. These findings suggests that the distribution of TL and the creation and maintenance of long TL memory T cells could be important for the persistence of long-lived T cell memory. PMID:23971624

Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival

PubMed Central

Datta, Antara; Silverman, Lee; Phipps, Andrew J; Hiraragi, Hajime; Ratner, Lee; Lairmore, Michael D

2007-01-01

Background Human T-lymphotropic virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma and is linked to a number of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13 and p30, whose roles are still being defined in the virus life cycle and in HTLV-1 virus-host cell interactions. Proviral clones of HTLV-1 with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. p30 expressed exogenously differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and while acting as a repressor of many genes including Tax, in part by blocking tax/rex RNA nuclear export, selectively enhances key gene pathways involved in T-cell signaling/activation. Results Herein, we analyzed the role of p30 in cell cycle regulation. Jurkat T-cells transduced with a p30 expressing lentivirus vector accumulated in the G2-M phase of cell cycle. We then analyzed key proteins involved in G2-M checkpoint activation. p30 expression in Jurkat T-cells resulted in an increase in phosphorylation at serine 216 of nuclear cell division cycle 25C (Cdc25C), had enhanced checkpoint kinase 1 (Chk1) serine 345 phosphorylation, reduced expression of polo-like kinase 1 (PLK1), diminished phosphorylation of PLK1 at tyrosine 210 and reduced phosphorylation of Cdc25C at serine 198. Finally, primary human lymphocyte derived cell lines immortalized by a HTLV-1 proviral clone defective in p30 expression were more susceptible to camptothecin induced apoptosis. Collectively these data are consistent with a cell survival role of p30 against genotoxic insults to HTLV-1 infected lymphocytes. Conclusion Collectively, our data are the first to indicate that HTLV-1 p30 expression results in activation of the G2-M cell cycle checkpoint, events that would promote early viral spread and T-cell survival. PMID:17634129

Expression of miR-17-92 enhances anti-tumor activity of T-cells transduced with the anti-EGFRvIII chimeric antigen receptor in mice bearing human GBM xenografts

PubMed Central

2013-01-01

Background Expression of miR-17-92 enhances T-cell survival and interferon (IFN)-γ production. We previously reported that miR-17-92 is down-regulated in T-cells derived from glioblastoma (GBM) patients. We hypothesized that transgene-derived co-expression of miR17-92 and chimeric antigen receptor (CAR) in T-cells would improve the efficacy of adoptive transfer therapy against GBM. Methods We constructed novel lentiviral vectors for miR-17-92 (FG12-EF1a-miR-17/92) and a CAR consisting of an epidermal growth factor receptor variant III (EGFRvIII)-specific, single-chain variable fragment (scFv) coupled to the T-cell receptor CD3ζ chain signaling module and co-stimulatory motifs of CD137 (4-1BB) and CD28 in tandem (pELNS-3C10-CAR). Human T-cells were transduced with these lentiviral vectors, and their anti-tumor effects were evaluated both in vitro and in vivo. Results CAR-transduced T-cells (CAR-T-cells) exhibited potent, antigen-specific, cytotoxic activity against U87 GBM cells that stably express EGFRvIII (U87-EGFRvIII) and, when co-transduced with miR-17-92, exhibited improved survival in the presence of temozolomide (TMZ) compared with CAR-T-cells without miR-17-92 co-transduction. In mice bearing intracranial U87-EGFRvIII xenografts, CAR-T-cells with or without transgene-derived miR-17-92 expression demonstrated similar levels of therapeutic effect without demonstrating any uncontrolled growth of CAR-T-cells. However, when these mice were re-challenged with U87-EGFRvIII cells in their brains, mice receiving co-transduced CAR-T-cells exhibited improved protection compared with mice treated with CAR-T-cells without miR-17-92 co-transduction. Conclusion These results warrant the development of novel CAR-T-cell strategies that incorporate miR-17-92 to improve therapeutic potency, especially in patients with GBM. PMID:24829757

The effect of convection on infrared detection by antennal warm cells in the bloodsucking bug Rhodnius prolixus.

PubMed

Tichy, Harald; Zopf, Lydia M

2015-04-01

Previous work revealed that bloodsucking bugs can discriminate between oscillating changes in infrared (IR) radiation and air temperature (T) using two types of warm cells located in peg-in-pit sensilla and tapered hairs (Zopf LM, Lazzari CR, Tichy H. J Neurophysiol 111: 1341-1349, 2014). These two stimuli are encoded and discriminated by the response quotient of the two warm cell types. IR radiation stimulates the warm cell in the peg-in-pit sensillum more strongly than that in the tapered hair. T stimuli evoke the reverse responses; they stimulate the latter more strongly than the former. In nature, IR and T cues are always present with certain radiation intensities and air temperatures, here referred to as background IR radiation and background T. In this article, we found that the response quotient permits the discrimination of IR and T oscillations even in the presence of different backgrounds. We show that the two warm cells respond well to IR oscillations if the background T operates by natural convection but poorly at forced convection, even if the background T is higher than at natural convection. Background IR radiation strongly affects the responses to T oscillations: the discharge rates of both warm cells are higher the higher the power of the IR background. We compared the warm cell responses with the T measured inside small model objects shaped like a cylinder, a cone, or a disc. The experiments indicate that passive thermal effects of the sense organs rather than intrinsic properties of the sensory cells are responsible for the observed results. Copyright © 2015 the American Physiological Society.

Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

PubMed Central

Unzueta, Ugutz; Céspedes, María Virtudes; Ferrer-Miralles, Neus; Casanova, Isolda; Cedano, Juan; Corchero, José Luis; Domingo-Espín, Joan; Villaverde, Antonio; Mangues, Ramón; Vázquez, Esther

2012-01-01

Background Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4) is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing. Results Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer. Conclusion Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles, and imaging agents. PMID:22923991

Development and Preclinical Application of an Immunocompetent Transplant Model of Basal Breast Cancer with Lung, Liver and Brain Metastases

PubMed Central

Hoenerhoff, Mark; Hixon, Julie A.; Durum, Scott K.; Qiu, Ting-hu; He, Siping; Burkett, Sandra; Liu, Zi-Yao; Swanson, Steven M.; Green, Jeffrey E.

2016-01-01

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is associated with a poor prognosis and for which no targeted therapies currently exist. In order to improve preclinical testing for TNBC that relies primarily on using human xenografts in immunodeficient mice, we have developed a novel immunocompetent syngeneic murine tumor transplant model for basal-like triple-negative breast cancer. The C3(1)/SV40-T/t-antigen (C3(1)/Tag) mouse mammary tumor model in the FVB/N background shares important similarities with human basal-like TNBC. However, these tumors or derived cell lines are rejected when transplanted into wt FVB/N mice, likely due to the expression of SV40 T-antigen. We have developed a sub-line of mice (designated REAR mice) that carry only one copy of the C3(1)/Tag-antigen transgene resulting from a spontaneous transgene rearrangement in the original founder line. Unlike the original C3(1)/Tag mice, REAR mice do not develop mammary tumors or other phenotypes observed in the original C3(1)/Tag transgenic mice. REAR mice are more immunologically tolerant to SV40 T-antigen driven tumors and cell lines in an FVB/N background (including prostate tumors from TRAMP mice), but are otherwise immunologically intact. This transplant model system offers the ability to synchronously implant the C3(1)/Tag tumor-derived M6 cell line or individual C3(1)/Tag tumors from various stages of tumor development into the mammary fat pads or tail veins of REAR mice. C3(1)/Tag tumors or M6 cells implanted into the mammary fat pads spontaneously metastasize at a high frequency to the lung and liver. M6 cells injected by tail vein can form brain metastases. We demonstrate that irradiated M6 tumor cells or the same cells expressing GM-CSF can act as a vaccine to retard tumor growth of implanted tumor cells in the REAR model. Preclinical studies performed in animals with an intact immune system should more authentically replicate treatment responses in human patients. PMID:27171183

High Frequency, Sustained T Cell Responses to PARV4 Suggest Viral Persistence In Vivo

PubMed Central

Simmons, Ruth; Sharp, Colin; Sims, Stuart; Kloverpris, Henrik; Goulder, Philip; Simmonds, Peter; Bowness, Paul; Klenerman, Paul

2011-01-01

Background. Parvovirus 4 (PARV4) is a recently identified human virus that has been found in livers of patients infected with hepatitis C virus (HCV) and in bone marrow of individuals infected with human immunodeficiency virus (HIV). T cells are important in controlling viruses but may also contribute to disease pathogenesis. The interaction of PARV4 with the cellular immune system has not been described. Consequently, we investigated whether T cell responses to PARV4 could be detected in individuals exposed to blood-borne viruses. Methods. Interferon γ (IFN-γ) enzyme-linked immunospot assay, intracellular cytokine staining, and a tetrameric HLA-A*0201–peptide complex were used to define the lymphocyte populations responding to PARV4 NS peptides in 88 HCV-positive and 13 HIV-positive individuals. Antibody responses were tested using a recently developed PARV4 enzyme-linked immunosorbent assay. Results. High-frequency T cell responses against multiple PARV4 NS peptides and antibodies were observed in 26% of individuals. Typical responses to the NS pools were >1000 spot-forming units per million peripheral blood mononuclear cells. Conclusions. PARV4 infection is common in individuals exposed to blood-borne viruses and elicits strong T cell responses, a feature typically associated with persistent, contained infections such as cytomegalovirus. Persistence of PARV4 viral antigen in tissue in HCV-positive and HIV-positive individuals and/or the associated activated antiviral T cell response may contribute to disease pathogenesis. PMID:21502079

Intermuscular and perimuscular fat expansion in obesity correlates with skeletal muscle T cell and macrophage infiltration and insulin resistance

PubMed Central

Khan, Ilvira M.; Dai Perrard, Xiao-Yuan; Brunner, Gerd; Lui, Hua; Sparks, Lauren M.; Smith, Steven R.; Wang, Xukui; Shi, Zheng-Zheng; Lewis, Dorothy E.; Wu, Huaizhu; Ballantyne, Christie M.

2015-01-01

Background/Objectives Limited numbers of studies demonstrated obesity-induced macrophage infiltration in skeletal muscle (SM), but dynamics of immune cell accumulation and contribution of T cells to SM insulin resistance are understudied. Subjects/Methods T cells and macrophage markers were examined in SM of obese humans by RT-PCR. Mice were fed high-fat diet (HFD) for 2–24 weeks, and time course of macrophage and T cell accumulation was assessed by flow cytometry and quantitative RT-PCR. Extramyocellular adipose tissue (EMAT) was quantified by high-resolution micro-CT, and correlation to T cell number in SM was examined. CD11a−/− mice and C57BL/6 mice were treated with CD11a-neutralizing antibody to determine the role of CD11a in T cell accumulation in SM. To investigate the involvement JAK/STAT, the major pathway for T helper I (TH1) cytokine IFNγ? in SM and adipose tissue inflammation and insulin resistance, mice were treated with a JAK1/JAK2 inhibitor, baricitinib. Results Macrophage and T cells markers were upregulated in SM of obese compared with lean humans. SM of obese mice had higher expression of inflammatory cytokines, with macrophages increasing by 2 weeks on HFD and T cells increasing by 8 weeks. The immune cells were localized in EMAT. Micro-CT revealed that EMAT expansion in obese mice correlated with T cell infiltration and insulin resistance. Deficiency or neutralization of CD11a reduced T cell accumulation in SM of obese mice. T cells polarized into a proinflammatory TH1 phenotype, with increased STAT1 phosphorylation in SM of obese mice. In vivo inhibition of JAK/STAT pathway with baricitinib reduced T cell numbers and activation markers in SM and adipose tissue and improved insulin resistance in obese mice. Conclusions Obesity-induced expansion of EMAT in SM was associated with accumulation and proinflammatory polarization of T cells, which may regulate SM metabolic functions through paracrine mechanisms. Obesity-associated SM “adiposopathy” may thus play an important role in development of insulin resistance and inflammation. PMID:26041698

CD4+ T cells spontaneously producing human immunodeficiency virus type I in breast milk from women with or without antiretroviral drugs

PubMed Central

2011-01-01

Background Transmission of human immunodeficiency virus type 1 (HIV-1) through breast-feeding may involve both cell-free and cell-associated virus. This latter viral reservoir remains, however, to be fully explored. CD4+ T cell-associated virus production in breast milk was therefore investigated. Methods The ex vivo spontaneous production of HIV-1 antigen and HIV-1 RNA by CD4+ T cells was measured in paired blood and breast milk samples from 15 HIV-1 infected women treated or not with antiretroviral drugs. Spontaneous antigen secreting cells (HIV-1-AgSCs) from breast milk and blood were enumerated by an ELISpot assay, and cell-associated HIV-1 RNA was quantified by real-time PCR in supernatants of CD4+ T cells cultured for 18 hours without addition of polyclonal activators. Results Among the CD4+ T cells present in breast milk, memory cells expressing high levels of cell-surface activation markers were predominant. Spontaneous HIV-1-AgSCs were detected and enumerated in the breast milk of all 15 women, with a median number of 13.0 and 9.5 HIV-1- AgSCs/106 CD4+ T cells in aviremic (n = 7) and viremic (n = 8) women, respectively. Cell- associated HIV-1 RNA was detected in cell-free supernatants from 4/7 aviremic and 5/8 viremic individuals at median levels of 190 and 245 copies/ml, respectively. Conclusions Activated CD4+ T cells producing HIV-1 are detected in the breast milk of untreated individuals as well as those receiving highly active antiretroviral therapy. This finding strongly suggests that HIV-1 replication occurs in latently infected CD4+ T cells that, upon spontaneous activation, revert to productively infected cells. These cells might be responsible for a residual breast milk transmission despite maternal highly active antiretroviral therapy. PMID:21569457

Occupational exposure to formaldehyde and alterations in lymphocyte subsets

PubMed Central

Hosgood, H. Dean; Zhang, Luoping; Tang, Xiaojiang; Vermeulen, Roel; Hao, Zhenyue; Shen, Min; Qiu, Chuangyi; Ge, Yichen; Hua, Ming; Ji, Zhiying; Li, Senhua; Xiong, Jun; Reiss, Boris; Liu, Songwang; Xin, Kerry X.; Azuma, Mariko; Xie, Yuxuan; Freeman, Laura Beane; Ruan, Xiaolin; Guo, Weihong; Galvan, Noe; Blair, Aaron; Li, Laiyu; Huang, Hanlin; Smith, Martyn T.; Rothman, Nathaniel; Lan, Qing

2012-01-01

Background Formaldehyde is used in many occupational settings, most notably in manufacturing, health care, and embalming. Formaldehyde has been classified as a human carcinogen, but its mechanism of action remains uncertain. Methods We carried out a cross-sectional study of 43 formaldehyde exposed-workers and 51 unexposed age and sex-matched controls in Guangdong, China to study formaldehyde’s early biologic effects. To follow-up our previous report that the total lymphocyte count was decreased in formaldehyde-exposed workers compared to controls, we evaluated each major lymphocyte subset (i.e., CD4+ T cells, CD8+ T cells, natural killer (NK) cells, and B cells) and T cell lymphocyte subset (CD4+ naïve and memory T cells, CD8+ naïve and memory T cells, and regulatory T cells). Linear regression of each subset was used to test for differences between exposed workers and controls, adjusting for potential confounders. Results Total NK cell and T cell counts were about 24% (p=0.037) and 16% (p=0.0042) lower, respectively, among exposed workers. Among certain T cell subsets, decreased counts among exposed workers were observed for CD8+ T cells (p=0.026), CD8+ effector memory T cells (p=0.018), and regulatory T cells (CD4+FoxP3+: p=0.04; CD25+FoxP3+: p=0.008). Conclusions Formaldehyde exposed-workers experienced decreased counts of NK cells, regulatory T cells, and CD8+ effector memory T cells; however, due to the small sample size these findings need to be confirmed in larger studies. PMID:22767408

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

PubMed Central

2009-01-01

Background Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. Methods A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Results Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene. Conclusion These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency. PMID:20042112

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

Khadjavi, Amina; Magnetto, Chiara; Panariti, Alice

Background: : In chronic wounds, efficient epithelial tissue repair is hampered by hypoxia, and balances between the molecules involved in matrix turn-over such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are seriously impaired. Intriguingly, new oxygenating nanocarriers such as 2H,3H-decafluoropentane-based oxygen-loaded nanodroplets (OLNs) might effectively target chronic wounds. Objective: : To investigate hypoxia and chitosan-shelled OLN effects on MMP/TIMP production by human keratinocytes. Methods: : HaCaT cells were treated for 24 h with 10% v/v OLNs both in normoxia or hypoxia. Cytotoxicity and cell viability were measured through biochemical assays; cellular uptake by confocal microscopy; and MMPmore » and TIMP production by enzyme-linked immunosorbent assay or gelatin zymography. Results: : Normoxic HaCaT cells constitutively released MMP-2, MMP-9, TIMP-1 and TIMP-2. Hypoxia strongly impaired MMP/TIMP balances by reducing MMP-2, MMP-9, and TIMP-2, without affecting TIMP-1 release. After cellular uptake by keratinocytes, nontoxic OLNs abrogated all hypoxia effects on MMP/TIMP secretion, restoring physiological balances. OLN abilities were specifically dependent on time-sustained oxygen diffusion from OLN core. Conclusion: : Chitosan-shelled OLNs effectively counteract hypoxia-dependent dysregulation of MMP/TIMP balances in human keratinocytes. Therefore, topical administration of exogenous oxygen, properly encapsulated in nanodroplet formulations, might be a promising adjuvant approach to promote healing processes in hypoxic wounds. - Highlights: • Hypoxia impairs MMP9/TIMP1 and MMP2/TIMP2 balances in HaCaT human keratinocytes. • Chitosan-shelled oxygen-loaded nanodroplets (OLNs) are internalised by HaCaT cells. • OLNs are not toxic to HaCaT cells. • OLNs effectively counteract hypoxia effects on MMP/TIMP balances in HaCaT cells. • OLNs appear as promising and cost-effective therapeutic tools for hypoxic wounds.« less

Curcumin Inhibits CD4+ T Cell Activation, but Augments CD69 Expression and TGF-β1-Mediated Generation of Regulatory T Cells at Late Phase

PubMed Central

Kim, Girak; Jang, Mi Seon; Son, Young Min; Seo, Min Ji; Ji, Sang Yun; Han, Seung Hyun; Jung, In Duk; Park, Yeong-Min; Jung, Hyun Jung; Yun, Cheol-Heui

2013-01-01

Background Curcumin is a promising candidate for a natural medicinal agent to treat chronic inflammatory diseases. Although CD4+ T cells have been implicated in the pathogenesis of chronic inflammation, whether curcumin directly regulates CD4+ T cells has not been definitively established. Here, we showed curcumin-mediated regulation of CD2/CD3/CD28-initiated CD4+ T cell activation in vitro. Methodology/Principal Findings Primary human CD4+ T cells were stimulated with anti-CD2/CD3/CD28 antibody-coated beads as an in vitro surrogate system for antigen presenting cell-T cell interaction and treated with curcumin. We found that curcumin suppresses CD2/CD3/CD28-initiated CD4+ T cell activation by inhibiting cell proliferation, differentiation and cytokine production. On the other hand, curcumin attenuated the spontaneous decline of CD69 expression and indirectly increased expression of CCR7, L-selectin and Transforming growth factor-β1 (TGF-β1) at the late phase of CD2/CD3/CD28-initiated T cell activation. Curcumin-mediated up-regulation of CD69 at late phase was associated with ERK1/2 signaling. Furthermore, TGF-β1 was involved in curcumin-mediated regulation of T cell activation and late-phase generation of regulatory T cells. Conclusions/Significance Curcumin not merely blocks, but regulates CD2/CD3/CD28-initiated CD4+ T cell activation by augmenting CD69, CCR7, L-selectin and TGF-β1 expression followed by regulatory T cell generation. These results suggest that curcumin could directly reduce T cell-dependent inflammatory stress by modulating CD4+ T cell activation at multiple levels. PMID:23658623

Escape is a more common mechanism than avidity reduction for evasion of CD8+ T cell responses in primary human immunodeficiency virus type 1 infection

PubMed Central

2011-01-01

Background CD8+ T cells play an important role in control of viral replication during acute and early human immunodeficiency virus type 1 (HIV-1) infection, contributing to containment of the acute viral burst and establishment of the prognostically-important persisting viral load. Understanding mechanisms that impair CD8+ T cell-mediated control of HIV replication in primary infection is thus of importance. This study addressed the relative extent to which HIV-specific T cell responses are impacted by viral mutational escape versus reduction in response avidity during the first year of infection. Results 18 patients presenting with symptomatic primary HIV-1 infection, most of whom subsequently established moderate-high persisting viral loads, were studied. HIV-specific T cell responses were mapped in each individual and responses to a subset of optimally-defined CD8+ T cell epitopes were followed from acute infection onwards to determine whether they were escaped or declined in avidity over time. During the first year of infection, sequence variation occurred in/around 26/33 epitopes studied (79%). In 82% of cases of intra-epitopic sequence variation, the mutation was confirmed to confer escape, although T cell responses were subsequently expanded to variant sequences in some cases. In contrast, < 10% of responses to index sequence epitopes declined in functional avidity over the same time-frame, and a similar proportion of responses actually exhibited an increase in functional avidity during this period. Conclusions Escape appears to constitute a much more important means of viral evasion of CD8+ T cell responses in acute and early HIV infection than decline in functional avidity of epitope-specific T cells. These findings support the design of vaccines to elicit T cell responses that are difficult for the virus to escape. PMID:21635736

Fragment Length of Circulating Tumor DNA.

PubMed

Underhill, Hunter R; Kitzman, Jacob O; Hellwig, Sabine; Welker, Noah C; Daza, Riza; Baker, Daniel N; Gligorich, Keith M; Rostomily, Robert C; Bronner, Mary P; Shendure, Jay

2016-07-01

Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134-144 bp vs. 167 bp, respectively). Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the BRAF V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132-145 bp vs. 165 bp, respectively). Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the EGFR T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA.

Factors affecting ultraviolet-A photon emission from β-irradiated human keratinocyte cells.

PubMed

Le, M; Mothersill, C E; Seymour, C B; Ahmad, S B; Armstrong, A; Rainbow, A J; McNeill, F E

2015-08-21

The luminescence intensity of 340±5 nm photons emitted from HaCaT (human keratinocyte) cells was investigated using a single-photon-counting system during cellular exposure to (90)Y β-particles. Multiple factors were assessed to determine their influence upon the quantity and pattern of photon emission from β-irradiated cells. Exposure of 1 x 10(4) cells/5 mL to 703 μCi resulted in maximum UVA photoemission at 44.8 x 10(3)±2.5 x 10(3) counts per second (cps) from live HaCaT cells (background: 1-5 cps); a 16-fold increase above cell-free controls. Significant biophoton emission was achieved only upon stimulation and was also dependent upon presence of cells. UVA luminescence was measured for (90)Y activities 14 to 703 μCi where a positive relationship between photoemission and (90)Y activity was observed. Irradiation of live HaCaT cells plated at various densities produced a distinct pattern of emission whereby luminescence increased up to a maximum at 1 x 10(4) cells/5 mL and thereafter decreased. However, this result was not observed in the dead cell population. Both live and dead HaCaT cells were irradiated and were found to demonstrate different rates of photon emission at low β activities (⩽400 μCi). Dead cells exhibited greater photon emission rates than live cells which may be attributable to metabolic processes taking place to modulate the photoemissive effect. The results indicate that photon emission from HaCaT cells is perturbed by external stimulation, is dependent upon the activity of radiation delivered, the density of irradiated cells, and cell viability. It is postulated that biophoton emission may be modulated by a biological or metabolic process.

UVB radiation and human monocyte accessory function: Differential effects on pre-mitotic events in T-cell activation

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

Krutmann, J.K.; Kammer, G.M.; Toossi, Z.

Purified T lymphocytes fail to proliferate in response to antigenic and mitogenic stimuli when cultured in the presence of accessory cells that have been exposed in vitro to sublethal doses of UVB radiation. Because proliferation represents a final stage in the T-cell activation process, the present study was conducted to determine whether T cells were able to progress through any of the pre-mitotic stages when UVB-irradiated monocytes were used as model accessory cells. In these experiments, monoclonal anti-CD3 antibodies were employed as the mitogenic stimulus. Culture of T cells with UVB-irradiated monocytes did allow the T cells to undergo anmore » increase in intracellular free calcium, which is one of the first steps in the activation sequence. The T cells expressed interleukin-2 receptors, although at a reduced level. However, T cells failed to produce interleukin-2 above background levels when they were placed in culture with monocytes exposed to UVB doses as low as 50 J/m2. Incubation of T cells with UVB-irradiated monocytes did not affect the subsequent capacity of T cells to proliferate, since they developed a normal proliferative response in secondary culture when restimulated with anti-CD3 antibodies and unirradiated monocytes. These studies indicate that T lymphocytes become partially activated when cultured with UVB-irradiated monocytes and mitogenic anti-CD3 monoclonal antibodies. In addition, they suggest that interleukin-2 production is the T-cell activation step most sensitive to inhibition when UVB-irradiated monocytes are employed as accessory cells.« less

A Reproducible Immunopotency Assay to Measure Mesenchymal Stromal Cell Mediated T cell Suppression

PubMed Central

Bloom, Debra D.; Centanni, John M.; Bhatia, Neehar; Emler, Carol A.; Drier, Diana; Leverson, Glen E.; McKenna, David H.; Gee, Adrian P.; Lindblad, Robert; Hei, Derek J.; Hematti, Peiman

2014-01-01

Background The T cell suppressive property of bone marrow derived mesenchymal stromal cells (MSCs) has been considered a major mode of action and basis for their utilization in a number of human clinical trials. However, there is no well-established reproducible assay to measure MSC-mediated T cell suppression. Methods At the University of Wisconsin-Madison Production Assistance for Cellular Therapy (PACT) Center we developed an in vitro quality control T cell suppression immunopotency assay (IPA) which utilizes anti-CD3 and anti-CD28 antibodies to stimulate T cell proliferation. We measured MSC-induced suppression of CD4+ T cell proliferation at various effector to target cell ratios using defined peripheral blood mononuclear cells and in parallel compared to a reference standard MSC product. We calculated an IPA value for suppression of CD4+ T cells for each MSC product. Results Eleven MSC products generated at three independent PACT centers were evaluated for cell surface phenotypic markers and T cell suppressive properties. Flow cytometry results demonstrated typical MSC cell surface marker profiles. There was significant variability in the level of suppression of T cell proliferation with IPA values ranging from 27% to 88%. However, MSC suppression did not correlate with HLA-DR expression. Discussion We have developed a reproducible immunopotency assay to measure allogeneic MSC-mediated suppression of CD4+ T cells. Additional studies may be warranted to determine how these in vitro assay results may correlate with other immunomodulatory properties of MSCs, in addition to evaluating the ability of this assay to predict in vivo efficacy. PMID:25455739

Bone marrow hematopoietic stem cells behavior with or without growth factors in trauma hemorrhagic shock

PubMed Central

Kumar, Manoj; Bhoi, Sanjeev; Mohanty, Sujata; Kamal, Vineet Kumar; Rao, D. N.; Mishra, Pravas; Galwankar, Sagar

2016-01-01

Background: Hemorrhagic shock (HS) is the major leading cause of death after trauma. Up to 50% of early deaths are due to massive hemorrhage. Excessive release of pro-inflammatory cytokine and hypercatecholamine induces hematopoietic progenitor cells (HPCs) apoptosis, leading to multiorgan failure and death. However, still, result remains elusive for hematopoietic stem cells (HSCs) behavior in trauma HS (T/HS). Objectives: Therefore, our aim was to evaluate the in vitro HSCs behavior with or without recombinant human erythropoietin (rhEPO), recombinant human granulocyte macrophage-colony-stimulating factor (rhGM-CSF), recombinant human interleukin-3 (rhIL-3) alone, and combination with rhEPO + rhGM-CSF + rhIL-3 (EG3) in T/HS patients. Methodology: Bone marrow (BM) aspirates (n = 14) were collected from T/HS patients, those survived on day 3. BM cells were cultured for HPCs: Colony-forming unit-erythroid (CFU-E), burst-forming unit-erythroid (BFU-E), and colony-forming unit-granulocyte, monocyte/macrophage colonies growth. HPCs were counted with or without rhEPO, rhGM-CSF, rhIL-3 alone, and combination with EG3 in T/HS patients. Results: BM HSCs growth significantly suppressed in T/HS when compared with control group (P < 0.05). In addition, CFU-E and BFU-E colony growth were increased with additional growth factor (AGF) (rhEPO, rhGM-CSF, and rhIL-3) as compared to baseline (without AGF) (P < 0.05). Conclusion: Suppressed HPCs may be reactivated by addition of erythropoietin, GM-CSF, IL-3 alone and with combination in T/HS. PMID:27722113

Inhibition of highly productive HIV-1 infection in T cells, primary human macrophages, microglia, and astrocytes by Sargassum fusiforme

PubMed Central

Paskaleva, Elena E; Lin, Xudong; Li, Wen; Cotter, Robin; Klein, Michael T; Roberge, Emily; Yu, Er K; Clark, Bruce; Veille, Jean-Claude; Liu, Yanze; Lee, David Y-W; Canki, Mario

2006-01-01

Background The high rate of HIV-1 mutation and increasing resistance to currently available antiretroviral (ART) therapies highlight the need for new antiviral agents. Products derived from natural sources have been shown to inhibit HIV-1 replication during various stages of the virus life cycle, and therefore represent a potential source of novel therapeutic agents. To expand our arsenal of therapeutics against HIV-1 infection, we investigated aqueous extract from Sargassum fusiforme (S. fusiforme) for ability to inhibit HIV-1 infection in the periphery, in T cells and human macrophages, and for ability to inhibit in the central nervous system (CNS), in microglia and astrocytes. Results S. fusiforme extract blocked HIV-1 infection and replication by over 90% in T cells, human macrophages and microglia, and it also inhibited pseudotyped HIV-1 (VSV/NL4-3) infection in human astrocytes by over 70%. Inhibition was mediated against both CXCR4 (X4) and CCR5 (R5)-tropic HIV-1, was dose dependant and long lasting, did not inhibit cell growth or viability, was not toxic to cells, and was comparable to inhibition by the nucleoside analogue 2', 3'-didoxycytidine (ddC). S. fusiforme treatment blocked direct cell-to-cell infection spread. To investigate at which point of the virus life cycle this inhibition occurs, we infected T cells and CD4-negative primary human astrocytes with HIV-1 pseudotyped with envelope glycoprotein of vesicular stomatitis virus (VSV), which bypasses the HIV receptor requirements. Infection by pseudotyped HIV-1 (VSV/NL4-3) was also inhibited in a dose dependant manner, although up to 57% less, as compared to inhibition of native NL4-3, indicating post-entry interferences. Conclusion This is the first report demonstrating S. fusiforme to be a potent inhibitor of highly productive HIV-1 infection and replication in T cells, in primary human macrophages, microglia, and astrocytes. Results with VSV/NL4-3 infection, suggest inhibition of both entry and post-entry events of the virus life cycle. Absence of cytotoxicity and high viability of treated cells also suggest that S. fusiforme is a potential source of novel naturally occurring antiretroviral compounds that inhibit HIV-1 infection and replication at more than one site of the virus life cycle. PMID:16725040

Hyper telomere recombination accelerates replicative senescence and may promote premature aging

PubMed Central

Hagelstrom, R. Tanner; Blagoev, Krastan B.; Niedernhofer, Laura J.; Goodwin, Edwin H.; Bailey, Susan M.

2010-01-01

Werner syndrome and Bloom syndrome result from defects in the RecQ helicases Werner (WRN) and Bloom (BLM), respectively, and display premature aging phenotypes. Similarly, XFE progeroid syndrome results from defects in the ERCC1-XPF DNA repair endonuclease. To gain insight into the origin of cellular senescence and human aging, we analyzed the dependence of sister chromatid exchange (SCE) frequencies on location [i.e., genomic (G-SCE) vs. telomeric (T-SCE) DNA] in primary human fibroblasts deficient in WRN, BLM, or ERCC1-XPF. Consistent with our other studies, we found evidence of elevated T-SCE in telomerase-negative but not telomerase-positive backgrounds. In telomerase-negative WRN-deficient cells, T-SCE—but not G-SCE—frequencies were significantly increased compared with controls. In contrast, SCE frequencies were significantly elevated in BLM-deficient cells irrespective of genome location. In ERCC1-XPF-deficient cells, neither T- nor G-SCE frequencies differed from controls. A theoretical model was developed that allowed an in silico investigation into the cellular consequences of increased T-SCE frequency. The model predicts that in cells with increased T-SCE, the onset of replicative senescence is dramatically accelerated even though the average rate of telomere loss has not changed. Premature cellular senescence may act as a powerful tumor-suppressor mechanism in telomerase-deficient cells with mutations that cause T-SCE levels to rise. Furthermore, T-SCE-driven premature cellular senescence may be a factor contributing to accelerated aging in Werner and Bloom syndromes, but not XFE progeroid syndrome. PMID:20798040

Complex antigen presentation pathway for an HLA-A*0201-restricted epitope from Chikungunya 6K protein

PubMed Central

Lemonnier, François A.; Esteban, Mariano

2017-01-01

Background The adaptive cytotoxic T lymphocyte (CTL)-mediated immune response is critical for clearance of many viral infections. These CTL recognize naturally processed short viral antigenic peptides bound to human leukocyte antigen (HLA) class I molecules on the surface of infected cells. This specific recognition allows the killing of virus-infected cells. The T cell immune T cell response to Chikungunya virus (CHIKV), a mosquito-borne Alphavirus of the Togaviridae family responsible for severe musculoskeletal disorders, has not been fully defined; nonetheless, the importance of HLA class I-restricted immune response in this virus has been hypothesized. Methodology/Principal findings By infection of HLA-A*0201-transgenic mice with a recombinant vaccinia virus that encodes the CHIKV structural polyprotein (rVACV-CHIKV), we identified the first human T cell epitopes from CHIKV. These three novel 6K transmembrane protein-derived epitopes are presented by the common HLA class I molecule, HLA-A*0201. One of these epitopes is processed and presented via a complex pathway that involves proteases from different subcellular locations. Specific chemical inhibitors blocked these events in rVACV-CHIKV-infected cells. Conclusions/Significance Our data have implications not only for the identification of novel Alphavirus and Togaviridae antiviral CTL responses, but also for analyzing presentation of antigen from viruses of different families and orders that use host proteinases to generate their mature envelope proteins. PMID:29084215

Induction of maturation and activation of human dendritic cells: A mechanism underlying the beneficial effect of Viscum album as complimentary therapy in cancer

PubMed Central

Elluru, Sri Ramulu; van Huyen, Jean-Paul Duong; Delignat, Sandrine; Kazatchkine, Michel D; Friboulet, Alain; Kaveri, Srini V; Bayry, Jagadeesh

2008-01-01

Background Viscum album (VA) preparations have been used as a complimentary therapy in cancer. In addition to their cytotoxic properties, they have also been shown to have immunostimulatory properties. In the present study, we examine the hypothesis that the VA preparations induce activation of human DC that facilitates effective tumor regression. Methods Four day old monocyte-derived immature DCs were treated with VA Qu Spez at 5, 10 and 15 μg/ml for 48 hrs. The expression of surface molecules was analyzed by flow cytometry. The ability of Qu Spez-educated DC to stimulate T cells was analyzed by allogeneic mixed lymphocyte reaction and activation of Melan-A/MART-1-specific M77-80 CD8+T cells. Cytokines in cell free culture supernatant was analyzed by cytokine bead array assay. Results VA Qu Spez stimulated DCs presented with increased expression of antigen presenting molecule HLA-DR and of co-stimulatory molecules CD40, CD80 and CD86. The VA Qu Spez also induced the secretion of inflammatory cytokines IL-6 and IL-8. Further, Qu Spez-educated DC stimulated CD4+T cells in a allogeneic mixed lymphocyte reaction and activated melanoma antigen Melan-A/MART-1-specific M77-80 CD8+T cells as evidenced by increased secretion of TNF-α and IFNγ. Conclusion The VA preparations stimulate the maturation and activation of human DCs, which may facilitate anti-tumoral immune responses. These results should assist in understanding the immunostimulatory properties of VA preparations and improving the therapeutic strategies. PMID:18533025

Platelet activation suppresses HIV-1 infection of T cells

PubMed Central

2013-01-01

Background Platelets, anucleate cell fragments abundant in human blood, can capture HIV-1 and platelet counts have been associated with viral load and disease progression. However, the impact of platelets on HIV-1 infection of T cells is unclear. Results We found that platelets suppress HIV-1 spread in co-cultured T cells in a concentration-dependent manner. Platelets containing granules inhibited HIV-1 spread in T cells more efficiently than degranulated platelets, indicating that the granule content might exert antiviral activity. Indeed, supernatants from activated and thus degranulated platelets suppressed HIV-1 infection. Infection was inhibited at the stage of host cell entry and inhibition was independent of the viral strain or coreceptor tropism. In contrast, blockade of HIV-2 and SIV entry was less efficient. The chemokine CXCL4, a major component of platelet granules, blocked HIV-1 entry and neutralization of CXCL4 in platelet supernatants largely abrogated their anti-HIV-1 activity. Conclusions Release of CXCL4 by activated platelets inhibits HIV-1 infection of adjacent T cells at the stage of virus entry. The inhibitory activity of platelet-derived CXCL4 suggests a role of platelets in the defense against infection by HIV-1 and potentially other pathogens. PMID:23634812

Breast Implant-Associated, ALK-Negative, T-Cell, Anaplastic, Large-Cell Lymphoma: Establishment and Characterization of a Model Cell Line (TLBR-1) for This Newly Emerging Clinical Entity

PubMed Central

Lechner, Melissa G.; Lade, Stephen; Liebertz, Daniel J.; Prince, H. Miles; Brody, Garry S.; Webster, Howard R.; Epstein, Alan L.

2014-01-01

BACKGROUND Primary lymphomas of the breast are very rare (0.2–1.5% of breast malignancies) and the vast majority (95%) are of B-cell origin. Recently, 40 cases of clinically indolent anaplastic large-cell kinase (ALK)-negative, T-cell, anaplastic, non-Hodgkin lymphomas (T-ALCL) have been reported worldwide. METHODS A tumor biopsy specimen from a patient in this series was obtained for characterization. By using a human stromal feeder layer and IL-2, a novel cell line, TLBR-1, was established from this biopsy and investigated by using cytogenetics and various biomolecular methods. RESULTS Immunoperoxidase staining of the tumor biopsy showed a CD30/CD8/CD4 coexpressing T-cell population that was epithelial membrane antigen (EMA)+ and perforin+. Multiplex polymerase chain reaction (PCR) of TCRγ genes showed monoclonality that suggested a T-cell origin, yet pan-T markers CD2/5/7, anaplastic large-cell kinase (ALK)-1, pancytokeratins, CD20, CD56, and Epstein-Barr virus (EBV) by in situ hybridization (ISH) were negative. TLBR-1 is IL-2 dependent, has a relatively long doubling time (55 hours), and displays different cellular shapes in culture. Cytogenetic analysis of tumor and TLBR-1 cells confirmed a highly anaplastic cell population with a modal number of 47 chromosomes lacking t(2;5). PCR screens for EBV and human T-lymphotropic virus types 1 and 2 (HTLV-1/2) were negative. Fluorescence-activated cell-sorting (FACS) analysis showed strong positivity for CD4/8, CD30, CD71, and CD26 expression, and antigen presentation (HLA-DR+CD80+CD86+), IL-2 signaling (CD25+CD122+), and NK (CD56+) markers, and Western blots demonstrated strong Notch1 expression. Severe combined immunodeficiency (SCID) mouse TLBR-1 heterotransplants recapitulated the histology and marker characteristics of the original tumor. CONCLUSIONS TLBR-1, a novel ALK-negative, T-cell, anaplastic, large-cell lymphoma, closely resembles the original biopsy and represents an important tool for studying this newly recognized disease entity. PMID:21425149

LKB1 tumor suppressor and salt-inducible kinases negatively regulate human T-cell leukemia virus type 1 transcription

PubMed Central

2013-01-01

Background Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). Treatment options are limited and prophylactic agents are not available. We have previously demonstrated an essential role for CREB-regulating transcriptional coactivators (CRTCs) in HTLV-1 transcription. Results In this study we report on the negative regulatory role of LKB1 tumor suppressor and salt-inducible kinases (SIKs) in the activation of HTLV-1 long terminal repeats (LTR) by the oncoprotein Tax. Activation of LKB1 and SIKs effectively blunted Tax activity in a phosphorylation-dependent manner, whereas compromising these kinases, but not AMP-dependent protein kinases, augmented Tax function. Activated LKB1 and SIKs associated with Tax and suppressed Tax-induced LTR activation by counteracting CRTCs and CREB. Enforced expression of LKB1 or SIK1 in cells transfected with HTLV-1 molecular clone pX1MT repressed proviral transcription. On the contrary, depletion of LKB1 in pX1MT-transfected cells and in HTLV-1-transformed T cells boosted the expression of Tax. Treatment of HTLV-1 transformed cells with metformin led to LKB1/SIK1 activation, reduction in Tax expression, and inhibition of cell proliferation. Conclusions Our findings revealed a new function of LKB1 and SIKs as negative regulators of HTLV-1 transcription. Pharmaceutical activation of LKB1 and SIKs might be considered as a new strategy in anti-HTLV-1 and anti-ATL therapy. PMID:23577667

First step in developing a 3D biodegradable fibrin scaffold for an artificial ovary

PubMed Central

2013-01-01

Background Although transplantation of cryopreserved ovarian tissue is a promising approach to restore fertility in cancer patients, it is not advisable for women at risk of ovarian involvement due to the threat of reintroducing malignant cells. The aim of this study was therefore to find an alternative for these patients by development of an artificial ovary. Methods For construction of the artificial ovary matrix, we used a central composite design to investigate nine combinations of fibrinogen (mg/ml) and thrombin (IU/mL) (F/T): F1/T4, F12.5/T1, F12.5/T20, F25/T0.1, F25/T4, F25/T500, F50/T1, F50/T20 and F100/T4. From the first qualitative analyses (handling and matrix size), five combinations (F12.5/T1, F25/T4, F50/T20, F50/T1 and F100/T4) yielded positive results. They were further evaluated in order to assess fibrin matrix degradation and homogeneous cell encapsulation (density), survival and proliferation (Ki67), and atresia (TUNEL) before and after 7 days of in vitro culture. To determine the best compromise between maximizing the dynamic density (Y1) and minimizing the apoptosis rate (Y2), we used the desirability function approach. Results Two combinations (F12.5/T1 and F25/T4) showed greater distribution of cells before in vitro culture, reproducible degradation of the fibrin network and adequate support for isolated human ovarian stromal cells, with a high proportion of Ki67-positive cells. SEM analysis revealed a network of fibers with regular pores and healthy stromal cells after in vitro culture with both F/T combinations. Conclusion This study reports two optimal F/T combinations that allow survival and proliferation of isolated human ovarian cells. Further studies are required to determine if such a scaffold will also be a suitable environment for isolated ovarian follicles. PMID:24274108

Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids.

PubMed

Seet, Christopher S; He, Chongbin; Bethune, Michael T; Li, Suwen; Chick, Brent; Gschweng, Eric H; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B; Baltimore, David; Crooks, Gay M; Montel-Hagen, Amélie

2017-05-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3 + TCR-αβ + single-positive CD8 + or CD4 + cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.

Osthole induces G2/M arrest and apoptosis in lung cancer A549 cells by modulating PI3K/Akt pathway

PubMed Central

2011-01-01

Background To explore the effects of Osthole on the proliferation, cell cycle and apoptosis of human lung cancer A549 cells. Methods Human lung cancer A549 cells were treated with Osthole at different concentrations. Cell proliferation was measured using the MTT assay. Cell cycle was evaluated using DNA flow cytometry analysis. Induction of apoptosis was determined by flow cytometry and fluorescent microscopy. The expressions of Cyclin B1, p-Cdc2, Bcl-2, Bax, t-Akt and p-Akt were evaluated by Western blotting. Results Osthole inhibited the growth of human lung cancer A549 cells by inducing G2/M arrest and apoptosis. Western blotting demonstrated that Osthole down-regulated the expressions of Cyclin B1, p-Cdc2 and Bcl-2 and up-regulated the expressions of Bax in A549 cells. Inhibition of PI3K/Akt signaling pathway was also observed after treating A549 cells with Osthole. Conclusions Our findings suggest that Osthole may have a therapeutic application in the treatment of human lung cancer. PMID:21447176

Activation of human T cells in hypertension: Studies of Humanized Mice and Hypertensive Humans

PubMed Central

Itani, Hana A.; McMaster, William G.; Saleh, Mohamed A.; Nazarewicz, Rafal R.; Mikolajczyk, Tomasz P.; Kaszuba, Anna; Konior, Anna; Prejbisz, Aleksander; Januszewicz, Andrzej; Norlander, Allison E.; Chen, Wei; Bonami, Rachel H.; Marshall, Andrew F.; Poffenberger, Greg; Weyand, Cornelia M.; Madhur, Meena S.; Moore, Daniel J.; Harrison, David G.; Guzik, Tomasz J.

2016-01-01

Emerging evidence supports an important role for T cells in the genesis of hypertension. Because this work has predominantly been performed in experimental animals, we sought to determine whether human T cells are activated in hypertension. We employed a humanized mouse model in which the murine immune system is replaced by the human immune system. Angiotensin II increased systolic pressure to 162 mm Hg vs. 116 mm Hg for sham treated animals. Flow cytometry of thoracic lymph nodes, thoracic aorta and kidney revealed increased infiltration of human leukocytes (CD45+) and T lymphocytes (CD3+ and CD4+) in response to angiotensin II infusion. Interestingly, there was also an increase in the memory T cells (CD3+/CD45RO+) in the aortas and lymph nodes. Prevention of hypertension using hydralazine and hydrochlorothiazide prevented the accumulation of T cells in these tissues. Studies of isolated human T cells and monocytes indicated that angiotensin II had no direct effect on cytokine production by T cells or the ability of dendritic cells to drive T cell proliferation. We also observed an increase in circulating IL-17A producing CD4+ T cells and both CD4+ and CD8+ T cells that produce IFN-γ in hypertensive compared to normotensive humans. Thus, human T cells become activated and invade critical end-organ tissues in response to hypertension in a humanized mouse model. This response likely reflects the hypertensive milieu encountered in vivo and is not a direct effect of the hormone angiotensin II. PMID:27217403

Activation of Human T Cells in Hypertension: Studies of Humanized Mice and Hypertensive Humans.

PubMed

Itani, Hana A; McMaster, William G; Saleh, Mohamed A; Nazarewicz, Rafal R; Mikolajczyk, Tomasz P; Kaszuba, Anna M; Konior, Anna; Prejbisz, Aleksander; Januszewicz, Andrzej; Norlander, Allison E; Chen, Wei; Bonami, Rachel H; Marshall, Andrew F; Poffenberger, Greg; Weyand, Cornelia M; Madhur, Meena S; Moore, Daniel J; Harrison, David G; Guzik, Tomasz J

2016-07-01

Emerging evidence supports an important role for T cells in the genesis of hypertension. Because this work has predominantly been performed in experimental animals, we sought to determine whether human T cells are activated in hypertension. We used a humanized mouse model in which the murine immune system is replaced by the human immune system. Angiotensin II increased systolic pressure to 162 versus 116 mm Hg for sham-treated animals. Flow cytometry of thoracic lymph nodes, thoracic aorta, and kidney revealed increased infiltration of human leukocytes (CD45(+)) and T lymphocytes (CD3(+) and CD4(+)) in response to angiotensin II infusion. Interestingly, there was also an increase in the memory T cells (CD3(+)/CD45RO(+)) in the aortas and lymph nodes. Prevention of hypertension using hydralazine and hydrochlorothiazide prevented the accumulation of T cells in these tissues. Studies of isolated human T cells and monocytes indicated that angiotensin II had no direct effect on cytokine production by T cells or the ability of dendritic cells to drive T-cell proliferation. We also observed an increase in circulating interleukin-17A producing CD4(+) T cells and both CD4(+) and CD8(+) T cells that produce interferon-γ in hypertensive compared with normotensive humans. Thus, human T cells become activated and invade critical end-organ tissues in response to hypertension in a humanized mouse model. This response likely reflects the hypertensive milieu encountered in vivo and is not a direct effect of the hormone angiotensin II. © 2016 American Heart Association, Inc.

Different features of Vδ2 T and NK cells in fatal and non-fatal human Ebola infections

PubMed Central

Cimini, Eleonora; Viola, Domenico; Cabeza-Cabrerizo, Mar; Romanelli, Antonella; Tumino, Nicola; Sacchi, Alessandra; Bordoni, Veronica; Casetti, Rita; Turchi, Federica; Martini, Federico; Bore, Joseph A.; Koundouno, Fara Raymond; Duraffour, Sophie; Michel, Janine; Holm, Tobias; Zekeng, Elsa Gayle; Cowley, Lauren; Garcia Dorival, Isabel; Doerrbecker, Juliane; Hetzelt, Nicole; Baum, Jonathan H. J.; Portmann, Jasmine; Wölfel, Roman; Gabriel, Martin; Miranda, Osvaldo; Díaz, Graciliano; Díaz, José E.; Fleites, Yoel A.; Piñeiro, Carlos A.; Castro, Carlos M.; Koivogui, Lamine; Magassouba, N’Faly; Diallo, Boubacar; Ruibal, Paula; Oestereich, Lisa; Wozniak, David M.; Lüdtke, Anja; Becker-Ziaja, Beate; Capobianchi, Maria R.; Ippolito, Giuseppe; Carroll, Miles W.; Günther, Stephan; Di Caro, Antonino; Muñoz-Fontela, César

2017-01-01

Background Human Ebola infection is characterized by a paralysis of the immune system. A signature of αβ T cells in fatal Ebola infection has been recently proposed, while the involvement of innate immune cells in the protection/pathogenesis of Ebola infection is unknown. Aim of this study was to analyze γδ T and NK cells in patients from the Ebola outbreak of 2014–2015 occurred in West Africa, and to assess their association with the clinical outcome. Methodology/Principal findings Nineteen Ebola-infected patients were enrolled at the time of admission to the Ebola Treatment Centre in Guinea. Patients were divided in two groups on the basis of the clinical outcome. The analysis was performed by using multiparametric flow cytometry established by the European Mobile Laboratory in the field. A low frequency of Vδ2 T-cells was observed during Ebola infection, independently from the clinical outcome. Moreover, Vδ2 T-cells from Ebola patients massively expressed CD95 apoptotic marker, suggesting the involvement of apoptotic mechanisms in Vδ2 T-cell loss. Interestingly, Vδ2 T-cells from survivors expressed an effector phenotype and presented a lower expression of the CTLA-4 exhaustion marker than fatalities, suggesting a role of effector Vδ2 T-cells in the protection. Furthermore, patients with fatal Ebola infection were characterized by a lower NK cell frequency than patients with non fatal infection. In particular, both CD56bright and CD56dim NK frequency were very low both in fatal and non fatal infections, while a higher frequency of CD56neg NK cells was associated to non-fatal infections. Finally, NK activation and expression of NKp46 and CD158a were independent from clinical outcome. Conclusions/Significances Altogether, the data suggest that both effector Vδ2 T-cells and NK cells may play a role in the complex network of protective response to EBOV infection. Further studies are required to characterize the protective effector functions of Vδ2 and NK cells. PMID:28558022

Fragment Length of Circulating Tumor DNA

PubMed Central

Underhill, Hunter R.; Kitzman, Jacob O.; Hellwig, Sabine; Welker, Noah C.; Daza, Riza; Gligorich, Keith M.; Rostomily, Robert C.; Shendure, Jay

2016-01-01

Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134–144 bp vs. 167 bp, respectively). Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the BRAF V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132–145 bp vs. 165 bp, respectively). Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the EGFR T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA. PMID:27428049

B and T Lymphocyte Attenuator Down-regulation by HIV-1 Depends on Type I Interferon and Contributes to T-Cell Hyperactivation

PubMed Central

Zhang, Zheng; Xu, Xiangsheng; Lu, Jiyun; Zhang, Shuye; Gu, Lanlan; Fu, Junliang; Jin, Lei; Li, Haiying; Zhao, Min; Zhang, Jiyuan; Wu, Hao; Su, Lishan; Fu, Yang-Xin

2011-01-01

Background. Nonspecific T-cell hyperactivation is the main driving force for human immunodeficiency virus (HIV)–1 disease progression, but the reasons why the excess immune response is not properly shut off are poorly defined. Methods. Eighty-five HIV-1–infected individuals were enrolled to characterize B and T lymphocyte attenuator (BTLA) expression and function. Infection and blockade assays were used to dissect the factors that influenced BTLA signaling in vitro. Results. BTLA expression on overall CD4+ and CD8+ T cells was progressively decreased in HIV-1 infection, which was directly correlated with disease progression and CD4+ T-cell differentiation and activation. BTLA+CD4+ T cells from HIV-1–infected patients also displayed an altered immune status, which was indicated by reduced expression of naive markers but increased activation and exhaustion markers. Cross-linking of BTLA can substantially decrease CD4+ T-cell activation in vitro. This responsiveness of CD4+ T cells to BTLA-mediated inhibitory signaling was further found to be impaired in HIV-1–infected patients. Furthermore, HIV-1 NL4-3 down-regulated BTLA expression on CD4+ T cells dependent on plasmacytoid dendritic cell (pDC)-derived interferon (IFN)-α. Blockade of IFN-α or depletion of pDCs prevents HIV-1-induced BTLA down-regulation. Conclusions. HIV-1 infection potentially impairs BTLA-mediated signaling dependent on pDC-derived IFN-α, which may contribute to broad T-cell hyperactivation induced by chronic HIV-1 infection. PMID:21592997

Whole genomic sequence analysis of Bacillus infantis: defining the genetic blueprint of strain NRRL B-14911, an emerging cardiopathogenic microbe

USDA-ARS?s Scientific Manuscript database

Background: We recently reported the identification of Bacillus sp. NRRL B-14911 that induces heart autoimmunity by generating cardiac-reactive T cells through molecular mimicry. This marine bacterium was originally isolated from the Gulf of Mexico, but no associations with human diseases were rep...

Selective destruction of mouse islet beta cells by human T lymphocytes in a newly-established humanized type 1 diabetic model

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

Zhao, Yong, E-mail: yongzhao@uic.edu; Guo, Chengshan; Hwang, David

2010-09-03

Research highlights: {yields} Establish a human immune-mediated type 1 diabetic model in NOD-scid IL2r{gamma}{sup null} mice. {yields} Using the irradiated diabetic NOD mouse spleen mononuclear cells as trigger. {yields} The islet {beta} cells were selectively destroyed by infiltrated human T cells. {yields} The model can facilitate translational research to find a cure for type 1 diabetes. -- Abstract: Type 1 diabetes (T1D) is caused by a T cell-mediated autoimmune response that leads to the loss of insulin-producing {beta} cells. The optimal preclinical testing of promising therapies would be aided by a humanized immune-mediated T1D model. We develop this model inmore » NOD-scid IL2r{gamma}{sup null} mice. The selective destruction of pancreatic islet {beta} cells was mediated by human T lymphocytes after an initial trigger was supplied by the injection of irradiated spleen mononuclear cells (SMC) from diabetic nonobese diabetic (NOD) mice. This resulted in severe insulitis, a marked loss of total {beta}-cell mass, and other related phenotypes of T1D. The migration of human T cells to pancreatic islets was controlled by the {beta} cell-produced highly conserved chemokine stromal cell-derived factor 1 (SDF-1) and its receptor C-X-C chemokine receptor (CXCR) 4, as demonstrated by in vivo blocking experiments using antibody to CXCR4. The specificity of humanized T cell-mediated immune responses against islet {beta} cells was generated by the local inflammatory microenvironment in pancreatic islets including human CD4{sup +} T cell infiltration and clonal expansion, and the mouse islet {beta}-cell-derived CD1d-mediated human iNKT activation. The selective destruction of mouse islet {beta} cells by a human T cell-mediated immune response in this humanized T1D model can mimic those observed in T1D patients. This model can provide a valuable tool for translational research into T1D.« less

Ana o 1 and Ana o 2 cashew allergens share cross-reactive CD4+ T-cell epitopes with other tree nuts

PubMed Central

Archila, Luis Diego; Chow, I-Ting; McGinty, John W.; Renand, Amedee; Jeong, David; Robinson, David; Farrington, Mary L.; Kwok, William.W.

2017-01-01

Background Allergies to cashew are increasing in prevalence, with clinical symptoms ranging from oral pruritus to fatal anaphylactic reaction. Yet, cashew-specific T-cell epitopes and T-cell cross-reactivity amongst cashew and other tree nut allergens in humans remain uncharacterized. Objectives In this study, we characterized cashew specific T-cell responses in cashew allergic subjects and examined cross-reactivity of these cashew specific cells toward other tree nut allergens. Methods CD154 up-regulation assay was used to determine immunodominance hierarchy among cashew major allergens at the T cell level. The phenotype, magnitude and functionality of cashew-specific T-cells was determined by utilizing ex vivo staining with MHC class II tetramers. Dual tetramer staining and proliferation experiments were used to determine cross-reactivity to other tree nuts. Results CD4+ T-cell responses were directed towards cashew allergens Ana o 1 and Ana o 2. Multiple Ana o 1 and Ana o 2 T-cell epitopes were then identified. These epitopes elicited either TH2 or TH2/TH17 responses in allergic subjects, which were either cashew unique epitope or cross-reactive epitopes. For clones that recognized the cross-reactive epitope, T-cell clones responded robustly to cashew, hazelnut and/or pistachio but not to walnut. Conclusions Phylogenetically diverse tree nut allergens can activate cashew reactive T-cells and elicit a TH2 type response at an epitope specific level. Clinical relevance Lack of cross-reactivity between walnut and cashew suggest that cashew peptide immunotherapy approach may not be most effective for walnut. PMID:27129138

Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival.

PubMed

Curiel, Tyler J; Coukos, George; Zou, Linhua; Alvarez, Xavier; Cheng, Pui; Mottram, Peter; Evdemon-Hogan, Melina; Conejo-Garcia, Jose R; Zhang, Lin; Burow, Matthew; Zhu, Yun; Wei, Shuang; Kryczek, Ilona; Daniel, Ben; Gordon, Alan; Myers, Leann; Lackner, Andrew; Disis, Mary L; Knutson, Keith L; Chen, Lieping; Zou, Weiping

2004-09-01

Regulatory T (T(reg)) cells mediate homeostatic peripheral tolerance by suppressing autoreactive T cells. Failure of host antitumor immunity may be caused by exaggerated suppression of tumor-associated antigen-reactive lymphocytes mediated by T(reg) cells; however, definitive evidence that T(reg) cells have an immunopathological role in human cancer is lacking. Here we show, in detailed studies of CD4(+)CD25(+)FOXP3(+) T(reg) cells in 104 individuals affected with ovarian carcinoma, that human tumor T(reg) cells suppress tumor-specific T cell immunity and contribute to growth of human tumors in vivo. We also show that tumor T(reg) cells are associated with a high death hazard and reduced survival. Human T(reg) cells preferentially move to and accumulate in tumors and ascites, but rarely enter draining lymph nodes in later cancer stages. Tumor cells and microenvironmental macrophages produce the chemokine CCL22, which mediates trafficking of T(reg) cells to the tumor. This specific recruitment of T(reg) cells represents a mechanism by which tumors may foster immune privilege. Thus, blocking T(reg) cell migration or function may help to defeat human cancer.

Immune Cells in the Normal Ovary and Spontaneous Ovarian Tumors in the Laying Hen (Gallus domesticus) Model of Human Ovarian Cancer

PubMed Central

Bradaric, Michael J.; Penumatsa, Krishna; Barua, Animesh; Edassery, Seby L.; Yu, Yi; Abramowicz, Jacques S.; Bahr, Janice M.; Luborsky, Judith L.

2013-01-01

Background Spontaneous ovarian cancer in chickens resembles human tumors both histologically and biochemically. The goal was to determine if there are differences in lymphocyte content between normal ovaries and ovarian tumors in chickens as a basis for further studies to understand the role of immunity in human ovarian cancer progression. Methods Hens were selected using grey scale and color Doppler ultrasound to determine if they had normal or tumor morphology. Cells were isolated from ovaries (n = 6 hens) and lymphocyte numbers were determined by flow cytometry using antibodies to avian CD4 and CD8 T and B (Bu1a) cells. Ovarian sections from another set of hens (n = 26) were assessed to verify tumor type and stage and to count CD4, CD8 and Bu1a immunostained cells by morphometric analysis. Results T and B cells were more numerous in ovarian tumors than in normal ovaries by flow cytometry and immunohistochemistry. There were less CD4+ cells than CD8+ and Bu1a+ cells in normal ovaries or ovarian tumors. CD8+ cells were the dominant T cell sub-type in both ovarian stroma and in ovarian follicles compared to CD4+ cells. Bu1a+ cells were consistently found in the stroma of normal ovaries and ovarian tumors but were not associated with follicles. The number of immune cells was highest in late stage serous tumors compared to endometrioid and mucinous tumors. Conclusions The results suggest that similar to human ovarian cancer there are comparatively more immune cells in chicken ovarian tumors than in normal ovaries, and the highest immune cell content occurs in serous tumors. Thus, this study establishes a foundation for further study of tumor immune responses in a spontaneous model of ovarian cancer which will facilitate studies of the role of immunity in early ovarian cancer progression and use of the hen in pre-clinical vaccine trials. PMID:24040191

Generation of mature T cells from human hematopoietic stem/progenitor cells in artificial thymic organoids

PubMed Central

Seet, Christopher S.; He, Chongbin; Bethune, Michael T.; Li, Suwen; Chick, Brent; Gschweng, Eric H.; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B.; Baltimore, David; Crooks, Gay M.; Montel-Hagen, Amélie

2017-01-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCRab+ single positive (SP) CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports highly efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naïve phenotypes, a diverse TCR repertoire, and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen specific cytotoxicity and near complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ loci. ATOs provide a robust tool for studying human T cell development and stem cell based approaches to engineered T cell therapies. PMID:28369043

Regulation of DNA Replication Timing on Human Chromosome by a Cell-Type Specific DNA Binding Protein SATB1

PubMed Central

Oda, Masako; Kanoh, Yutaka; Watanabe, Yoshihisa; Masai, Hisao

2012-01-01

Background Replication timing of metazoan DNA during S-phase may be determined by many factors including chromosome structures, nuclear positioning, patterns of histone modifications, and transcriptional activity. It may be determined by Mb-domain structures, termed as “replication domains”, and recent findings indicate that replication timing is under developmental and cell type-specific regulation. Methodology/Principal Findings We examined replication timing on the human 5q23/31 3.5-Mb segment in T cells and non-T cells. We used two independent methods to determine replication timing. One is quantification of nascent replicating DNA in cell cycle-fractionated stage-specific S phase populations. The other is FISH analyses of replication foci. Although the locations of early- and late-replicating domains were common between the two cell lines, the timing transition region (TTR) between early and late domains were offset by 200-kb. We show that Special AT-rich sequence Binding protein 1 (SATB1), specifically expressed in T-cells, binds to the early domain immediately adjacent to TTR and delays the replication timing of the TTR. Measurement of the chromosome copy number along the TTR during synchronized S phase suggests that the fork movement may be slowed down by SATB1. Conclusions Our results reveal a novel role of SATB1 in cell type-specific regulation of replication timing along the chromosome. PMID:22879953

Optimization of methods for the genetic modification of human T cells.

PubMed

Bilal, Mahmood Y; Vacaflores, Aldo; Houtman, Jon Cd

2015-11-01

CD4(+) T cells are not only critical in the fight against parasitic, bacterial and viral infections, but are also involved in many autoimmune and pathological disorders. Studies of protein function in human T cells are confined to techniques such as RNA interference (RNAi) owing to ethical reasons and relative simplicity of these methods. However, introduction of RNAi or genes into primary human T cells is often hampered by toxic effects from transfection or transduction methods that yield cell numbers inadequate for downstream assays. Additionally, the efficiency of recombinant DNA expression is frequently low because of multiple factors including efficacy of the method and strength of the targeting RNAs. Here, we describe detailed protocols that will aid in the study of primary human CD4(+) T cells. First, we describe a method for development of effective microRNA/shRNAs using available online algorithms. Second, we illustrate an optimized protocol for high efficacy retroviral or lentiviral transduction of human T-cell lines. Importantly, we demonstrate that activated primary human CD4(+) T cells can be transduced efficiently with lentiviruses, with a highly activated population of T cells receiving the largest number of copies of integrated DNA. We also illustrate a method for efficient lentiviral transduction of hard-to-transduce un-activated primary human CD4(+) T cells. These protocols will significantly assist in understanding the activation and function of human T cells and will ultimately aid in the development or improvement of current drugs that target human CD4(+) T cells.

MicroRNA-320c inhibits tumorous behaviors of bladder cancer by targeting Cyclin-dependent kinase 6

PubMed Central

2014-01-01

Background Increasing evidence has suggested that dysregulation of microRNAs (miRNAs) could contribute to human disease including cancer. Previous miRNA microarray analysis illustrated that miR-320c is down-regulated in various cancers. However, the roles of miR-320c in human bladder cancer have not been well elucidated. Therefore, this study was performed to investigate the biological functions and molecular mechanisms of miR-320c in human bladder cancer cell lines, discussing whether it could be a therapeutic biomarker of bladder cancer in the future. Methods Two human bladder cancer cell lines and samples from thirteen patients with bladder cancer were analyzed for the expression of miR-320c by quantitative RT-PCR. Over-expression of miR-320c was established by transfecting mimics into T24 and UM-UC-3. Cell proliferation and cell cycle were assessed by cell viability assay, flow cytometry and colony formation assay. Cell motility ability was evaluated by transwell assay. The target gene of miR-320c was determined by luciferase assay, quantitative RT-PCR and western blot. The regulation of cell cycle and mobility by miR-320c was analyzed by western blot. Results We observed that miR-320c was down-regulated in human bladder cancer tissues and bladder cancer cell lines T24 and UM-UC-3. Over-expression of miR-320c could induce G1 phase arrest in UM-UC-3 and T24 cells, and subsequently inhibited cell growth. We also indentified miR-320c could impair UM-UC-3 and T24 cell motility. In addition, we identified CDK6, a cell cycle regulator, as a novel target of miR-320c. Moreover, we demonstrated miR-320c could induce bladder cancer cell cycle arrest and mobility via regulating CDK6. We also observed that inhibition of miR-320c or restoration of CDK6 in miR-320c-over-expressed bladder cancer cells partly reversed the suppressive effects of miR-320c. Conclusions miR-320c could inhibit the proliferation, migration and invasion of bladder cancer cells via regulating CDK6. Our study revealed that miR-320c could be a therapeutic biomarker of bladder cancer in the future. PMID:25178497

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

PubMed Central

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

2011-01-01

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

T-cell receptor transfer into human T cells with ecotropic retroviral vectors.

PubMed

Koste, L; Beissert, T; Hoff, H; Pretsch, L; Türeci, Ö; Sahin, U

2014-05-01

Adoptive T-cell transfer for cancer immunotherapy requires genetic modification of T cells with recombinant T-cell receptors (TCRs). Amphotropic retroviral vectors (RVs) used for TCR transduction for this purpose are considered safe in principle. Despite this, TCR-coding and packaging vectors could theoretically recombine to produce replication competent vectors (RCVs), and transduced T-cell preparations must be proven free of RCV. To eliminate the need for RCV testing, we transduced human T cells with ecotropic RVs so potential RCV would be non-infectious for human cells. We show that transfection of synthetic messenger RNA encoding murine cationic amino-acid transporter 1 (mCAT-1), the receptor for murine retroviruses, enables efficient transient ecotropic transduction of human T cells. mCAT-1-dependent transduction was more efficient than amphotropic transduction performed in parallel, and preferentially targeted naive T cells. Moreover, we demonstrate that ecotropic TCR transduction results in antigen-specific restimulation of primary human T cells. Thus, ecotropic RVs represent a versatile, safe and potent tool to prepare T cells for the adoptive transfer.

No genetic evidence for involvement of Deltaretroviruses in adult patients with precursor and mature T-cell neoplasms

PubMed Central

Burmeister, Thomas; Schwartz, Stefan; Hummel, Michael; Hoelzer, Dieter; Thiel, Eckhard

2007-01-01

Background The Deltaretrovirus genus comprises viruses that infect humans (HTLV), various simian species (STLV) and cattle (BLV). HTLV-I is the main causative agent in adult T-cell leukemia in endemic areas and some of the simian T-cell lymphotropic viruses have been implicated in the induction of malignant lymphomas in their hosts. BLV causes enzootic bovine leukosis in infected cattle or sheep. During the past few years several new Deltaretrovirus isolates have been described in various primate species. Two new HTLV-like viruses in humans have recently been identified and provisionally termed HTLV-III and HTLV-IV. In order to identify a broad spectrum of Deltaretroviruses by a single PCR approach we have established a novel consensus PCR based on nucleotide sequence data obtained from 42 complete virus isolates (HTLV-I/-II, STLV-I/-II/-III, BLV). The primer sequences were based on highly interspecies-conserved virus genome regions. We used this PCR to detect Deltaretroviruses in samples from adult patients with a variety of rare T-cell neoplasms in Germany. Results The sensitivity of the consensus PCR was at least between 10-2 and 10-3 with 100% specificity as demonstrated by serial dilutions of cell lines infected with either HTLV-I, HTLV-II or BLV. Fifty acute T-cell lymphoblastic leukemia (T-ALL) samples and 33 samples from patients with various rare mature T-cell neoplasms (T-PLL, Sézary syndrome and other T-NHL) were subsequently investigated. There were no cases with HTLV-I, HTLV-II or any other Deltaretroviruses. Conclusion The results rule out a significant involvement of HTLV-I or HTLV-II in these disease entities and show that other related Deltaretroviruses are not likely to be involved. The newly established Deltaretrovirus PCR may be a useful tool for identifying new Deltaretroviruses. PMID:17284327

Functional impairment of Tax-specific but not cytomegalovirus-specific CD8+ T lymphocytes in a minor population of asymptomatic human T-cell leukemia virus type 1-carriers

PubMed Central

2011-01-01

Background Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a small percentage of infected individuals. ATL is often associated with general immune suppression and an impaired HTLV-1-specific T-cell response, an important host defense system. We previously found that a small fraction of asymptomatic HTLV-1-carriers (AC) already showed impaired T-cell responses against the major target antigen, Tax. However, it is unclear whether the impaired HTLV-1 Tax-specific T-cell response in these individuals is an HTLV-1-specific phenomenon, or merely reflects general immune suppression. In this study, in order to characterize the impaired HTLV-1-specific T-cell response, we investigated the function of Tax-specific CD8+ T-cells in various clinical status of HTLV-1 infection. Results By using tetramers consisting of HLA-A*0201, -A*2402, or -A*1101, and corresponding Tax epitope peptides, we detected Tax-specific CD8+ T-cells in the peripheral blood from 87.0% of ACs (n = 20/23) and 100% of HAM/TSP patients (n = 18/18) tested. We also detected Tax-specific CD8+ T-cells in 38.1% of chronic type ATL (cATL) patients (n = 8/21), although its frequencies in peripheral blood CD8+ T cells were significantly lower than those of ACs or HAM/TSP patients. Tax-specific CD8+ T-cells detected in HAM/TSP patients proliferated well in culture and produced IFN-γ when stimulated with Tax peptides. However, such functions were severely impaired in the Tax-specific CD8+ T-cells detected in cATL patients. In ACs, the responses of Tax-specific CD8+ T-cells were retained in most cases. However, we found one AC sample whose Tax-specific CD8+ T-cells hardly produced IFN-γ, and failed to proliferate and express activation (CD69) and degranulation (CD107a) markers in response to Tax peptide. Importantly, the same AC sample contained cytomegalovirus (CMV) pp65-specific CD8+ T-cells that possessed functions upon CMV pp65 peptide stimulation. We further examined additional samples of two smoldering type ATL patients and found that they also showed dysfunctions of Tax-specific but not CMV-specific CD8+ T-cells. Conclusions These findings indicated that Tax-specific CD8+ T-cells were scarce and dysfunctional not only in ATL patients but also in a limited AC population, and that the dysfunction was selective for HTLV-1-specifc CD8+ T-cells in early stages. PMID:22151736

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

PubMed Central

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

2016-01-01

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

Morphologic study of the effect of iron on pseudocyst formation in Trichomonas vaginalis and its interaction with human epithelial cells

PubMed Central

Dias-Lopes, Geovane; Saboia-Vahia, Leonardo; Margotti, Eliane Trindade; Fernandes, Nilma de Souza; Castro, Cássia Luana de Faria; Oliveira, Francisco Odencio; Peixoto, Juliana Figueiredo; Britto, Constança; Silva, Fernando Costa e; Cuervo, Patricia; de Jesus, José Batista

2017-01-01

BACKGROUND Trichomonas vaginalis is the aetiological agent of human trichomoniasis, which is one of the most prevalent sexually transmitted diseases in humans. Iron is an important element for the survival of this parasite and the colonisation of the host urogenital tract. OBJECTIVES In this study, we investigated the effects of iron on parasite proliferation in the dynamics of pseudocyst formation and morphologically characterised iron depletion-induced pseudocysts. METHODS We performed structural and ultrastructural analyses using light microscopy, scanning electron microscopy and transmission electron microscopy. FINDINGS It was observed that iron depletion (i) interrupts the proliferation of T. vaginalis, (ii) induces morphological changes in typical multiplicative trophozoites to spherical non-proliferative, non-motile pseudocysts, and (iii) induces the arrest of cell division at different stages of the cell cycle; (iv) iron is the fundamental element for the maintenance of typical trophozoite morphology; (v) pseudocysts induced by iron depletion are viable and reversible forms; and, finally, (vi) we demonstrated that pseudocysts induced by iron depletion are able to interact with human epithelial cells maintaining their spherical forms. MAIN CONCLUSIONS Together, these data suggest that pseudocysts could be induced as a response to iron nutritional stress and could have a potential role in the transmission and infection of T. vaginalis. PMID:28953994

Human skin is protected by four functionally and phenotypically discrete populations of resident and recirculating memory T cells

PubMed Central

Watanabe, Rei; Gehad, Ahmed; Yang, Chao; Campbell, Laura; Teague, Jessica E.; Schlapbach, Christoph; Elco, Christopher; Huang, Victor; Matos, Tiago R.; Kupper, Thomas S.; Clark, Rachael A.

2015-01-01

The skin of an adult human contains approximately 20 billion memory T cells. Epithelial barrier tissues are infiltrated by a combination of resident and recirculating T cells in mice but the relative proportions and functional activities of resident versus recirculating T cells have not been evaluated in human skin. We discriminated resident from recirculating T cells in human engrafted mice and lymphoma patients using alemtuzumab, a medication that depletes recirculating T cells from skin, and then analyzed these T cell populations in healthy human skin. All non-recirculating resident memory T cells (TRM) expressed CD69, but the majority were CD4+, CD103− and located in the dermis, in contrast to studies in mice. Both CD4+ and CD8+ CD103+ TRM were enriched in the epidermis, had potent effector functions and had a limited proliferative capacity compared to CD103− TRM. TRM of both types had more potent effector functions than recirculating T cells. Induction of CD103 on human T cells was enhanced by keratinocyte contact, depended on TGFβ and was independent of T cell keratinocyte adhesive interactions. We observed two distinct populations of recirculating T cells, CCR7+/L-selectin+ central memory T cells (TCM) and CCR7+/L-selectin− T cells, which we term migratory memory T cells (TMM). Circulating skin-tropic TMM were intermediate in cytokine production between TCM and effector memory T cells. In patients with cutaneous T cell lymphoma, malignant TCM and TMM induced distinct inflammatory skin lesions and TMM were depleted more slowly from skin after alemtuzumab, suggesting TMM may recirculate more slowly. In summary, human skin is protected by four functionally distinct populations of T cells, two resident and two recirculating, with differing territories of migration and distinct functional activities. PMID:25787765

Cancer immunotherapy and immunological memory.

PubMed

Murata, Kenji; Tsukahara, Tomohide; Torigoe, Toshihiko

2016-01-01

Human immunological memory is the key distinguishing hallmark of the adaptive immune system and plays an important role in the prevention of morbidity and the severity of infection. The differentiation system of T cell memory has been clarified using mouse models. However, the human T cell memory system has great diversity induced by natural antigens derived from many pathogens and tumor cells throughout life, and profoundly differs from the mouse memory system constructed using artificial antigens and transgenic T cells. We believe that only human studies can elucidate the human immune system. The importance of immunological memory in cancer immunotherapy has been pointed out, and the trafficking properties and long-lasting anti-tumor capacity of memory T cells play a crucial role in the control of malignant tumors. Adoptive cell transfer of less differentiated T cells has consistently demonstrated superior anti-tumor capacity relative to more differentiated T cells. Therefore, a human T cell population with the characteristics of stem cell memory is thought to be attractive for peptide vaccination and adoptive cell transfer. A novel human memory T cell population that we have identified is closer to the naive state than previous memory T cells in the T cell differentiation lineage, and has the characteristics of stem-like chemoresistance. Here we introduce this novel population and describe the fundamentals of immunological memory in cancer immunotherapy.

Dendritic Cells Program Non-Immunogenic Prostate-Specific T Cell Responses Beginning at Early Stages of Prostate Tumorigenesis

PubMed Central

Mihalyo, Marianne A.; Hagymasi, Adam T.; Slaiby, Aaron M.; Nevius, Erin E.; Adler, Adam J.

2010-01-01

BACKGROUND Prostate cancer promotes the development of T cell tolerance towards prostatic antigens, potentially limiting the efficacy of prostate cancer vaccines targeting these antigens. Here, we sought to determine the stage of disease progression when T cell tolerance develops, as well as the role of steady state dendritic cells (DC) and CD4+CD25+ T regulatory cells (Tregs) in programming tolerance. METHODS The response of naïve HA-specific CD4+ T cells were analyzed following adoptive transfer into Pro-HA × TRAMP transgenic mice harboring variably-staged HA-expressing prostate tumors on two genetic backgrounds that display different patterns and kinetics of tumorigenesis. The role of DC and Tregs in programming HA-specific CD4 cell responses were assessed via depletion. RESULTS HA-specific CD4 cells underwent non-immunogenic responses at all stages of tumorigenesis in both genetic backgrounds. These responses were completely dependent on DC, but not appreciably influenced by Tregs. CONCLUSIONS These results suggest that tolerogenicity is an early and general property of prostate tumors. PMID:17221844

Characterization of the CD8{sup +} T cell responses directed against respiratory syncytial virus during primary and secondary infection in C57BL/6 mice

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

Lukens, Michael V.; Claassen, Erwin A.W.; Graaff, Patricia M.A. de

2006-08-15

The BALB/c mouse model for human respiratory syncytial virus infection has contributed significantly to our understanding of the relative role for CD4{sup +} and CD8{sup +} T cells to immune protection and pathogenic immune responses. To enable comparison of RSV-specific T cell responses in different mouse strains and allow dissection of immune mechanisms by using transgenic and knockout mice that are mostly available on a C57BL/6 background, we characterized the specificity, level and functional capabilities of CD8{sup +} T cells during primary and secondary responses in lung parenchyma, airways and spleens of C57BL/6 mice. During the primary response, epitopes weremore » recognized originating from the matrix, fusion, nucleo- and attachment proteins, whereas the secondary response focused predominantly on the matrix epitope. C57BL/6 mice are less permissive for hRSV infection than BALB/c mice, yet we found CD8{sup +} T cell responses in the lungs and bronchoalveolar lavage, comparable to the responses described for BALB/c mice.« less

Mapping of the minimal inorganic phosphate transporting unit of human PiT2 suggests a structure universal to PiT-related proteins from all kingdoms of life

PubMed Central

2011-01-01

Background The inorganic (Pi) phosphate transporter (PiT) family comprises known and putative Na+- or H+-dependent Pi-transporting proteins with representatives from all kingdoms. The mammalian members are placed in the outer cell membranes and suggested to supply cells with Pi to maintain house-keeping functions. Alignment of protein sequences representing PiT family members from all kingdoms reveals the presence of conserved amino acids and that bacterial phosphate permeases and putative phosphate permeases from archaea lack substantial parts of the protein sequence when compared to the mammalian PiT family members. Besides being Na+-dependent Pi (NaPi) transporters, the mammalian PiT paralogs, PiT1 and PiT2, also are receptors for gamma-retroviruses. We have here exploited the dual-function of PiT1 and PiT2 to study the structure-function relationship of PiT proteins. Results We show that the human PiT2 histidine, H502, and the human PiT1 glutamate, E70, - both conserved in eukaryotic PiT family members - are critical for Pi transport function. Noticeably, human PiT2 H502 is located in the C-terminal PiT family signature sequence, and human PiT1 E70 is located in ProDom domains characteristic for all PiT family members. A human PiT2 truncation mutant, which consists of the predicted 10 transmembrane (TM) domain backbone without a large intracellular domain (human PiT2ΔR254-V483), was found to be a fully functional Pi transporter. Further truncation of the human PiT2 protein by additional removal of two predicted TM domains together with the large intracellular domain created a mutant that resembles a bacterial phosphate permease and an archaeal putative phosphate permease. This human PiT2 truncation mutant (human PiT2ΔL183-V483) did also support Pi transport albeit at very low levels. Conclusions The results suggest that the overall structure of the Pi-transporting unit of the PiT family proteins has remained unchanged during evolution. Moreover, in combination, our studies of the gene structure of the human PiT1 and PiT2 genes (SLC20A1 and SLC20A2, respectively) and alignment of protein sequences of PiT family members from all kingdoms, along with the studies of the dual functions of the human PiT paralogs show that these proteins are excellent as models for studying the evolution of a protein's structure-function relationship. PMID:21586110

Cysteine-dependent immune regulation by TRX and MIF/GIF family proteins.

PubMed

Kondo, Norihiko; Ishii, Yasuyuki; Son, Aoi; Sakakura-Nishiyama, Junko; Kwon, Yong-Won; Tanito, Masaki; Nishinaka, Yumiko; Matsuo, Yoshiyuki; Nakayama, Toshinori; Taniguchi, Masaru; Yodoi, Junji

2004-03-29

Thioredoxin (TRX) superfamily proteins that contain a conserved redox-active site -Cys-Xa.a.-Xa.a.-Cys- includes proinflammatory cytokine, macrophage migration inhibiting factor (MIF) and the immune regulatory cytokine, glycosylation inhibiting factor (GIF) in which Cys-60 is cysteinylated. In this report, we have analyzed the functional interaction between TRX and MIF/GIF. The stable Jurkat T cell line transfected with human TRX gene (TRX-transfectant) was highly resistant to hydrogen peroxide-induced apoptosis, but not the cell line transfected with vector (mock-transfectant). The expression level of MIF/GIF protein of TRX-transfectant was lower than that of mock-transfectant. Conversely, the expression level of intracellular TRX protein in CD4(+)-T cells derived from MIF -/- mice were significantly higher than that from background BALB/c mice. These findings collectively suggest that oxidative stress-induced apoptosis on T lymphocytes might be protected by the reciprocal regulation of TRX and MIF/GIF expression.

The hydroxyflavone, fisetin, suppresses mast cell activation induced by interaction with activated T cell membranes

PubMed Central

Nagai, K; Takahashi, Y; Mikami, I; Fukusima, T; Oike, H; Kobori, M

2009-01-01

Background and purpose: Cell-to-cell interactions between mast cells and activated T cells are increasingly recognized as a possible mechanism in the aetiology of allergic or non-allergic inflammatory disorders. To determine the anti-allergic effect of fisetin, we examined the ability of fisetin to suppress activation of the human mast cell line, HMC-1, induced by activated Jurkat T cell membranes. Experimental approach: HMC-1 cells were incubated with or without fisetin for 15 min and then co-cultured with Jurkat T cell membranes activated by phorbol-12-myristate 13-acetate for 16 h. We determined gene expression in activated HMC-1 cells by DNA microarray and quantitative reverse transcription (RT)-PCR analysis. We also examined activation of the transcription factor NF-κB and MAP kinases (MAPKs) in activated HMC-1 cells. Key results: Fisetin suppresses cell spreading and gene expression in HMC-1 cells stimulated by activated T cell membranes. Additionally, we show that these stimulated HMC-1 cells expressed granzyme B. The stimulatory interaction also induced activation of NF-κB and MAPKs; these activations were suppressed by fisetin. Fisetin also reduced the amount of cell surface antigen CD40 and intercellular adhesion molecule-1 (ICAM-1) on activated HMC-1 cells. Conclusions and implications: Fisetin suppressed activation of HMC-1 cells by activated T cell membranes by interfering with cell-to-cell interaction and inhibiting the activity of NF-κB and MAPKs and thereby suppressing gene expression. Fisetin may protect against the progression of inflammatory diseases by limiting interactions between mast cells and activated T cells. PMID:19702784

Immunosuppressive Mesenchymal Stromal Cells Derived from Human-Induced Pluripotent Stem Cells Induce Human Regulatory T Cells In Vitro and In Vivo

PubMed Central

Roux, Clémence; Saviane, Gaëlle; Pini, Jonathan; Belaïd, Nourhène; Dhib, Gihen; Voha, Christine; Ibáñez, Lidia; Boutin, Antoine; Mazure, Nathalie M.; Wakkach, Abdelilah; Blin-Wakkach, Claudine; Rouleau, Matthieu

2018-01-01

Despite mesenchymal stromal cells (MSCs) are considered as a promising source of cells to modulate immune functions on cells from innate and adaptive immune systems, their clinical use remains restricted (few number, limited in vitro expansion, absence of a full phenotypic characterization, few insights on their in vivo fate). Standardized MSCs derived in vitro from human-induced pluripotent stem (huIPS) cells, remediating part of these issues, are considered as well as a valuable tool for therapeutic approaches, but their functions remained to be fully characterized. We generated multipotent MSCs derived from huiPS cells (huiPS-MSCs), and focusing on their immunosuppressive activity, we showed that human T-cell activation in coculture with huiPS-MSCs was significantly reduced. We also observed the generation of functional CD4+ FoxP3+ regulatory T (Treg) cells. Further tested in vivo in a model of human T-cell expansion in immune-deficient NSG mice, huiPS-MSCs immunosuppressive activity prevented the circulation and the accumulation of activated human T cells. Intracytoplasmic labeling of cytokines produced by the recovered T cells showed reduced percentages of human-differentiated T cells producing Th1 inflammatory cytokines. By contrast, T cells producing IL-10 and FoxP3+-Treg cells, absent in non-treated animals, were detected in huiPS-MSCs treated mice. For the first time, these results highlight the immunosuppressive activity of the huiPS-MSCs on human T-cell stimulation with a concomitant generation of human Treg cells in vivo. They may favor the development of new tools and strategies based on the use of huiPS cells and their derivatives for the induction of immune tolerance. PMID:29422893

Immunosuppressive Mesenchymal Stromal Cells Derived from Human-Induced Pluripotent Stem Cells Induce Human Regulatory T Cells In Vitro and In Vivo.

PubMed

Roux, Clémence; Saviane, Gaëlle; Pini, Jonathan; Belaïd, Nourhène; Dhib, Gihen; Voha, Christine; Ibáñez, Lidia; Boutin, Antoine; Mazure, Nathalie M; Wakkach, Abdelilah; Blin-Wakkach, Claudine; Rouleau, Matthieu

2017-01-01

Despite mesenchymal stromal cells (MSCs) are considered as a promising source of cells to modulate immune functions on cells from innate and adaptive immune systems, their clinical use remains restricted (few number, limited in vitro expansion, absence of a full phenotypic characterization, few insights on their in vivo fate). Standardized MSCs derived in vitro from human-induced pluripotent stem (huIPS) cells, remediating part of these issues, are considered as well as a valuable tool for therapeutic approaches, but their functions remained to be fully characterized. We generated multipotent MSCs derived from huiPS cells (huiPS-MSCs), and focusing on their immunosuppressive activity, we showed that human T-cell activation in coculture with huiPS-MSCs was significantly reduced. We also observed the generation of functional CD4 + FoxP3 + regulatory T (Treg) cells. Further tested in vivo in a model of human T-cell expansion in immune-deficient NSG mice, huiPS-MSCs immunosuppressive activity prevented the circulation and the accumulation of activated human T cells. Intracytoplasmic labeling of cytokines produced by the recovered T cells showed reduced percentages of human-differentiated T cells producing Th1 inflammatory cytokines. By contrast, T cells producing IL-10 and FoxP3 + -Treg cells, absent in non-treated animals, were detected in huiPS-MSCs treated mice. For the first time, these results highlight the immunosuppressive activity of the huiPS-MSCs on human T-cell stimulation with a concomitant generation of human Treg cells in vivo . They may favor the development of new tools and strategies based on the use of huiPS cells and their derivatives for the induction of immune tolerance.

Sterile Immunity to Malaria after DNA Prime/Adenovirus Boost Immunization Is Associated with Effector Memory CD8+T Cells Targeting AMA1 Class I Epitopes

PubMed Central

Sedegah, Martha; Hollingdale, Michael R.; Farooq, Fouzia; Ganeshan, Harini; Belmonte, Maria; Kim, Yohan; Peters, Bjoern; Sette, Alessandro; Huang, Jun; McGrath, Shannon; Abot, Esteban; Limbach, Keith; Shi, Meng; Soisson, Lorraine; Diggs, Carter; Chuang, Ilin; Tamminga, Cindy; Epstein, Judith E.; Villasante, Eileen; Richie, Thomas L.

2014-01-01

Background Fifteen volunteers were immunized with three doses of plasmid DNA encoding P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) and boosted with human adenovirus-5 (Ad) expressing the same antigens (DNA/Ad). Four volunteers (27%) demonstrated sterile immunity to controlled human malaria infection and, overall, protection was statistically significantly associated with ELISpot and CD8+ T cell IFN-γ activities to AMA1 but not CSP. DNA priming was required for protection, as 18 additional subjects immunized with Ad alone (AdCA) did not develop sterile protection. Methodology/Principal Findings We sought to identify correlates of protection, recognizing that DNA-priming may induce different responses than AdCA alone. Among protected volunteers, two and three had higher ELISpot and CD8+ T cell IFN-γ responses to CSP and AMA1, respectively, than non-protected volunteers. Unexpectedly, non-protected volunteers in the AdCA trial showed ELISpot and CD8+ T cell IFN-γ responses to AMA1 equal to or higher than the protected volunteers. T cell functionality assessed by intracellular cytokine staining for IFN-γ, TNF-α and IL-2 likewise did not distinguish protected from non-protected volunteers across both trials. However, three of the four protected volunteers showed higher effector to central memory CD8+ T cell ratios to AMA1, and one of these to CSP, than non-protected volunteers for both antigens. These responses were focused on discrete regions of CSP and AMA1. Class I epitopes restricted by A*03 or B*58 supertypes within these regions of AMA1 strongly recalled responses in three of four protected volunteers. We hypothesize that vaccine-induced effector memory CD8+ T cells recognizing a single class I epitope can confer sterile immunity to P. falciparum in humans. Conclusions/Significance We suggest that better understanding of which epitopes within malaria antigens can confer sterile immunity and design of vaccine approaches that elicit responses to these epitopes will increase the potency of next generation gene-based vaccines. PMID:25211344

Human Naive T Cells Express Functional CXCL8 and Promote Tumorigenesis.

PubMed

Crespo, Joel; Wu, Ke; Li, Wei; Kryczek, Ilona; Maj, Tomasz; Vatan, Linda; Wei, Shuang; Opipari, Anthony W; Zou, Weiping

2018-05-25

Naive T cells are thought to be functionally quiescent. In this study, we studied and compared the phenotype, cytokine profile, and potential function of human naive CD4 + T cells in umbilical cord and peripheral blood. We found that naive CD4 + T cells, but not memory T cells, expressed high levels of chemokine CXCL8. CXCL8 + naive T cells were preferentially enriched CD31 + T cells and did not express T cell activation markers or typical Th effector cytokines, including IFN-γ, IL-4, IL-17, and IL-22. In addition, upon activation, naive T cells retained high levels of CXCL8 expression. Furthermore, we showed that naive T cell-derived CXCL8 mediated neutrophil migration in the in vitro migration assay, supported tumor sphere formation, and promoted tumor growth in an in vivo human xenograft model. Thus, human naive T cells are phenotypically and functionally heterogeneous and can carry out active functions in immune responses. Copyright © 2018 by The American Association of Immunologists, Inc.

A Single Amino Acid Substitution Prevents Recognition of a Dominant Human Aquaporin-4 Determinant in the Context of HLA-DRB1*03:01 by a Murine TCR

PubMed Central

Arellano, Benjamine; Hussain, Rehana; Miller-Little, William A.; Herndon, Emily; Lambracht-Washington, Doris; Eagar, Todd N.; Lewis, Robert; Healey, Don; Vernino, Steven; Greenberg, Benjamin M.; Stüve, Olaf

2016-01-01

Background Aquaporin 4 (AQP4) is considered a putative autoantigen in patients with Neuromyelitis optica (NMO), an autoinflammatory disorder of the central nervous system (CNS). HLA haplotype analyses of patients with NMO suggest a positive association with HLA-DRB1* 03:01. We previously showed that the human (h) AQP4 peptide 281–300 is the dominant immunogenic determinant of hAQP4 in the context of HLA-DRB1*03:01. This immunogenic peptide stimulates a strong Th1 and Th17 immune response. AQP4281-300-specific encephalitogenic CD4+ T cells should initiate CNS inflammation that results in a clinical phenotype in HLA-DRB1*03:01 transgenic mice. Methods Controlled study with humanized experimental animals. HLA-DRB1*03:01 transgenic mice were immunized with hAQP4281-300, or whole-length hAQP4 protein emulsified in complete Freund’s adjuvant. Humoral immune responses to both antigens were assessed longitudinally. In vivo T cell frequencies were assessed by tetramer staining. Mice were followed clinically, and the anterior visual pathway was tested by pupillometry. CNS tissue was examined histologically post-mortem. Flow cytometry was utilized for MHC binding assays and to immunophenotype T cells, and T cell frequencies were determined by ELISpot assay. Results Immunization with hAQP4281-300 resulted in an in vivo expansion of antigen-specific CD4+ T cells, and an immunoglobulin isotype switch. HLA-DRB1*03:01 TG mice actively immunized with hAQP4281-300, or with whole-length hAQP4 protein were resistant to developing a neurological disease that resembles NMO. Experimental mice show no histological evidence of CNS inflammation, nor change in pupillary responses. Subsequent analysis reveals that a single amino acid substitution from aspartic acid in hAQP4 to glutamic acid in murine (m)AQP4 at position 290 prevents the recognition of hAQP4281-300 by the murine T cell receptor (TCR). Conclusion Induction of a CNS inflammatory autoimmune disorder by active immunization of HLA-DRB1*03:01 TG mice with human hAQP4281-300 will be complex due to a single amino acid substitution. The pathogenic role of T cells in this disorder remains critical despite these observations. PMID:27054574

Human skin is protected by four functionally and phenotypically discrete populations of resident and recirculating memory T cells.

PubMed

Watanabe, Rei; Gehad, Ahmed; Yang, Chao; Scott, Laura L; Teague, Jessica E; Schlapbach, Christoph; Elco, Christopher P; Huang, Victor; Matos, Tiago R; Kupper, Thomas S; Clark, Rachael A

2015-03-18

The skin of an adult human contains about 20 billion memory T cells. Epithelial barrier tissues are infiltrated by a combination of resident and recirculating T cells in mice, but the relative proportions and functional activities of resident versus recirculating T cells have not been evaluated in human skin. We discriminated resident from recirculating T cells in human-engrafted mice and lymphoma patients using alemtuzumab, a medication that depletes recirculating T cells from skin, and then analyzed these T cell populations in healthy human skin. All nonrecirculating resident memory T cells (TRM) expressed CD69, but most were CD4(+), CD103(-), and located in the dermis, in contrast to studies in mice. Both CD4(+) and CD8(+) CD103(+) TRM were enriched in the epidermis, had potent effector functions, and had a limited proliferative capacity compared to CD103(-) TRM. TRM of both types had more potent effector functions than recirculating T cells. We observed two distinct populations of recirculating T cells, CCR7(+)/L-selectin(+) central memory T cells (TCM) and CCR7(+)/L-selectin(-) T cells, which we term migratory memory T cells (TMM). Circulating skin-tropic TMM were intermediate in cytokine production between TCM and effector memory T cells. In patients with cutaneous T cell lymphoma, malignant TCM and TMM induced distinct inflammatory skin lesions, and TMM were depleted more slowly from skin after alemtuzumab, suggesting that TMM may recirculate more slowly. In summary, human skin is protected by four functionally distinct populations of T cells, two resident and two recirculating, with differing territories of migration and distinct functional activities. Copyright © 2015, American Association for the Advancement of Science.

Ta1, a novel 105 KD human T cell activation antigen defined by a monoclonal antibody.

PubMed

Fox, D A; Hussey, R E; Fitzgerald, K A; Acuto, O; Poole, C; Palley, L; Daley, J F; Schlossman, S F; Reinherz, E L

1984-09-01

By using a murine monoclonal antibody produced against an IL 2-dependent human T cell line, we defined a T lineage-specific molecule, termed Ta1, that is expressed strongly on activated T lymphocytes of both the T4 and T8 subsets, as well as on T cell lines and clones, but only weakly on a fraction of resting T cells. SDS-PAGE analysis of immunoprecipitates from 125I-labeled, activated T cells demonstrates a single major band of apparent m.w. 105 KD under both reducing and nonreducing conditions. Unlike anti-IL 2 receptor antibodies, anti-Ta1 does not inhibit T cell proliferative responses to mitogen, antigen, or IL 2-containing medium. Moreover, anti-Ta1 has no effect on T cell-mediated cytotoxicity. Ta1 appears to be a novel human T cell-specific activation antigen that may serve as a useful marker of T cell activation in human disease.

Holistic systems biology approaches to molecular mechanisms of human helper T cell differentiation to functionally distinct subsets.

PubMed

Chen, Z; Lönnberg, T; Lahesmaa, R

2013-08-01

Current knowledge of helper T cell differentiation largely relies on data generated from mouse studies. To develop therapeutical strategies combating human diseases, understanding the molecular mechanisms how human naïve T cells differentiate to functionally distinct T helper (Th) subsets as well as studies on human differentiated Th cell subsets is particularly valuable. Systems biology approaches provide a holistic view of the processes of T helper differentiation, enable discovery of new factors and pathways involved and generation of new hypotheses to be tested to improve our understanding of human Th cell differentiation and immune-mediated diseases. Here, we summarize studies where high-throughput systems biology approaches have been exploited to human primary T cells. These studies reveal new factors and signalling pathways influencing T cell differentiation towards distinct subsets, important for immune regulation. Such information provides new insights into T cell biology and into targeting immune system for therapeutic interventions. © 2013 John Wiley & Sons Ltd.

Control of T lymphocyte morphology by the GTPase Rho

NASA Technical Reports Server (NTRS)

Woodside, Darren G.; Wooten, David K.; Teague, T. Kent; Miyamoto, Yuko J.; Caudell, Eva G.; Udagawa, Taturo; Andruss, Bernard F.; McIntyre, Bradley W.

2003-01-01

BACKGROUND: Rho family GTPase regulation of the actin cytoskeleton governs a variety of cell responses. In this report, we have analyzed the role of the GTPase Rho in maintenance of the T lymphocyte actin cytoskeleton. RESULTS: Inactivation of the GTPase Rho in the human T lymphocytic cell line HPB-ALL does not inhibit constitutively high adhesion to the integrin beta1 substrate fibronectin. It did however result in the aberrant extension of finger-like dendritic processes on the substrates VCAM-1, Fn, and mAb specific to beta1 integrins. Time-lapse video microscopy demonstrated that C3 induced extensions were primarily the result of an altered pseudopod elongation rather than retraction. Once the stellate pseudopodia extended, none retracted, and cells became completely immobile. Filipodial structures were absent and the dendritic-like processes in C3 treated cells were rich in filamentous actin. Immunolocalization of RhoA in untreated HPB-ALL cells spreading on fibronectin demonstrated a diffuse staining pattern within the pseudopodia. In C3 treated cells, clusters of RhoA were pronounced and localized within the altered extensions. CONCLUSIONS: GTPase Rho is actively involved in the regulation of T lymphocyte morphology and motility.

Development of operational models of receptor activation including constitutive receptor activity and their use to determine the efficacy of the chemokine CCL17 at the CC chemokine receptor CCR4

PubMed Central

Slack, RJ; Hall, DA

2012-01-01

BACKGROUND AND PURPOSE The operational model provides a key conceptual framework for the analysis of pharmacological data. However, this model does not include constitutive receptor activity, a frequent phenomenon in modern pharmacology, particularly in recombinant systems. Here, we developed extensions of the operational model which include constitutive activity and applied them to effects of agonists at the chemokine receptor CCR4. EXPERIMENTAL APPROACH The effects of agonists of CCR4 on [35S]GTPγS binding to recombinant cell membranes and on the filamentous (F-) actin content of human CD4+ CCR4+ T cells were determined. The basal [35S]GTPγS binding was changed by varying the GDP concentration whilst the basal F-actin contents of the higher expressing T cell populations were elevated, suggesting constitutive activity of CCR4. Both sets of data were analysed using the mathematical models. RESULTS The affinity of CCL17 (also known as TARC) derived from analysis of the T cell data (pKa= 9.61 ± 0.17) was consistent with radioligand binding experiments (9.50 ± 0.11) while that from the [35S]GTPγS binding experiments was lower (8.27 ± 0.09). Its intrinsic efficacy differed between the two systems (110 in T cells vs. 11). CONCLUSIONS AND IMPLICATIONS The presence of constitutive receptor activity allows the absolute intrinsic efficacy of agonists to be determined without a contribution from the signal transduction system. Intrinsic efficacy estimated in this way is consistent with Furchgott's definition of this property. CCL17 may have a higher intrinsic efficacy at CCR4 in human T cells than that expressed recombinantly in CHO cells. PMID:22335621

Unique and Common Features of Innate-Like Human Vδ2+ γδT Cells and Mucosal-Associated Invariant T Cells.

PubMed

Provine, Nicholas M; Binder, Benedikt; FitzPatrick, Michael E B; Schuch, Anita; Garner, Lucy C; Williamson, Kate D; van Wilgenburg, Bonnie; Thimme, Robert; Klenerman, Paul; Hofmann, Maike

2018-01-01

Mucosal-associated invariant T (MAIT) cells are innate-like T cells abundant in humans that can be activated in a TCR-independent manner by inflammatory and antiviral cytokines. In humans, the capacity for TCR-independent activation is functionally linked to a transcriptional program that can be identified by the expression of the C-type lectin receptor, CD161. In addition to MAIT cells, it has been demonstrated that a subset of γδT cells expresses CD161 and can be activated by TCR-independent cytokine stimulation. In this study, we sought to clarify the nature of cytokine-responsive human γδT cells. We could link CD161 expression on Vδ2 + versus Vδ1 + γδT cells to the observation that Vδ2 + γδT cells, but not Vδ1 + γδT cells, robustly produced IFN-γ upon stimulation with a variety of cytokine combinations. Interestingly, both CD161 + and CD161 - Vδ2 + γδT cells responded to these stimuli, with increased functionality within the CD161 + subset. This innate-like responsiveness corresponded to high expression of PLZF and IL-18Rα, analogous to MAIT cells. Vδ2 + γδT cells in human duodenum and liver maintained a CD161 + IL-18Rα + phenotype and produced IFN-γ in response to IL-12 and IL-18 stimulation. In contrast to MAIT cells, we could not detect IL-17A production but observed higher steady-state expression of Granzyme B by Vδ2 + γδT cells. Finally, we investigated the frequency and functionality of γδT cells in the context of chronic hepatitis C virus infection, as MAIT cells are reduced in frequency in this disease. By contrast, Vδ2 + γδT cells were maintained in frequency and displayed unimpaired IFN-γ production in response to cytokine stimulation. In sum, human Vδ2 + γδT cells are a functionally distinct population of cytokine-responsive innate-like T cells that is abundant in blood and tissues with similarities to human MAIT cells.

Quantitative [Fe]MRI of PSMA-targeted SPIONs specifically discriminates among prostate tumor cell types based on their PSMA expression levels.

PubMed

Sillerud, Laurel O

2016-01-01

We report the development, experimental verification, and application of a general theory called [Fe]MRI (pronounced fem-ree) for the non-invasive, quantitative molecular magnetic resonance imaging (MRI) of added magnetic nanoparticles or other magnetic contrast agents in biological tissues and other sites. [Fe]MRI can easily be implemented on any MRI instrument, requiring only measurements of the background nuclear magnetic relaxation times (T1, T2) of the tissue of interest, injection of the magnetic particles, and the subsequent acquisition of a pair of T1-weighted and T2-weighted images. These images, converted into contrast images, are subtracted to yield a contrast difference image proportional to the absolute nanoparticle, iron concentration, ([Fe]) image. [Fe]MRI was validated with the samples of superparamagnetic iron oxide nanoparticles (SPIONs) both in agarose gels and bound to human prostate tumor cells. The [Fe]MRI measurement of the binding of anti-prostate specific membrane antigen (PSMA) conjugated SPIONs to PSMA-positive LNCaP and PSMA-negative DU145 cells in vitro allowed a facile discrimination among prostate tumor cell types based on their PSMA expression level. The low [Fe] detection limit of ~2 μM for SPIONs allows sensitive MRI of added iron at concentrations considerably below the US Food and Drug Administration's human iron dosage guidelines (<90 μM, 5 mg/kg).

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

Takahashi, Takeshi, E-mail: takeshi-takahashi@ciea.or.jp; Katano, Ikumi; Ito, Ryoji

Highlights: • β-Lactoglobulin (BLG) specific TCR genes were introduced to human HSC by retrovirus. • Human HSC with BLG-specific TCR were transplanted into NOG-HLA-DR4 I-A{sup −/−} mice. • BLG-specific TCR induced positive selection of thymocytes. • BLG-specific TCR positive CD4{sup +} T cells mediated immune responses in humanized mice. - Abstract: The development of severe immunodeficient mouse strains containing various human genes, including cytokines or HLA, has enabled the reconstitution of functional human immune systems after transplantation of human hematopoietic stem cells (HSC). Accumulating evidence has suggested that HLA-restricted antigen-specific human T-cell responses can be generated in these humanized mice.more » To directly monitor immune responses of human CD4{sup +} T cells, we introduced β-lactoglobulin (BLG)-specific T cell receptor (TCR) genes derived from CD4{sup +} T-cell clones of cow-milk allergy patients into HSCs, and subsequently transplanted them into NOG-HLA-DR4 transgenic/I-Aβ deficient mice (NOG-DR4/I-A{sup o}). In the thymus, thymocytes with BLG-specific TCR preferentially differentiated into CD4{sup +}CD8{sup −} single-positive cells. Adoptive transfer of mature CD4{sup +} T cells expressing the TCR into recipient NOG-DR4/I-A{sup o} mice demonstrated that human CD4{sup +} T cells proliferated in response to antigenic stimulation and produced IFN-γ in vivo, suggesting that functional T-cell reactions (especially Th1-skewed responses) were induced in humanized mice.« less

An amelogenin mutation leads to disruption of the odontogenic apparatus and aberrant expression of Notch I

PubMed Central

Chen, Xu; Li, Yong; Alawi, Faizan; Bouchard, Jessica R.; Kulkarni, Ashok B.; Gibson, Carolyn W.

2012-01-01

BACKGROUND Amelogenins are highly conserved proteins secreted by ameloblasts in the dental organ of developing teeth. These proteins regulate dental enamel thickness and structure in humans and mice. Mice that express an amelogenin transgene with a P70T mutation (TgP70T) develop abnormal epithelial proliferation in an amelogenin null (KO) background. Some of these cellular masses have the appearance of proliferating stratum intermedium, which is the layer adjacent to the ameloblasts in unerupted teeth. As Notch proteins are thought to constitute the developmental switch that separates ameloblasts from stratum intermedium, these signaling proteins were evaluated in normal and proliferating tissues. METHODS Mandibles were dissected for histology and immunohistochemistry using Notch I antibodies. Molar teeth were dissected for western blotting and RT-PCR for evaluation of Notch levels through imaging and statistical analyses. RESULTS Notch I was immunolocalized to ameloblasts of TgP70TKO mice, KO ameloblasts stained, but less strongly, and wild-type teeth had minimal staining. Cells within the proliferating epithelial cell masses were positive for Notch I and had an appearance reminiscent of calcifying epithelial odontogenic tumor with amyloid-like deposits. Notch I protein and mRNA were elevated in molar teeth from TgP70TKO mice. CONCLUSION Expression of TgP70T leads to abnormal structures in mandibles and maxillae of mice with the KO genetic background and these mice have elevated levels of Notch I in developing molars. As cells within the masses also express transgenic amelogenins, development of the abnormal proliferations suggests communication between amelogenin producing cells and the proliferating cells, dependent on the presence of the mutated amelogenin protein. PMID:20923441

Extremely low-frequency electromagnetic field influences the survival and proliferation effect of human adipose derived stem cells

PubMed Central

Razavi, Shahnaz; Salimi, Marzieh; Shahbazi-Gahrouei, Daryoush; Karbasi, Saeed; Kermani, Saeed

2014-01-01

Background: Extremely low-frequency electromagnetic fields (ELF-EMF) can effect on biological systems and alters some cell functions like proliferation rate. Therefore, we aimed to attempt the evaluation effect of ELF-EMF on the growth of human adipose derived stem cells (hADSCs). Materials and Methods: ELF-EMF was generated by a system including autotransformer, multi-meter, solenoid coils, teslameter and its probe. We assessed the effect of ELF-EMF with intensity of 0.5 and 1 mT and power line frequency 50 Hz on the survival of hADSCs for 20 and 40 min/day for 7 days by MTT assay. One-way analysis of variance was used to assessment the significant differences in groups. Results: ELF-EMF has maximum effect with intensity of 1 mT for 20 min/day on proliferation of hADSCs. The survival and proliferation effect (PE) in all exposure groups were significantly higher than that in sham groups (P < 0.05) except in group of 1 mT and 40 min/day. Conclusion: Our results show that between 0.5 m and 1 mT ELF-EMF could be enhances survival and PE of hADSCs conserving the duration of exposure. PMID:24592372

Mu opioid receptor expression is increased in inflammatory bowel diseases: implications for homeostatic intestinal inflammation

PubMed Central

Philippe, D; Chakass, D; Thuru, X; Zerbib, P; Tsicopoulos, A; Geboes, K; Bulois, P; Breisse, M; Vorng, H; Gay, J; Colombel, J‐F; Desreumaux, P; Chamaillard, M

2006-01-01

Background and aims Recent studies with μ opioid receptor (MOR) deficient mice support a physiological anti‐inflammatory effect of MOR at the colon interface. To better understand the potential pharmacological effect of certain opiates in inflammatory bowel diseases (IBD), we (1) evaluated the regulation in vivo and in vitro of human MOR expression by inflammation; and (2) tested the potential anti‐inflammatory function of a specific opiate (DALDA) in inflamed and resting human mucosa. Patients and methods Expression of MOR mRNA and protein was evaluated in healthy and inflamed small bowel and colonic tissues, isolated peripheral blood mononuclear cells and purified monocytes, and CD4+ and CD8+ T cells from healthy donors and IBD patients. The effect of cytokines and nuclear factor κB (NFκB) activation on MOR expression in lymphocyte T and monocytic human cell lines was assessed. Finally, DALDA induced anti‐inflammatory effect was investigated in mucosal explants from controls and IBD patients. Results MOR was expressed in ileal and colonic enteric neurones as well as in immunocytes such as myeloid cells and CD4+ and CD8+ T cells. Overexpressed in active IBD mucosa, MOR was significantly enhanced by cytokines and repressed by NFκB inhibitor in myeloid and lymphocytic cell lines. Furthermore, ex vivo DALDA treatment dampened tumour necrosis factor α mRNA expression in the colon of active IBD patients. Conclusions Given the increased expression of MOR and the ex vivo beneficial effect of DALDA in active IBD, natural and/or synthetic opioid agonists could help to prevent overt pathological intestinal inflammation. PMID:16299031

Human T-Cell Lymphotropic Virus Types 1 and 2 Seropositivity among Blood Donors at Mbarara Regional Blood Bank, South Western Uganda.

PubMed

Uchenna Tweteise, Patience; Natukunda, Bernard; Bazira, Joel

2016-01-01

Background. The human T-cell lymphotropic virus types 1 and 2 (HTLV 1/2) are retroviruses associated with different pathologies. HTLV-1 causes adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP); HTLV-2 is not clearly associated with a known clinical disease. Both viruses may be transmitted by whole blood transfusion, from mother to child predominantly through breastfeeding, and by sexual contact. Presently, none of the regional blood banks in Uganda perform routine pretransfusion screening for HTLV. The aim of this study was to determine the prevalence of anti-human T-cell lymphotropic virus types 1/2 (HTLV-1/2) antibodies among blood donors at Mbarara Regional Blood Bank in South Western Uganda. A cross-sectional study was conducted between June 2014 and September 2014. Methodology. Consecutive blood samples of 368 blood donors were screened for anti-HTLV-1/2 antibodies using an enzyme linked immunosorbent assay (ELISA). Samples reactive on a first HTLV-1/2 ELISA were further retested in duplicate using the same ELISA. Of the three hundred and sixty-eight blood donors (229 (62.2%) males and 139 (37.8%) females), only two male donors aged 20 and 21 years were HTLV-1/2 seropositive, representing a prevalence of 0.54%. Conclusion. HTLV-1/2 prevalence is low among blood donors at Mbarara Regional Blood Bank. Studies among other categories of people at risk for HTLV 1/2 infection should be carried out.

Human memory CD8 T cell effector potential is epigenetically preserved during in vivo homeostasis.

PubMed

Abdelsamed, Hossam A; Moustaki, Ardiana; Fan, Yiping; Dogra, Pranay; Ghoneim, Hazem E; Zebley, Caitlin C; Triplett, Brandon M; Sekaly, Rafick-Pierre; Youngblood, Ben

2017-06-05

Antigen-independent homeostasis of memory CD8 T cells is vital for sustaining long-lived T cell-mediated immunity. In this study, we report that maintenance of human memory CD8 T cell effector potential during in vitro and in vivo homeostatic proliferation is coupled to preservation of acquired DNA methylation programs. Whole-genome bisulfite sequencing of primary human naive, short-lived effector memory (T EM ), and longer-lived central memory (T CM ) and stem cell memory (T SCM ) CD8 T cells identified effector molecules with demethylated promoters and poised for expression. Effector-loci demethylation was heritably preserved during IL-7- and IL-15-mediated in vitro cell proliferation. Conversely, cytokine-driven proliferation of T CM and T SCM memory cells resulted in phenotypic conversion into T EM cells and was coupled to increased methylation of the CCR7 and Tcf7 loci. Furthermore, haploidentical donor memory CD8 T cells undergoing in vivo proliferation in lymphodepleted recipients also maintained their effector-associated demethylated status but acquired T EM -associated programs. These data demonstrate that effector-associated epigenetic programs are preserved during cytokine-driven subset interconversion of human memory CD8 T cells. © 2017 Abdelsamed et al.

Humanized CD7 nanobody-based immunotoxins exhibit promising anti-T-cell acute lymphoblastic leukemia potential

PubMed Central

Yu, Yuan; Li, Jialu; Zhu, Xuejun; Tang, Xiaowen; Bao, Yangyi; Sun, Xiang; Huang, Yuhui; Tian, Fang; Liu, Xiaomei; Yang, Lin

2017-01-01

Background Nanobodies, named as VHHs (variable domain of heavy chain of HCAb [heavy-chain antibodies]), are derived from heavy-chain-only antibodies that circulate in sera of camelids. Their exceptional physicochemical properties, possibility of humanization, and unique antigen recognition properties make them excellent candidates for targeted delivery of biologically active components, including immunotoxins. In our previous efforts, we have successfully generated the monovalent and bivalent CD7 nanobody-based immunotoxins, which can effectively trigger the apoptosis of CD7-positive malignant cells. To pursue the possibility of translating those immunotoxins into clinics, we humanized the nanobody sequences (designated as dhuVHH6) as well as further truncated the Pseudomonas exotoxin A (PE)-derived PE38 toxin to produce a more protease-resistant form, which is named as PE-LR, by deleting majority of PE domain II. Methods and results Three new types of immunotoxins, dhuVHH6-PE38, dVHH6-PE-LR, and dhuVHH6-PE-LR, were successfully constructed. These recombinant immunotoxins were expressed in Escherichia coli and showed that nanobody immunotoxins have the benefits of easy soluble expression in a prokaryotic expression system. Flow cytometry results revealed that all immunotoxins still maintained the ability to bind specifically to CD7-positive T lymphocyte strains without binding to CD7-negative control cells. Laser scanning confocal microscopy revealed that these proteins can be endocytosed into the cytoplasm after binding with CD7-positive cells and that this phenomenon was not observed in CD7-negative cells. WST-8 experiments showed that all immunotoxins retained the highly effective and specific growth inhibition activity in CD7-positive cell lines and primary T-cell acute lymphoblastic leukemia (T-ALL) cells. Further in vivo animal model experiments showed that humanized dhuVHH6-PE38 immunotoxin can tolerate higher doses and extend the survival of NOD-Prkdcem26Il2rgem26Nju (NCG) mice transplanted with CEM cells without any obvious decrease in body weight. Further studies on NCG mice model with patient-derived T-ALL cells, dhuVHH6-PE38 treatment, significantly prolonged mice survival with ~40% survival improvement. However, it was also noticed that although dhuVHH6-PE-LR showed strong antitumor effect in vitro, its in vivo antitumor efficacy was disappointing. Conclusion We have successfully constructed a targeted CD7 molecule-modified nanobody (CD7 molecule-improved nanobody) immunotoxin dhuVHH6-PE38 and demonstrated its potential for treating CD7-positive malignant tumors, especially T-cell acute lymphoblastic leukemia. PMID:28331319

Enforced IL-10 Expression Confers Type 1 Regulatory T Cell (Tr1) Phenotype and Function to Human CD4+ T Cells

PubMed Central

Andolfi, Grazia; Fousteri, Georgia; Rossetti, Maura; Magnani, Chiara F; Jofra, Tatiana; Locafaro, Grazia; Bondanza, Attilio; Gregori, Silvia; Roncarolo, Maria-Grazia

2012-01-01

Type 1 regulatory T (Tr1) cells are an inducible subset of CD4+ Tr cells characterized by high levels of interleukin (IL)-10 production and regulatory properties. Several protocols to generate human Tr1 cells have been developed in vitro. However, the resulting population includes a significant fraction of contaminating non-Tr1 cells, representing a major bottleneck for clinical application of Tr1 cell therapy. We generated an homogeneous IL-10–producing Tr1 cell population by transducing human CD4+ T cells with a bidirectional lentiviral vector (LV) encoding for human IL-10 and the marker gene, green fluorescent protein (GFP), which are independently coexpressed. The resulting GFP+ LV-IL-10–transduced human CD4+ T (CD4LV-IL-10) cells expressed, upon T-cell receptor (TCR) activation, high levels of IL-10 and concomitant low levels of IL-4, and markers associated with IL-10. Moreover, CD4LV-IL-10 T cells displayed typical Tr1 features: the anergic phenotype, the IL-10, and transforming growth factor (TGF)-β dependent suppression of allogeneic T-cell responses, and the ability to suppress in a cell-to-cell contact independent manner in vitro. CD4LV-IL-10 T cells were able to control xeno graft-versus-host disease (GvHD), demonstrating their suppressive function in vivo. These results show that constitutive over-expression of IL-10 in human CD4+ T cells leads to a stable cell population that recapitulates the phenotype and function of Tr1 cells. PMID:22692497

Antiretroviral therapy suppressed participants with low CD4+ T-cell counts segregate according to opposite immunological phenotypes

PubMed Central

Pérez-Santiago, Josué; Ouchi, Dan; Urrea, Victor; Carrillo, Jorge; Cabrera, Cecilia; Villà-Freixa, Jordi; Puig, Jordi; Paredes, Roger; Negredo, Eugènia; Clotet, Bonaventura; Massanella, Marta; Blanco, Julià

2016-01-01

Background: The failure to increase CD4+ T-cell counts in some antiretroviral therapy suppressed participants (immunodiscordance) has been related to perturbed CD4+ T-cell homeostasis and impacts clinical evolution. Methods: We evaluated different definitions of immunodiscordance based on CD4+ T-cell counts (cutoff) or CD4+ T-cell increases from nadir value (ΔCD4) using supervised random forest classification of 74 immunological and clinical variables from 196 antiretroviral therapy suppressed individuals. Unsupervised clustering was performed using relevant variables identified in the supervised approach from 191 individuals. Results: Cutoff definition of CD4+ cell count 400 cells/μl performed better than any other definition in segregating immunoconcordant and immunodiscordant individuals (85% accuracy), using markers of activation, nadir and death of CD4+ T cells. Unsupervised clustering of relevant variables using this definition revealed large heterogeneity between immunodiscordant individuals and segregated participants into three distinct subgroups with distinct production, programmed cell-death protein-1 (PD-1) expression, activation and death of T cells. Surprisingly, a nonnegligible number of immunodiscordant participants (22%) showed high frequency of recent thymic emigrants and low CD4+ T-cell activation and death, very similar to immunoconcordant participants. Notably, human leukocyte antigen - antigen D related (HLA-DR) PD-1 and CD45RA expression in CD4+ T cells allowed reproducing subgroup segregation (81.4% accuracy). Despite sharp immunological differences, similar and persistently low CD4+ values were maintained in these participants over time. Conclusion: A cutoff value of CD4+ T-cell count 400 cells/μl classified better immunodiscordant and immunoconcordant individuals than any ΔCD4 classification. Immunodiscordance may present several, even opposite, immunological patterns that are identified by a simple immunological follow-up. Subgroup classification may help clinicians to delineate diverse approaches that may be needed to boost CD4+ T-cell recovery. PMID:27427875

HIV-1 infection, response to treatment and establishment of viral latency in a novel humanized T cell-only mouse (TOM) model.

PubMed

Honeycutt, Jenna B; Wahl, Angela; Archin, Nancie; Choudhary, Shailesh; Margolis, David; Garcia, J Victor

2013-10-24

The major targets of HIV infection in humans are CD4⁺ T cells. CD4⁺ T cell depletion is a hallmark of AIDS. Previously, the SCID-hu thy/liv model was used to study the effect of HIV on thymopoeisis in vivo. However, these mice did not develop high levels of peripheral T cell reconstitution and required invasive surgery for infection and analysis. Here, we describe a novel variant of this model in which thy/liv implantation results in systemic reconstitution with human T cells in the absence of any other human hematopoietic lineages. NOD/SCID-hu thy/liv and NSG-hu thy/liv mice were created by implanting human fetal thymus and liver tissues under the kidney capsule of either NOD/SCID or NSG mice. In contrast to NOD/SCID-hu thy/liv mice that show little or no human cells in peripheral blood or tissues, substantial systemic human reconstitution occurs in NSG-hu thy/liv. These mice are exclusively reconstituted with human T cells (i.e. T-cell only mice or TOM). Despite substantial levels of human T cells no signs of graft-versus-host disease (GVHD) were noted in these mice over a period of 14 months. TOM are readily infected after parenteral exposure to HIV-1. HIV replication is sustained in peripheral blood at high levels and results in modest reduction of CD4⁺ T cells. HIV-1 replication in TOM responds to daily administration of combination antiretroviral therapy (ART) resulting in strong suppression of virus replication as determined by undetectable viral load in plasma. Latently HIV infected resting CD4⁺ T cells can be isolated from suppressed mice that can be induced to express HIV ex-vivo upon activation demonstrating the establishment of latency in vivo. NSG-hu thy/liv mice are systemically reconstituted with human T cells. No other human lymphoid lineages are present in these mice (i.e. monocytes/macrophages, B cells and DC are all absent). These T cell only mice do not develop GVHD, are susceptible to HIV-1 infection and can efficiently maintain virus replication. HIV infected TOM undergoing ART harbor latently infected, resting CD4+ T cells.

Selective bispecific T cell recruiting antibody and antitumor activity of adoptive T cell transfer.

PubMed

Kobold, Sebastian; Steffen, Julius; Chaloupka, Michael; Grassmann, Simon; Henkel, Jonas; Castoldi, Raffaella; Zeng, Yi; Chmielewski, Markus; Schmollinger, Jan C; Schnurr, Max; Rothenfußer, Simon; Schendel, Dolores J; Abken, Hinrich; Sustmann, Claudio; Niederfellner, Gerhard; Klein, Christian; Bourquin, Carole; Endres, Stefan

2015-01-01

One bottleneck for adoptive T cell therapy (ACT) is recruitment of T cells into tumors. We hypothesized that combining tumor-specific T cells, modified with a marker antigen and a bispecific antibody (BiAb) that selectively recognizes transduced T cells and tumor cells would improve T cell recruitment to tumors and enhance therapeutic efficacy. SV40 T antigen-specific T cells from T cell receptor (TCR)-I-transgenic mice were transduced with a truncated human epidermal growth factor receptor (EGFR) as a marker protein. Targeting and killing by combined ACT and anti-EGFR-anti-EpCAM BiAb therapy was analyzed in C57Bl/6 mice (n = six to 12 per group) carrying subcutaneous tumors of the murine gastric cancer cell line GC8 (SV40(+) and EpCAM(+)). Anti-EGFR x anti-c-Met BiAb was used for targeting of human tumor-specific T cells to c-Met(+) human tumor cell lines. Differences between experimental conditions were analyzed using the Student's t test, and differences in tumor growth with two-way analysis of variance. Overall survival was analyzed by log-rank test. All statistical tests were two-sided. The BiAb linked EGFR-transduced T cells to tumor cells and enhanced tumor cell lysis. In vivo, the combination of ACT and Biab produced increased T cell infiltration of tumors, retarded tumor growth, and prolonged survival compared with ACT with a control antibody (median survival 95 vs 75 days, P < .001). In human cells, this strategy enhanced recruitment of human EGFR-transduced T cells to immobilized c-Met and recognition of tyrosinase(+) melanoma cells by TCR-, as well as of CEA(+) colon cancer cells by chimeric antigen receptor (CAR)-modified T cells. BiAb recruitment of tumor-specific T cells transduced with a marker antigen to tumor cells may enhance efficacy of ACT. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

PubMed

Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

1992-05-15

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes.

T cells establish and maintain CNS viral infection in HIV-infected humanized mice.

PubMed

Honeycutt, Jenna B; Liao, Baolin; Nixon, Christopher C; Cleary, Rachel A; Thayer, William O; Birath, Shayla L; Swanson, Michael D; Sheridan, Patricia; Zakharova, Oksana; Prince, Francesca; Kuruc, JoAnn; Gay, Cynthia L; Evans, Chris; Eron, Joseph J; Wahl, Angela; Garcia, J Victor

2018-06-04

The human brain is an important site of HIV replication and persistence during antiretroviral therapy (ART). Direct evaluation of HIV infection in the brains of otherwise healthy individuals is not feasible; therefore, we performed a large-scale study of bone marrow/liver/thymus (BLT) humanized mice as an in vivo model to study HIV infection in the brain. Human immune cells, including CD4+ T cells and macrophages, were present throughout the BLT mouse brain. HIV DNA, HIV RNA, and/or p24+ cells were observed in the brains of HIV-infected animals, regardless of the HIV isolate used. HIV infection resulted in decreased numbers of CD4+ T cells, increased numbers of CD8+ T cells, and a decreased CD4+/CD8+ T cell ratio in the brain. Using humanized T cell-only mice (ToM), we demonstrated that T cells establish and maintain HIV infection of the brain in the complete absence of human myeloid cells. HIV infection of ToM resulted in CD4+ T cell depletion and a reduced CD4+/CD8+ T cell ratio. ART significantly reduced HIV levels in the BLT mouse brain, and the immune cell populations present were indistinguishable from those of uninfected controls, which demonstrated the effectiveness of ART in controlling HIV replication in the CNS and returning cellular homeostasis to a pre-HIV state.

The Toxoplasma gondii ME-49 strain upregulates levels of A20 that inhibit NF-κB activation and promotes apoptosis in human leukaemia T-cell lines.

PubMed

Chen, Qian; Pang, Min-Hui; Ye, Xiao-Hong; Yang, Guang; Lin, Chen

2018-05-18

Acute T-lymphocyte leukaemia is a form of haematological malignancy with abnormal activation of NF-κB pathway, which results in high expression of A20 and ABIN1, which constitute a negative feedback mechanism for the regulation of NF-κB activation. Clinical studies have found that acute T-lymphocyte leukaemia patients are susceptible to Toxoplasma gondii infection; however, the effect of T. gondii on the proliferation and apoptosis of human leukaemia T-cells remains unclear. Here, we used the T. gondii ME-49 strain to infect human leukaemia T-cell lines Jurkat and Molt-4, to explore the effect of T. gondii on proliferation and apoptosis, which is mediated by NF-κB in human leukaemia T-cells. The Tunel assay was used to detect cell apoptosis. Cell Counting Kit-8 was used to detect cell proliferation viability. The apoptosis level and the expression level of NF-κB related proteins in human leukaemia T-cells were detected by flow cytometry and Western blotting. Western blotting analyses revealed that the T. gondii ME-49 strain increased the expression of A20 and decreased both ABIN1 expression and NF-κB p65 phosphorylation. By constructing a lentiviral-mediated shRNA to knockdown the A20 gene in Jurkat T-cells and Molt-4 T-cells, the apoptosis levels of the two cell lines decreased after T. gondii ME-49 infection, and levels of NF-κB p65 phosphorylation and ABIN1 were higher than in the non-konckdown group. After knockingdown ABIN1 gene expression by constructing the lentiviral-mediated shRNA and transfecting the recombinant expression plasmid containing the ABIN1 gene into two cell lines, apoptosis levels and cleaved caspase-8 expression increased or decreased in response to T. gondii ME-49 infection, respectively. Our data suggest that ABIN1 protects human leukaemia T-cells by allowing them to resist the apoptosis induced by T. gondii ME-49 and that the T. gondii ME-49 strain induces the apoptosis of human leukaemia T-cells via A20-mediated downregulation of ABIN1 expression.

Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase

PubMed Central

Agúndez, Leticia; González-Prieto, Coral; Machón, Cristina; Llosa, Matxalen

2012-01-01

Background Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering. Methodology/Principal Findings We have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome. Conclusions/Significance The results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells. PMID:22292089

Adoptive cell therapy: genetic modification to redirect effector cell specificity.

PubMed

Morgan, Richard A; Dudley, Mark E; Rosenberg, Steven A

2010-01-01

Building on the principals that the adoptive transfer of T cells can lead to the regression of established tumors in humans, investigators are now further manipulating these cells using genetic engineering. Two decades of human gene transfer experiments have resulted in the translation of laboratory technology into robust clinical applications. The purpose of this review is to give the reader an introduction to the 2 major approaches being developed to redirect effector T-cell specificity. Primary human T cells can be engineered to express exogenous T-cell receptors or chimeric antigen receptors directed against multiple human tumor antigens. Initial clinical trial results have demonstrated that both T-cell receptor- and chimeric antigen receptor-engineered T cells can be administered to cancer patients and mediate tumor regression.

Generation and application of human induced-stem cell memory T (iTSCM ) cells for adoptive immunotherapy.

PubMed

Kondo, Taisuke; Imura, Yuuki; Chikuma, Shunsuke; Hibino, Sana; Omata-Mise, Setsuko; Ando, Makoto; Akanuma, Takashi; Iizuka, Mana; Sakai, Ryota; Morita, Rimpei; Yoshimura, Akihiko

2018-05-23

Adoptive T cell therapy is an effective strategy for cancer immunotherapy. However, infused T cells frequently become functionally exhausted, and consequently offer a poor prognosis after transplantation into patients. Adoptive transfer of tumor antigen-specific stem cell memory T (T SCM ) cells is expected to overcome this shortcoming since T SCM cells are close to naïve T cells, but are also highly proliferative, long-lived, and produce a large number of effector T cells in response to antigen stimulation. We previously reported that activated effector T cells can be converted into T SCM -like cells (iT SCM ) by co-culturing with OP9 cells expressing Notch ligand, Delta-like 1 (OP9-hDLL1). Here we show the methodological parameters of human CD8 + iT SCM cell generation and their application to adoptive cancer immunotherapy. Regardless of the stimulation by anti-CD3/CD28 antibodies or by antigen-presenting cells, human iT SCM cells were more efficiently induced from central memory type T cells than from effector memory T cells. During the induction phase by co-culture with OP9-hDLL1 cells, IL-7 and IL-15 (but not IL-2 or IL-21) could efficiently generate iT SCM cells. Epstein Barr (EB) virus-specific iT SCM cells showed much stronger antitumor potentials than conventionally activated T cells did in humanized EB virus transformed-tumor model mice. Thus, adoptive T cell therapy with iT SCM offers a promising therapeutic strategy for cancer immunotherapy. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

1,25D3 enhances antitumor activity of gemcitabine and cisplatin in human bladder cancer models

PubMed Central

Ma, Yingyu; Yu, Wei-Dong; Trump, Donald L.; Johnson, Candace S.

2010-01-01

Background 1,25 dihydroxyvitamin D3 (1,25D3) potentiates the cytotoxic effects of several common chemotherapeutic agents. The combination of gemcitabine and cisplatin (GC) is a current standard chemotherapy regimen for bladder cancer. We investigated whether 1,25D3 could enhance the antitumor activity of GC in bladder cancer model systems. Methods Human bladder cancer T24 and UMUC3 cells were pretreated with 1,25D3 followed by GC. Apoptosis were assessed by annexin V staining. Caspase activation was examined by immunoblot analysis and substrate-based caspase activity assay. The cytotoxic effects were examined using MTT and in vitro clonogenic assay. p73 protein levels were assessed by immunoblot analysis. Knockdown of p73 was achieved by siRNA. The in vivo antitumor activity was assessed by in vivo excision clonogenic assay and tumor regrowth delay in the T24 xenograft model. Results 1,25D3 pretreatment enhanced GC-induced apoptosis and the activities of caspases- 8, 9 and 3 in T24 and UMUC3 cells. 1,25D3 synergistically reduced GC-suppressed surviving fraction in T24 cells. 1,25D3, gemcitabine, or cisplatin induced p73 accumulation, which was enhanced by GC or 1,25D3 and GC. p73 expression was lower in human primary bladder tumor tissue compared with adjacent normal tissue. Knockdown of p73 increased clonogenic capacity of T24 cells treated with 1,25D3, GC or 1,25D3 and GC. 1,25D3 and GC combination enhanced tumor regression compared with 1,25D3 or GC alone. Conclusions 1,25D3 potentiates GC-mediated growth inhibition in human bladder cancer models in vitro and in vivo, which involves p73 induction and apoptosis. PMID:20564622

Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells

PubMed Central

Kebriaei, Partow; Singh, Harjeet; Huls, M. Helen; Figliola, Matthew J.; Bassett, Roland; Olivares, Simon; Jena, Bipulendu; Dawson, Margaret J.; Kumaresan, Pappanaicken R.; Su, Shihuang; Maiti, Sourindra; Dai, Jianliang; Moriarity, Branden; Forget, Marie-Andrée; Senyukov, Vladimir; Orozco, Aaron; Liu, Tingting; McCarty, Jessica; Jackson, Rineka N.; Moyes, Judy S.; Rondon, Gabriela; Qazilbash, Muzaffar; Ciurea, Stefan; Alousi, Amin; Nieto, Yago; Rezvani, Katy; Marin, David; Popat, Uday; Hosing, Chitra; Shpall, Elizabeth J.; Kantarjian, Hagop; Keating, Michael; Wierda, William; Do, Kim Anh; Largaespada, David A.; Lee, Dean A.; Hackett, Perry B.; Champlin, Richard E.; Cooper, Laurence J.N.

2016-01-01

BACKGROUND. T cells expressing antigen-specific chimeric antigen receptors (CARs) improve outcomes for CD19-expressing B cell malignancies. We evaluated a human application of T cells that were genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific CAR. METHODS. T cells were genetically modified using DNA plasmids from the SB platform to stably express a second-generation CD19-specific CAR and selectively propagated ex vivo with activating and propagating cells (AaPCs) and cytokines. Twenty-six patients with advanced non-Hodgkin lymphoma and acute lymphoblastic leukemia safely underwent hematopoietic stem cell transplantation (HSCT) and infusion of CAR T cells as adjuvant therapy in the autologous (n = 7) or allogeneic settings (n = 19). RESULTS. SB-mediated genetic transposition and stimulation resulted in 2,200- to 2,500-fold ex vivo expansion of genetically modified T cells, with 84% CAR expression, and without integration hotspots. Following autologous HSCT, the 30-month progression-free and overall survivals were 83% and 100%, respectively. After allogeneic HSCT, the respective 12-month rates were 53% and 63%. No acute or late toxicities and no exacerbation of graft-versus-host disease were observed. Despite a low antigen burden and unsupportive recipient cytokine environment, CAR T cells persisted for an average of 201 days for autologous recipients and 51 days for allogeneic recipients. CONCLUSIONS. CD19-specific CAR T cells generated with SB and AaPC platforms were safe, and may provide additional cancer control as planned infusions after HSCT. These results support further clinical development of this nonviral gene therapy approach. TRIAL REGISTRATION. Autologous, NCT00968760; allogeneic, NCT01497184; long-term follow-up, NCT01492036. FUNDING. National Cancer Institute, private foundations, and institutional funds. Please see Acknowledgments for details. PMID:27482888

Tissue reservoirs of antiviral T cell immunity in persistent human CMV infection

PubMed Central

Gordon, Claire L.; Thome, Joseph J.C.; Igarashi, Suzu

2017-01-01

T cell responses to viruses are initiated and maintained in tissue sites; however, knowledge of human antiviral T cells is largely derived from blood. Cytomegalovirus (CMV) persists in most humans, requires T cell immunity to control, yet tissue immune responses remain undefined. Here, we investigated human CMV-specific T cells, virus persistence and CMV-associated T cell homeostasis in blood, lymphoid, mucosal and secretory tissues of 44 CMV seropositive and 28 seronegative donors. CMV-specific T cells were maintained in distinct distribution patterns, highest in blood, bone marrow (BM), or lymph nodes (LN), with the frequency and function in blood distinct from tissues. CMV genomes were detected predominantly in lung and also in spleen, BM, blood and LN. High frequencies of activated CMV-specific T cells were found in blood and BM samples with low virus detection, whereas in lung, CMV-specific T cells were present along with detectable virus. In LNs, CMV-specific T cells exhibited quiescent phenotypes independent of virus. Overall, T cell differentiation was enhanced in sites of viral persistence with age. Together, our results suggest tissue T cell reservoirs for CMV control shaped by both viral and tissue-intrinsic factors, with global effects on homeostasis of tissue T cells over the lifespan. PMID:28130404

Tissue reservoirs of antiviral T cell immunity in persistent human CMV infection.

PubMed

Gordon, Claire L; Miron, Michelle; Thome, Joseph J C; Matsuoka, Nobuhide; Weiner, Joshua; Rak, Michael A; Igarashi, Suzu; Granot, Tomer; Lerner, Harvey; Goodrum, Felicia; Farber, Donna L

2017-03-06

T cell responses to viruses are initiated and maintained in tissue sites; however, knowledge of human antiviral T cells is largely derived from blood. Cytomegalovirus (CMV) persists in most humans, requires T cell immunity to control, yet tissue immune responses remain undefined. Here, we investigated human CMV-specific T cells, virus persistence and CMV-associated T cell homeostasis in blood, lymphoid, mucosal and secretory tissues of 44 CMV seropositive and 28 seronegative donors. CMV-specific T cells were maintained in distinct distribution patterns, highest in blood, bone marrow (BM), or lymph nodes (LN), with the frequency and function in blood distinct from tissues. CMV genomes were detected predominantly in lung and also in spleen, BM, blood and LN. High frequencies of activated CMV-specific T cells were found in blood and BM samples with low virus detection, whereas in lung, CMV-specific T cells were present along with detectable virus. In LNs, CMV-specific T cells exhibited quiescent phenotypes independent of virus. Overall, T cell differentiation was enhanced in sites of viral persistence with age. Together, our results suggest tissue T cell reservoirs for CMV control shaped by both viral and tissue-intrinsic factors, with global effects on homeostasis of tissue T cells over the lifespan. @Gordon et al.

Polarized Th2 like cells, in the absence of Th0 cells, are responsible for lymphocyte produced IL-4 in high IgE-producer schistosomiasis patients

PubMed Central

Dutra, Walderez O; Correa-Oliveira, Rodrigo; Dunne, David; Cecchini, Luiza Fosenca; Fraga, Lúcia; Roberts, Morven; Soares-Silveira, Alda Maria; Webster, Michelle; Yssel, Hans; Gollob, Kenneth J

2002-01-01

Background Human resistance to re-infection with S. mansoni is correlated with high levels of anti-soluble adult worm antigens (SWAP) IgE. Although it has been shown that IL-4 and IL-5 are crucial in establishing IgE responses in vitro, the active in vivo production of these cytokines by T cells, and the degree of polarization of Th2 vs. Th0 in human schistosomiasis is not known. To address this question, we determined the frequency of IL-4 and IFN-γ or IL-5 and IL-2 producing lymphocytes from schistosomiasis patients with high or low levels of IgE anti-SWAP. Results Our analysis showed that high and low IgE-producers responded equally to schistosomiasis antigens as determined by proliferation. Moreover, patients from both groups displayed similar percentages of circulating lymphocytes. However, high IgE-producers had an increased percentage of activated CD4+ T cells as compared to the low IgE-producers. Moreover, intracellular cytokine analysis, after short-term stimulation with anti-CD3/CD28 mAbs, showed that IgE high-producers display an increase in the percentage of T lymphocytes expressing IL-4 and IL-5 as compared to IgE low-responders. A coordinate control of the frequency of IL-4 and IL-5 producing lymphocytes in IgE high, but not IgE low-responders, was observed. Conclusions High IgE phenotype human schistosomiasis patients exhibit a coordinate regulation of IL-4 and IL-5 producing cells and the lymphocyte derived IL-4 comes from true polarized Th2 like cells, in the absence of measurable Th0 cells as measured by co-production of IL-4 and IFN-γ. PMID:12100735

T lymphocyte recruitment by interleukin-8 (IL-8). IL-8-induced degranulation of neutrophils releases potent chemoattractants for human T lymphocytes both in vitro and in vivo.

PubMed Central

Taub, D D; Anver, M; Oppenheim, J J; Longo, D L; Murphy, W J

1996-01-01

IL-8 has been shown to be a human neutrophil and T cell chemoattractant in vitro. In an effort to assess the in vivo effects of IL-8 on human leukocyte migration, we examined the ability of rhIL-8 to induce human T cell infiltration using a human/mouse model in which SCID mice were administered human peripheral blood lymphocytes intraperitoneally, followed by subcutaneous injections of rhIL-8. rhIL-8 induced predominantly murine neutrophil accumulation by 4 h after administration while recombinant human macrophage inflammatory protein-1beta (rhMIP-1beta) induced both murine monocytes and human T cell infiltration during the same time period as determined by immunohistology. Interestingly, 72 h after chemokine administration, a marked human T cell infiltrate was observed in the IL-8 injection site suggesting that rhIL-8 may be acting indirectly possibly through a murine neutrophil-derived T cell chemoattractant. This hypothesis was confirmed using granulocyte-depleted SCID mice. Moreover, human neutrophils stimulated in vitro with IL-8 were found to release granule-derived factor(s) that induce in vitro T cell and monocyte chemotaxis and chemokinesis. This T cell and monocyte chemotactic activity was detected in extracts of both azurophilic and specific granules. Together, these results demonstrate that neutrophils store and release, upon stimulation with IL-8 or other neutrophil activators, chemoattractants that mediate T cell and monocyte accumulation at sites of inflammation. PMID:8621778

A third generation vaccine for human visceral leishmaniasis and post kala azar dermal leishmaniasis: First-in-human trial of ChAd63-KH

PubMed Central

Osman, Mohamed; Mistry, Anoop; Keding, Ada; Cook, Elizabeth; Wiggins, Rebecca; Di Marco, Stefania; Colloca, Stefano; Siani, Loredana; Smith, Deborah F.; Aebischer, Toni; Lacey, Charles J.

2017-01-01

Background Visceral leishmaniasis (VL or kala azar) is the most serious form of human leishmaniasis, responsible for over 20,000 deaths annually, and post kala azar dermal leishmaniasis (PKDL) is a stigmatizing skin condition that often occurs in patients after successful treatment for VL. Lack of effective or appropriately targeted cell mediated immunity, including CD8+ T cell responses, underlies the progression of VL and progression to PKDL, and can limit the therapeutic efficacy of anti-leishmanial drugs. Hence, in addition to the need for prophylactic vaccines against leishmaniasis, the development of therapeutic vaccines for use alone or in combined immuno-chemotherapy has been identified as an unmet clinical need. Here, we report the first clinical trial of a third-generation leishmaniasis vaccine, developed intentionally to induce Leishmania-specific CD8+ T cells. Methods We conducted a first-in-human dose escalation Phase I trial in 20 healthy volunteers to assess the safety, tolerability and immunogenicity of a prime-only adenoviral vaccine for human VL and PKDL. ChAd63-KH is a replication defective simian adenovirus expressing a novel synthetic gene (KH) encoding two Leishmania proteins KMP-11 and HASPB. Uniquely, the latter was engineered to reflect repeat domain polymorphisms and arrangements identified from clinical isolates. We monitored innate immune responses by whole blood RNA-Seq and antigen specific CD8+ T cell responses by IFNγ ELISPOT and intracellular flow cytometry. Findings ChAd63-KH was safe at intramuscular doses of 1x1010 and 7.5x1010 vp. Whole blood transcriptomic profiling indicated that ChAd63-KH induced innate immune responses characterized by an interferon signature and the presence of activated dendritic cells. Broad and quantitatively robust CD8+ T cell responses were induced by vaccination in 100% (20/20) of vaccinated subjects. Conclusion The results of this study support the further development of ChAd63-KH as a novel third generation vaccine for VL and PKDL. Trial registration This clinical trial (LEISH1) was registered at EudraCT (2012-005596-14) and ISRCTN (07766359). PMID:28498840

Epigallocatechin-3-gallate (EGCG) up-regulates miR-15b expression thus attenuating store operated calcium entry (SOCE) into murine CD4+ T cells and human leukaemic T cell lymphoblasts.

PubMed

Zhang, Shaqiu; Al-Maghout, Tamer; Bissinger, Rosi; Zeng, Ni; Pelzl, Lisann; Salker, Madhuri S; Cheng, Anchun; Singh, Yogesh; Lang, Florian

2017-10-27

CD4 + T cells are key elements in immune responses and inflammation. Activation of T cell receptors in CD4 + T cells triggers cytosolic Ca 2+ release with subsequent store operated Ca 2+ entry (SOCE), which is accomplished by the pore forming Ca 2+ release activated Ca 2+ (CRAC) channel Orai1 and its regulator stromal cell-interaction molecule 2 (STIM2). Green tea polyphenol epigallocatechin-3-gallate (EGCG) acts as a potent anti-inflammatory and anti-oxidant agent for various types of cells including immune cells. However, how post-transcriptional gene regulators such as miRNAs are involved in the regulation of Ca 2+ influx into murine CD4 + T cells and human Jurkat T cells through EGCG is not defined. EGCG treatment of murine CD4 + T cells significantly down-regulated the expression of STIM2 and Orai1 both at mRNA and protein levels. Furthermore, EGCG significantly decreased SOCE in both murine and human T cells. EGCG treatment increased miRNA-15b (miR-15b) abundance in both murine and human T cells. Bioinformatics analysis reveals that miR-15b, which has a STIM2 binding site, is involved in the down-regulation of SOCE. Overexpression of miR-15b significantly decreased the mRNA and protein expression of STIM2 and Orai1 in murine T cells. Treatment of Jurkat T cells with 10 μM EGCG further decreased mTOR and PTEN protein levels. EGCG decreased mitochondrial membrane potential (MMP) in both human and murine T cells. In conclusion, the observations suggest that EGCG inhibits the Ca 2+ entry into murine and human T cells, an effect accomplished at least in part by up-regulation of miR-15b.

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

PubMed Central

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

2013-01-01

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

Complete suppression of in vivo growth of human leukemia cells by specific immunotoxins: nude mouse models

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

Hara, H.; Seon, B.K.

1987-05-01

In this study, immunotoxins containing monoclonal anti-human T-cell leukemia antibodies are shown to be capable of completely suppressing the tumor growth of human T-cell leukemia cells in vivo without any overt undersirable toxicity. These immunotoxins were prepared by conjugating ricin A chain (RA) with our monoclonal antibodies, SN1 and SN2, directed specifically to the human T-cell leukemia cell surface antigens TALLA and GP37, respectively. The authors have shown that these monoclonal antibodies are highly specific for human T-cell leukemia cells and do not react with various normal cells including normal T and B cells, thymocytes, and bone marrow cells. Asciticmore » and solid human T-cell leukemia cell tumors were generated in nude mice. The ascitic tumor was generated by transplanting Ichikawa cells (a human T-cell leukemia cell) i.p. into nude mice, whereas the solid tumor was generated by transplanting s.c. MOLT-4 cells (a human T-cell leukemia cell line) and x-irradiated human fibrosarcoma cells into x-irradiated nude mice. To investigate the efficacy of specific immunotoxins in suppression the in vivo growth of the ascitic tumor, they divided 40 nude mice that were injected with Ichikawa cells into four groups. None of the mice in group 4 that were treated with SN1-RA and SN2-RA showed any signs of a tumor or undesirable toxic effects for the 20 weeks that they were followed after the transplantation. Treatment with SN1-RA plus SN2-RA completely suppressed solid tumor growth in 4 of 10 nude mice carrying solid tumors and partially suppressed the tumor growth in the remaining 6 nude mice. These results strongly suggest that SN1-RA and SN2-RA may be useful for clinical treatment.« less

Primary Central Nervous System T-Cell Lymphoma With Aberrant Expression of CD20 and CD79a: A Diagnostic Pitfall.

PubMed

Gupta, Neha; Nasim, Mansoor; Spitzer, Silvia G; Zhang, Xinmin

2017-10-01

Primary central nervous system T-cell lymphoma (PCNSTCL) is rare, accounting for 2% of CNS lymphomas. We report the first case of PCNSTCL with aberrant expression of CD20 and CD79a in an 81-year-old man with a left periventricular brain mass. A biopsy revealed dense lymphoid infiltrate consisting of medium-sized cells in a background of gliosis and many histiocytes. The lymphoid cells were positive for CD2, CD3, CD7, CD8, T-cell intracellular antigen-1, granzyme B, CD20, and CD79a and negative for CD4, CD5, PAX-5, OCT-2, BOB-1, human herpes virus-8, and Epstein-Barr virus-encoded small RNAs. Molecular studies revealed clonal TCR-β and TCR-γ gene rearrangements and negative immunoglobulin gene rearrangements. The patient was treated with chemotherapy (vincristine and methotrexate) and rituximab, but he died 1 month after the diagnosis. This is a unique case that emphasizes the use of a multimodal approach, including a broad immunohistochemical panel and molecular studies in lineage determination for lymphomas with ambiguous phenotype.

Type 1 diabetes vaccine candidates promote human Foxp3+Treg induction in humanized mice

PubMed Central

Serr, Isabelle; Fürst, Rainer W.; Achenbach, Peter; Scherm, Martin G.; Gökmen, Füsun; Haupt, Florian; Sedlmeier, Eva-Maria; Knopff, Annette; Shultz, Leonard; Willis, Richard A.; Ziegler, Anette-Gabriele; Daniel, Carolin

2016-01-01

Immune tolerance is executed partly by Foxp3+regulatory T (Treg) cells, which suppress autoreactive T cells. In autoimmune type 1 diabetes (T1D) impaired tolerance promotes destruction of insulin-producing β-cells. The development of autoantigen-specific vaccination strategies for Foxp3+Treg-induction and prevention of islet autoimmunity in patients is still in its infancy. Here, using human haematopoietic stem cell-engrafted NSG-HLA-DQ8 transgenic mice, we provide direct evidence for human autoantigen-specific Foxp3+Treg-induction in vivo. We identify HLA-DQ8-restricted insulin-specific CD4+T cells and demonstrate efficient human insulin-specific Foxp3+Treg-induction upon subimmunogenic vaccination with strong agonistic insulin mimetopes in vivo. Induced human Tregs are stable, show increased expression of Treg signature genes such as Foxp3, CTLA4, IL-2Rα and TIGIT and can efficiently suppress effector T cells. Such Foxp3+Treg-induction does not trigger any effector T cells. These T1D vaccine candidates could therefore represent an expedient improvement in the challenge to induce human Foxp3+Tregs and to develop novel precision medicines for prevention of islet autoimmunity in children at risk of T1D. PMID:26975663

Human Effector Memory T Helper Cells Engage with Mouse Macrophages and Cause Graft-versus-Host-Like Pathology in Skin of Humanized Mice Used in a Nonclinical Immunization Study.

PubMed

Sundarasetty, Balasai; Volk, Valery; Theobald, Sebastian J; Rittinghausen, Susanne; Schaudien, Dirk; Neuhaus, Vanessa; Figueiredo, Constanca; Schneider, Andreas; Gerasch, Laura; Mucci, Adele; Moritz, Thomas; von Kaisenberg, Constantin; Spineli, Loukia M; Sewald, Katherina; Braun, Armin; Weigt, Henning; Ganser, Arnold; Stripecke, Renata

2017-06-01

Humanized mice engrafted with human hematopoietic stem cells and developing functional human T-cell adaptive responses are in critical demand to test human-specific therapeutics. We previously showed that humanized mice immunized with long-lived induced-dendritic cells loaded with the pp65 viral antigen (iDCpp65) exhibited a faster development and maturation of T cells. Herein, we evaluated these effects in a long-term (36 weeks) nonclinical model using two stem cell donors to assess efficacy and safety. Relative to baseline, iDCpp65 immunization boosted the output of effector memory CD4 + T cells in peripheral blood and lymph nodes. No weight loss, human malignancies, or systemic graft-versus-host (GVH) disease were observed. However, for one reconstitution cohort, some mice immunized with iDCpp65 showed GVH-like signs on the skin. Histopathology analyses of the inflamed skin revealed intrafollicular and perifollicular human CD4 + cells near F4/80 + mouse macrophages around hair follicles. In spleen, CD4 + cells formed large clusters surrounded by mouse macrophages. In plasma, high levels of human T helper 2-type inflammatory cytokines were detectable, which activated in vitro the STAT5 pathway of murine macrophages. Despite this inflammatory pattern, human CD8 + T cells from mice with GVH reacted against the pp65 antigen in vitro. These results uncover a dynamic cross-species interaction between human memory T cells and mouse macrophages in the skin and lymphatic tissues of humanized mice. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Analyses of variant human papillomavirus type-16 E5 proteins for their ability to induce mitogenesis of murine fibroblasts

PubMed Central

Nath, Rahul; Mant, Christine A; Kell, Barbara; Cason, John; Bible, Jon M

2006-01-01

Background Human papillomavirus type 16 (HPV-16) E5 protein co-operates with epidermal growth factor to stimulate mitogenesis of murine fibroblasts. Currently, little is known about which viral amino acids are involved in this process. Using sequence variants of HPV-16 E5 we have investigated their effects upon E5 transcription, cell-cycling and cell-growth of murine fibroblasts. Results We demonstrate that: (i) introduction of Thr64 into the reference E5 sequence of HPV-16 abrogates mitogenic activity: both were poorly transcribed in NIH-3T3 cells; (ii) substitution of Leu44Val65 or, Thr37Leu44Val65 into the HPV-16 E5 reference backbone resulted in high transcription in NIH-3T3 cells, enhanced cell-cycle progression and high cell-growth; and, (iii) inclusion of Tyr8 into the Leu44Val65 backbone inhibited E5 induced cell-growth and repression of p21 expression, despite high transcription levels. Conclusion The effects of HPV-16 E5 variants upon mitosis help to explain why Leu44Val65 HPV-16 E5 variants are most prevalent in 'wild' pathogenic viral populations in the UK. PMID:16899131

Biliary epithelium and liver B cells exposed to bacteria activate intrahepatic MAIT cells through MR1

PubMed Central

Jeffery, Hannah C.; van Wilgenburg, Bonnie; Kurioka, Ayako; Parekh, Krishan; Stirling, Kathryn; Roberts, Sheree; Dutton, Emma E.; Hunter, Stuart; Geh, Daniel; Braitch, Manjit K.; Rajanayagam, Jeremy; Iqbal, Tariq; Pinkney, Thomas; Brown, Rachel; Withers, David R.; Adams, David H.; Klenerman, Paul; Oo, Ye H.

2016-01-01

Background & Aims Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells characterised by the invariant TCR-chain, Vα7.2-Jα33, and are restricted by MR1, which presents bacterial vitamin B metabolites. They are important for antibacterial immunity at mucosal sites; however, detailed characteristics of liver-infiltrating MAIT (LI-MAIT) and their role in biliary immune surveillance remain unexplored. Methods The phenotype and intrahepatic localisation of human LI-MAIT cells was examined in diseased and normal livers. MAIT cell activation in response to E. coli-exposed macrophages, biliary epithelial cells (BEC) and liver B cells was assessed with/without anti-MR1. Results Intrahepatic MAIT cells predominantly localised to bile ducts in the portal tracts. Consistent with this distribution, they expressed biliary tropic chemokine receptors CCR6, CXCR6, and integrin αEβ7. LI-MAIT cells were also present in the hepatic sinusoids and possessed tissue-homing chemokine receptor CXCR3 and integrins LFA-1 and VLA-4, suggesting their recruitment via hepatic sinusoids. LI-MAIT cells were enriched in the parenchyma of acute liver failure livers compared to chronic diseased livers. LI-MAIT cells had an activated, effector memory phenotype, expressed α4β7 and receptors for IL-12, IL-18, and IL-23. Importantly, in response to E. coli-exposed macrophages, liver B cells and BEC, MAIT cells upregulated IFN-γ and CD40 Ligand and degranulated in an MR1-dependent, cytokine-independent manner. In addition, diseased liver MAIT cells expressed T-bet and RORγt and the cytokines IFN-γ, TNF-α, and IL-17. Conclusions Our findings provide the first evidence of an immune surveillance effector response for MAIT cells towards BEC in human liver; thus they could be manipulated for treatment of biliary disease in the future. PMID:26743076

Differences in Mouse and Human Non-Memory B Cell Pools1

PubMed Central

Benitez, Abigail; Weldon, Abby J.; Tatosyan, Lynnette; Velkuru, Vani; Lee, Steve; Milford, Terry-Ann; Francis, Olivia L.; Hsu, Sheri; Nazeri, Kavoos; Casiano, Carlos M.; Schneider, Rebekah; Gonzalez, Jennifer; Su, Rui-Jun; Baez, Ineavely; Colburn, Keith; Moldovan, Ioana; Payne, Kimberly J.

2014-01-01

Identifying cross-species similarities and differences in immune development and function is critical for maximizing the translational potential of animal models. Co-expression of CD21 and CD24 distinguishes transitional and mature B cell subsets in mice. Here, we validate these markers for identifying analogous subsets in humans and use them to compare the non-memory B cell pools in mice and humans, across tissues, during fetal/neonatal and adult life. Among human CD19+IgM+ B cells, the CD21/CD24 schema identifies distinct populations that correspond to T1 (transitional 1), T2 (transitional 2), FM (follicular mature), and MZ (marginal zone) subsets identified in mice. Markers specific to human B cell development validate the identity of MZ cells and the maturation status of human CD21/CD24 non-memory B cell subsets. A comparison of the non-memory B cell pools in bone marrow (BM), blood, and spleen in mice and humans shows that transitional B cells comprise a much smaller fraction in adult humans than mice. T1 cells are a major contributor to the non-memory B cell pool in mouse BM where their frequency is more than twice that in humans. Conversely, in spleen the T1:T2 ratio shows that T2 cells are proportionally ∼8 fold higher in humans than mouse. Despite the relatively small contribution of transitional B cells to the human non-memory pool, the number of naïve FM cells produced per transitional B cell is 3-6 fold higher across tissues than in mouse. These data suggest differing dynamics or mechanisms produce the non-memory B cell compartments in mice and humans. PMID:24719464

Surveillance Study of Influenza Occurrence and Immunity in a Wisconsin Cohort During the 2009 Pandemic

PubMed Central

Lo, Chia-Yun; Strobl, Susan L.; Dunham, Kimberly; Wang, Wei; Stewart, Lucy; Misplon, Julia A.; Garcia, Mayra; Gao, Jin; Ozawa, Tatsuhiko; Price, Graeme E.; Navidad, Jose; Gradus, Steve; Bhattacharyya, Sanjib; Viboud, Cecile; Eichelberger, Maryna C.; Weiss, Carol D.; Gorski, Jack

2017-01-01

Abstract Background. Antibody and T-cell immunity to conserved influenza virus antigens can protect animals against infection with diverse influenza strains. Although immunity against conserved antigens occurs in humans, whether such responses provide cross-protection in humans and could be harnessed as the basis for universal influenza vaccines is controversial. The 2009 pandemic provided an opportunity to investigate whether pre-existing cross-reactive immunity affected susceptibility to infection. Methods. In 2009, we banked sera and peripheral blood mononuclear cells (PBMC) from blood donors, then monitored them for pandemic influenza infection (pH1N1) by polymerase chain reaction or seroconversion. Antibodies to hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix 2 (M2), and HA-pseudotypes were measured in sera. T-cell inteferon-γ enzyme-linked immunospot responses were measured in PBMC. Results. There were 13 infections in 117 evaluable donors. Pre-existing T-cell reactivity to pH1N1 was substantial (of 153 donors tested, 146 had >100 spot-forming cells/106 cells). Antibodies reactive with pH1N1 were common: anti-NP (all donors) and anti-M2 (44% of donors). Pseudotype-neutralizing antibodies to H1 were detected, but not to highly conserved HA epitopes. Unexpectedly, donors with symptomatic pH1N1 infection had sharp rises in HA pseudotype-neutralizing antibodies, not only pH1N1 but also against multiple seasonal H1s. In addition, an exploratory study of a T-cell marker (response to NP418-426) identified probable infection missed by standard criteria. Conclusions. Although the number of infections was inadequate for conclusions about mechanisms of protection, this study documents the wide variety of pre-existing, cross-reactive, humoral and cellular immune responses to pandemic influenza virus antigens in humans. These responses can be compared with results of other studies and explored in universal influenza vaccine studies. PMID:28730155

Cytotoxic activity of ten algae from the Persian Gulf and Oman Sea on human breast cancer cell lines; MDA-MB-231, MCF-7, and T-47D

PubMed Central

Erfani, Nasrollah; Nazemosadat, Zahra; Moein, Mahmoodreza

2015-01-01

Background: Seaweeds have proven to be a promising natural source of bioactive metabolites for drug development. Objective: This study aimed to monitor the ethanol extract of ten algae from the Persian Gulf and Oman Sea, for their in vitro cytotoxic activity on three human breast cancer cell lines. Materials and Methods: Three human breast cancer cell lines including MDA-MB-231(ER−), MCF-7(ER+), and T-47D (ER+) were treated by different concentrations of total ethanol (90%) algae extracts and the cytotoxic effects were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Doxorubicin (Ebewe, Austria) was used as a positive control. After 72 h of incubation, the cytotoxic effect of the algae was calculated and presented as 50%-inhibitory concentration (IC50). Results: The results indicated Gracilaria foliifera and Cladophoropsis sp. to be the most active algae in terms of cytotoxic effects on the investigated cancer cell lines. The IC50 values against MDA-MB-231, MCF-7, and T-47D cells were, respectively, 74.89 ± 21.71, 207.81 ± 12.07, and 203.25 ± 30.98 µg/ml for G. foliifera and 66.48 ± 4.96, 150.86 ± 51.56 and >400 µg/ml for Cladophoropsis sp. The rest of the algal extracts were observed not to have significant cytotoxic effects in the concentration range from 6.25 µg/ml to 400 µg/ml. Conclusion: Our data conclusively suggest that G. foliifera and Cladophoropsis sp. may be good candidates for further fractionation to obtain novel anticancer substances. Moreover, stronger cytotoxic effects on estrogen negative breast cancer cell line (MDA-MB-231(ER−)) in comparison to estrogen positive cells (MCF-7 and T-47D) suggest that the extract of G. foliifera and Cladophoropsis sp. may have an estrogen receptor/progesterone receptor-independent mechanism for their cellular growth inhibition. PMID:25829786

Separation of concanavalin A-induced human suppressor and helper T cells by the autologous erythrocyte rosette technique.

PubMed

Sakane, T; Honda, M; Taniguchi, Y; Kotani, H

1981-08-01

Very few normal human peripheral blood T cells are capable of binding autologous erythrocytes to form rosettes, whereas in the T cell population activated by concanavalin A (Con A) the autorosette levels are markedly enhanced. Fractionation of the Con A-activated T cells with autologous erythrocytes into autorosetting and nonrosetting cells demonstrates that suppressor, but not helper, activity resides in the autorosetting population, whereas the reverse is true of the nonrosetting population. Both these activities are found to be Con A dependent. The Con A-induced human suppressor cells can be identified and separated from the Con A-induced human helper cells by the autorosette technique. Studies on the surface properties of autorosetting and nonrosetting T cells indicate that there is little correlation between the activated suppressor and helper T cell subsets defined by autorosette technique and either those defined by monoclonal antibodies (which are able to distinguish these subsets in the resting but not activated T cells) or those defined by Fc receptors. Since the autorosetting T cell population (which acts as suppressor cells) bears receptors for peanut agglutinin, the nature of Con A-induced human suppressor cells appears to be analogous to that of Con A-induced murine suppressor cells.

Positive Selection in CD8+ T-Cell Epitopes of Influenza Virus Nucleoprotein Revealed by a Comparative Analysis of Human and Swine Viral Lineages

PubMed Central

Machkovech, Heather M.; Bedford, Trevor; Suchard, Marc A.

2015-01-01

ABSTRACT Numerous experimental studies have demonstrated that CD8+ T cells contribute to immunity against influenza by limiting viral replication. It is therefore surprising that rigorous statistical tests have failed to find evidence of positive selection in the epitopes targeted by CD8+ T cells. Here we use a novel computational approach to test for selection in CD8+ T-cell epitopes. We define all epitopes in the nucleoprotein (NP) and matrix protein (M1) with experimentally identified human CD8+ T-cell responses and then compare the evolution of these epitopes in parallel lineages of human and swine influenza viruses that have been diverging since roughly 1918. We find a significant enrichment of substitutions that alter human CD8+ T-cell epitopes in NP of human versus swine influenza virus, consistent with the idea that these epitopes are under positive selection. Furthermore, we show that epitope-altering substitutions in human influenza virus NP are enriched on the trunk versus the branches of the phylogenetic tree, indicating that viruses that acquire these mutations have a selective advantage. However, even in human influenza virus NP, sites in T-cell epitopes evolve more slowly than do nonepitope sites, presumably because these epitopes are under stronger inherent functional constraint. Overall, our work demonstrates that there is clear selection from CD8+ T cells in human influenza virus NP and illustrates how comparative analyses of viral lineages from different hosts can identify positive selection that is otherwise obscured by strong functional constraint. IMPORTANCE There is a strong interest in correlates of anti-influenza immunity that are protective against diverse virus strains. CD8+ T cells provide such broad immunity, since they target conserved viral proteins. An important question is whether T-cell immunity is sufficiently strong to drive influenza virus evolution. Although many studies have shown that T cells limit viral replication in animal models and are associated with decreased symptoms in humans, no studies have proven with statistical significance that influenza virus evolves under positive selection to escape T cells. Here we use comparisons of human and swine influenza viruses to rigorously demonstrate that human influenza virus evolves under pressure to fix mutations in the nucleoprotein that promote escape from T cells. We further show that viruses with these mutations have a selective advantage since they are preferentially located on the “trunk” of the phylogenetic tree. Overall, our results show that CD8+ T cells targeting nucleoprotein play an important role in shaping influenza virus evolution. PMID:26311880

Human T cells expressing BEND3 on their surface represent a novel subpopulation that preferentially produces IL-6 and IL-8.

PubMed

Shiheido, Hirokazu; Kitagori, Koji; Sasaki, Chiyomi; Kobayashi, Shio; Aoyama, Takane; Urata, Kozue; Oku, Takuma; Hirayama, Yoshitaka; Yoshitomi, Hiroyuki; Hikida, Masaki; Yoshifuji, Hajime; Mimori, Tsuneyo; Watanabe, Takeshi; Shimizu, Jun

2014-06-01

BEN domain-containing protein 3 (BEND3) has no transmembrane region, is localized in the cytoplasm, and is involved in chromatin function and transcription. We here identified a novel subpopulation of human T cells that expressed BEND3 on their cell surface (BEND3(+) T cells). BEND3(+) T cells consisted of approximately 3% of T cells in the peripheral blood, were present in both CD4(+) and CD8(+) T cells, and were also observed in cord blood. The stimulation of BEND3(+) T cells through the TCR/CD3 complex led to the production of various kinds of cytokines; however, the levels of IL-6 and IL-8 produced by BEND3(+) T cells were higher than those by BEND3(-) T cells. The proportion of BEND3(+) T cells was also increased in some patients with inflammatory diseases. Taken together, these results indicate that BEND3(+) T cells are a new subpopulation of T cells in terms of their cytokine profile. Further analyses on BEND3(+) T cells may be of importance and useful in understanding human T cell immunology.

Human T cells expressing BEND3 on their surface represent a novel subpopulation that preferentially produces IL-6 and IL-8

PubMed Central

Shiheido, Hirokazu; Kitagori, Koji; Sasaki, Chiyomi; Kobayashi, Shio; Aoyama, Takane; Urata, Kozue; Oku, Takuma; Hirayama, Yoshitaka; Yoshitomi, Hiroyuki; Hikida, Masaki; Yoshifuji, Hajime; Mimori, Tsuneyo; Watanabe, Takeshi; Shimizu, Jun

2014-01-01

BEN domain-containing protein 3 (BEND3) has no transmembrane region, is localized in the cytoplasm, and is involved in chromatin function and transcription. We here identified a novel subpopulation of human T cells that expressed BEND3 on their cell surface (BEND3+ T cells). BEND3+ T cells consisted of approximately 3% of T cells in the peripheral blood, were present in both CD4+ and CD8+ T cells, and were also observed in cord blood. The stimulation of BEND3+ T cells through the TCR/CD3 complex led to the production of various kinds of cytokines; however, the levels of IL-6 and IL-8 produced by BEND3+ T cells were higher than those by BEND3− T cells. The proportion of BEND3+ T cells was also increased in some patients with inflammatory diseases. Taken together, these results indicate that BEND3+ T cells are a new subpopulation of T cells in terms of their cytokine profile. Further analyses on BEND3+ T cells may be of importance and useful in understanding human T cell immunology. PMID:25400923

Direct anti-inflammatory effects of granulocyte colony-stimulating factor (G-CSF) on activation and functional properties of human T cell subpopulations in vitro.

PubMed

Malashchenko, Vladimir Vladimirovich; Meniailo, Maxsim Evgenievich; Shmarov, Viacheslav Anatolievich; Gazatova, Natalia Dinislamovna; Melashchenko, Olga Borisovna; Goncharov, Andrei Gennadievich; Seledtsova, Galina Victorovna; Seledtsov, Victor Ivanovich

2018-03-01

We investigated the direct effects of human granulocyte colony-stimulating factor (G-CSF) on functionality of human T-cell subsets. CD3 + T-lymphocytes were isolated from blood of healthy donors by positive magnetic separation. T cell activation with particles conjugated with antibodies (Abs) to human CD3, CD28 and CD2 molecules increased the proportion of cells expressing G-CSF receptor (G-CSFR, CD114) in all T cell subpopulations studied (CD45RA + /CD197 + naive T cells, CD45RA - /CD197 + central memory T cells, CD45RA - /CD197 - effector memory T cells and CD45RA + /CD197 - terminally differentiated effector T cells). Upon T-cell activation in vitro, G-CSF (10.0 ng/ml) significantly and specifically enhanced the proportion of CD114 + T cells in central memory CD4 + T cell compartment. A dilution series of G-CSF (range, 0.1-10.0 ng/ml) was tested, with no effect on the expression of CD25 (interleukin-2 receptor α-chain) on activated T cells. Meanwhile, G-CSF treatment enhanced the proportion of CD38 + T cells in CD4 + naïve T cell, effector memory T cell and terminally differentiated effector T cell subsets, as well as in CD4 - central memory T cells and terminally differentiated effector T cells. G-CSF did not affect IL-2 production by T cells; relatively low concentrations of G-CSF down-regulated INF-γ production, while high concentrations of this cytokine up-regulated IL-4 production in activated T cells. The data obtained suggests that G-CSF could play a significant role both in preventing the development of excessive and potentially damaging inflammatory reactivity, and in constraining the expansion of potentially cytodestructive T cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Foxp3-dependent transformation of human primary CD4+ T lymphocytes by the retroviral protein tax.

PubMed

Chen, Li; Liu, Dan; Zhang, Yang; Zhang, Huan; Cheng, Hua

2015-10-23

The retroviral Tax proteins of human T cell leukemia virus type 1 and 2 (HTLV-1 and -2) are highly homologous viral transactivators. Both viral proteins can immortalize human primary CD4+ memory T cells, but when expressed alone they rarely transform T cells. In the present study, we found that the Tax proteins displayed a differential ability to immortalize human CD4+Foxp3+ T cells with characteristic expression of CTLA-4 and GITR. Because epidermal growth factor receptor (EGFR) was reportedly expressed and activated in a subset of CD4+Foxp3+ T cells, we introduced an activated EGFR into Tax-immortalized CD4+Foxp3+ T cells. We observed that these modified cells were grown independently of exogenous IL-2, correlating with a T cell transformation phenotype. In Tax-immortalized CD4+Foxp3- T cells, ectopic expression of Foxp3 was a prerequisite for Tax transformation of T cells. Accordingly, treatment of the transformed T cells with erlotinib, a selective inhibitor of EGFR, induced degradation of EGFR in lysosome, consequently causing T cell growth inhibition. Further, we identified autophagy as a crucial cellular survival pathway for the transformed T cells. Silencing key autophagy molecules including Beclin1, Atg5 and PI3 kinase class III (PI3KC3) resulted in drastic impairment of T cell growth. Our data, therefore, unveiled a previously unidentified role of Foxp3 in T cell transformation, providing a molecular basis for HTLV-1 transformation of CD4+Foxp3+ T cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Foxp3-dependent Transformation of Human Primary CD4+ T Lymphocytes by the Retroviral Protein Tax

PubMed Central

Chen, Li; Liu, Dan; Zhang, Yang; Zhang, Huan; Cheng, Hua

2015-01-01

The retroviral Tax proteins of human T cell leukemia virus type 1 and 2 (HTLV-1 and -2) are highly homologous viral transactivators. Both viral proteins can immortalize human primary CD4+ memory T cells, but when expressed alone they rarely transform T cells. In the present study, we found that the Tax proteins displayed a differential ability to immortalize human CD4+Foxp3+ T cells with characteristic expression of CTLA-4 and GITR. Because epidermal growth factor receptor (EGFR) was reportedly expressed and activated in a subset of CD4+Foxp3+ T cells, we introduced an activated EGFR into Tax-immortalized CD4+Foxp3+ T cells. We observed that these modified cells were grown independently of exogenous IL-2, correlating with a T cell transformation phenotype. In Tax-immortalized CD4+Foxp3- T cells, ectopic expression of Foxp3 was a prerequisite for Tax transformation of T cells. Accordingly, treatment of the transformed T cells with erlotinib, a selective inhibitor of EGFR, induced degradation of EGFR in lysosome, consequently causing T cell growth inhibition. Further, we identified autophagy as a crucial cellular survival pathway for the transformed T cells. Silencing key autophagy molecules including Beclin1, Atg5 and PI3 kinase class III (PI3KC3) resulted in drastic impairment of T cell growth. Our data, therefore, unveiled a previously unidentified role of Foxp3 in T cell transformation, providing a molecular basis for HTLV-1 transformation of CD4+Foxp3+ T cells. PMID:26381169

Human CD1c+ dendritic cells drive the differentiation of CD103+ CD8+ mucosal effector T cells via the cytokine TGF-β

PubMed Central

Yu, Chun I; Becker, Christian; Wang, Yuanyuan; Marches, Florentina; Helft, Julie; Leboeuf, Marylene; Anguiano, Esperanza; Pourpe, Stephane; Goller, Kristina; Pascual, Virginia; Banchereau, Jacques; Merad, Miriam; Palucka, Karolina

2013-01-01

Summary In comparison to murine dendritic cells (DCs), less is known about the function of human DCs in tissues. Here, we analyzed, using lung tissues from humans and humanized mice, the role of human CD1c+ and CD141+ DCs in determining the type of CD8+ T cell immunity generated to live-attenuated influenza virus (LAIV) vaccine. We found that both lung DC subsets acquired influenza antigens in vivo and expanded specific cytotoxic CD8+ T cells in vitro. However, lung-tissue-resident CD1c+ DCs but not CD141+ DCs were able to drive CD103 expression on CD8+ T cells and promoted CD8+ T cell accumulation in lung epithelia in vitro and in vivo. CD1c+ DCs induction of CD103 expression was dependent on membrane-bound cytokine TGF-β1. Thus, CD1c+ and CD141+ DCs generate CD8+ T cells with different properties, and CD1c+ DCs specialize in the regulation of mucosal CD8+ T cells. PMID:23562160

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

PubMed

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

2015-09-01

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

Rifaximin has a Marginal Impact on Microbial Translocation, T-cell Activation and Inflammation in HIV-Positive Immune Non-responders to Antiretroviral Therapy – ACTG A5286

PubMed Central

Tenorio, Allan R.; Chan, Ellen S.; Bosch, Ronald J.; Macatangay, Bernard J. C.; Read, Sarah W.; Yesmin, Suria; Taiwo, Babafemi; Margolis, David M.; Jacobson, Jeffrey M.; Landay, Alan L.; Wilson, Cara C.; Mellors, John W.; Keshavarzian, Ali; Rodriguez, Benigno; Aziz, Mariam; Presti, Rachel; Deeks, Steven; Ebiasah, Ruth; Myers, Laurie; Borowski, LuAnn; Plants, Jill; Palm, David A.; Weibel, Derek; Putnam, Beverly; Lindsey, Elizabeth; Player, Amy; Albrecht, Mary; Kershaw, Andrea; Sax, Paul; Keenan, Cheryl; Walton, Patricia; Baum, Jane; Stroberg, Todd; Hughes, Valery; Coster, Laura; Kumar, Princy N.; Yin, Michael T.; Noel-Connor, Jolene; Tebas, Pablo; Thomas, Aleshia; Davis, Charles E.; Redfield, Robert R.; Sbrolla, Amy; Flynn, Teri; Davis, Traci; Whitely, Kim; Singh, Baljinder; Swaminathan, Shobha; McGregor, Donna; Palella, Frank; Aberg, Judith; Cavanagh, Karen; Santana Bagur, Jorge L.; Flores, Olga Méndez; Fritsche, Janice; Sha, Beverly; Slamowitz, Debbie; Valle, Sandra; Tashima, Karen; Patterson, Helen; Harber, Heather; Para, Michael; Eaton, Molly; Maddox, Dale; Currier, Judith; Cajahuaringa, Vanessa; Luetkemeyer, Annie; Dwyer, Jay; Fichtenbaum, Carl J.; Saemann, Michelle; Ray, Graham; Campbell, Thomas; Fischl, Margaret A.; Bolivar, Hector; Oakes, Jonathan; Chicurel-Bayard, Miriam; Tripoli, Christine; Weinman, D. Renee; Adams, Mary; Hurley, Christine; Dunaway, Shelia; Storey, Sheryl; Klebert, Michael; Royal, Michael

2015-01-01

Background. Rifaximin, a nonabsorbable antibiotic that decreases lipopolysaccharide (LPS) in cirrhotics, may decrease the elevated levels of microbial translocation, T-cell activation and inflammation in human immunodeficiency virus (HIV)-positive immune nonresponders to antiretroviral therapy (ART). Methods. HIV-positive adults receiving ART for ≥96 weeks with undetectable viremia for ≥48 weeks and CD4+ T-cell counts <350 cells/mm3 were randomized 2:1 to rifaximin versus no study treatment for 4 weeks. T-cell activation, LPS, and soluble CD14 were measured at baseline and at weeks 2, 4, and 8. Wilcoxon rank sum tests compared changes between arms. Results. Compared with no study treatment (n = 22), rifaximin (n = 43) use was associated with a significant difference between study arms in the change from baseline to week 4 for CD8+T-cell activation (median change, 0.0% with rifaximin vs +0.6% with no treatment; P = .03). This difference was driven by an increase in the no-study-treatment arm because there was no significant change within the rifaximin arm. Similarly, although there were significant differences between study arms in change from baseline to week 2 for LPS and soluble CD14, there were no significant changes within the rifaximin arm. Conclusions. In immune nonresponders to ART, rifaximin minimally affected microbial translocation and CD8+T-cell activation. Trial registration number. NCT01466595. PMID:25214516

Progenitor cell dose determines the pace and completeness of engraftment in a xenograft model for cord blood transplantation

PubMed Central

Liu, Congxiao; Chen, Benny J.; DeOliveira, Divinomar; Sempowski, Gregory D.; Chao, Nelson J.

2010-01-01

Two critical concerns in clinical cord blood transplantation are the initial time to engraftment and the subsequent restoration of immune function. These studies measured the impact of progenitor cell dose on both the pace and strength of hematopoietic reconstitution by transplanting nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor-gamma–null (NSγ) mice with lineage-depleted aldehyde dehydrogenase-bright CD34+ human cord blood progenitors. The progress of each transplant was monitored over an extended time course by repeatedly analyzing the peripheral blood for human hematopoietic cells. In vivo human hematopoietic development was complete. After long-term transplantation assays (≥ 19 weeks), human T-cell development was documented within multiple tissues in 16 of 32 NSγ mice. Human T-cell differentiation was active within NSγ thymuses, as documented by the presence of CD4+ CD8+ T-cell progenitors as well as T-cell receptor excision circles. It is important to note that although myeloid and B-cell engraftment was detected as early as 4 weeks after transplantation, human T-cell development was exclusively late onset. High progenitor cell doses were associated with a robust human hematopoietic chimerism that accelerated both initial time to engraftment and subsequent T-cell development. At lower progenitor cell doses, the chimerism was weak and the human hematopoietic lineage development was frequently incomplete. PMID:20833978

Exploring biological and social networks to better understand and treat diabetes mellitus. Comment on "Network science of biological systems at different scales: A review" by Gosak et al.

NASA Astrophysics Data System (ADS)

Belgardt, Bengt-Frederik; Jarasch, Alexander; Lammert, Eckhard

2018-03-01

Improvements and breakthroughs in computational sciences in the last 20 years have paralleled the rapid gain of influence of social networks on our daily life. As timely reviewed by Perc and colleagues [1], understanding and treating complex human diseases, such as type 2 diabetes (T2D), from which already more than 5% of the global population suffer, will necessitate analyzing and understanding the multi-layered and interconnected networks that usually keep physiological functions intact, but are disturbed in disease states. These networks range from intra- and intercellular networks influencing cell behavior (e.g., secretion of insulin in response to food intake and anabolic response to insulin) to social networks influencing human behavior (e.g., food intake and physical activity). This commentary first expands on the background of pancreatic beta cell networks in human health and T2D, briefly introduces exosomes as novel signals exchanged between distant cellular networks, and finally discusses potential pitfalls and chances in network analyses with regards to experimental data acquisition and processing.

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

PubMed

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

2018-05-07

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

IL-15 induces antigen-independent expansion and differentiation of human naive CD8+ T cells in vitro.

PubMed

Alves, Nuno L; Hooibrink, Berend; Arosa, Fernando A; van Lier, René A W

2003-10-01

Recent studies in mice have shown that although interleukin 15 (IL-15) plays an important role in regulating homeostasis of memory CD8+ T cells, it has no apparent function in controlling homeostatic proliferation of naive T cells. We here assessed the influence of IL-15 on antigen-independent expansion and differentiation of human CD8+ T cells. Both naive and primed human T cells divided in response to IL-15. In this process, naive CD8+ T cells successively down-regulated CD45RA and CD28 but maintained CD27 expression. Concomitant with these phenotypic changes, naive cells acquired the ability to produce interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha), expressed perforin and granzyme B, and acquired cytotoxic properties. Primed CD8+ T cells, from both noncytotoxic (CD45RA-CD27+) and cytotoxic (CD45RA+CD27-) subsets, responded to IL-15 and yielded ample numbers of cytokine-secreting and cytotoxic effector cells. In summary, all human CD8+ T-cell subsets had the ability to respond to IL-15, which suggests a generic influence of this cytokine on CD8+ T-cell homeostasis in man.

Flavonoids Activated Caspases for Apoptosis in Human Glioblastoma T98G and U87MG Cells But Not in Human Normal Astrocytes

PubMed Central

Das, Arabinda; Banik, Naren L.; Ray, Swapan K.

2011-01-01

BACKGROUND Human glioblastoma is a deadly brain cancer that continues to defy all current therapeutic strategies. We induced apoptosis in human glioblastoma T98G and U87MG cells following treatment with apigenin (APG), (−)-epigallocatechin (EGC), (−)-epigallocatechin-3-gallate (EGCG), and genistein (GST) that did not induce apoptosis in human normal astrocytes (HNA). METHODS Induction of apoptosis was examined using Wright staining and ApopTag assay. Production of reactive oxygen species (ROS) and increase in intracellular free [Ca2+] were measured by fluoresent probes. Analysis of mRNA and Western blotting indicated increases in expression and activities of the stress kinases and cysteine proteases for apoptosis. JC-1 showed changes in mitochondrial membrane potential (ΔΨm) and use of specific inhibitors confirmed activation of kinases and proteases in apoptosis. RESULTS Treatment of glioblastoma cells with APG, EGC, EGCG, or GST triggered ROS production that induced apoptosis with phosphorylation of p38 MAPK and activation of the redox-sensitive JNK1 pathway. Pretreatment of cells with ascorbic acid attenuated ROS production and p38 MAPK phosphorylation. Increases in intracellular free [Ca2+] and activation of caspase-4 indicated involvement of endoplasmic reticulum stress in apoptosis. Other events in apoptosis included overexpression of Bax, loss of ΔΨm, mitochondrial release of cytochrome c and Smac into the cytosol, down regulation of baculoviral inhibitor-of-apoptosis repeat containing proteins, and activation of calpain, caspase-9, and caspase-3. EGC and EGCG also induced caspase-8 activity. APG, EGC, EGCG, or GST did not induce apoptosis in HNA. CONCLUSION Results strongly suggest that flavonoids are potential therapeutic agents for induction of apoptosis in human glioblastoma cells. PMID:19894226

CD19xCD3 DART protein mediates human B-cell depletion in vivo in humanized BLT mice

PubMed Central

Tsai, Perry; Thayer, William O; Liu, Liqin; Silvestri, Guido; Nordstrom, Jeffrey L; Garcia, J Victor

2016-01-01

Novel therapeutic strategies are needed for the treatment of hematologic malignancies; and bispecific antibody-derived molecules, such as dual-affinity re-targeting (DART) proteins, are being developed to redirect T cells to kill target cells expressing tumor or viral antigens. Here we present our findings of specific and systemic human B-cell depletion by a CD19xCD3 DART protein in humanized BLT mice. Administration of the CD19xCD3 DART protein resulted in a dramatic sustained depletion of human CD19+ B cells from the peripheral blood, as well as a dramatic systemic reduction of human CD19+ B-cell levels in all tissues (bone marrow, spleen, liver, lung) analyzed. When human CD8+ T cells were depleted from the mice, no significant B-cell depletion was observed in response to CD19xCD3 DART protein treatment, confirming that human CD8+ T cells are the primary effector cells in this in vivo model. These studies validate the use of BLT humanized mice for the in vivo evaluation and preclinical development of bispecific molecules that redirect human T cells to selectively deplete target cells. PMID:27119115

IL13Rα2 expression identifies tissue-resident IL-22 producing PLZF+ innate T cells in the human liver.

PubMed

Paquin-Proulx, Dominic; Greenspun, Benjamin C; Pasquet, Lise; Strunz, Benedikt; Aleman, Soo; Falconer, Karolin; Terabe, Masaki; Berzofsky, Jay A; Sandberg, Johan K; Melum, Espen; Nixon, Douglas F; Björkström, Niklas K

2018-04-20

Innate lymphocytes are selectively enriched in the liver where they have important roles in liver immunology. Murine studies have shown that type I NKT cells can promote liver inflammation whereas type II NKT cells have an anti-inflammatory role. In humans, type II NKT cells were found to accumulate in the gut during inflammation and IL13Rα2 was proposed as a marker for these cells. In the human liver, less is known about type I and II NKT cells. Here, we studied the phenotype and function of human liver T cells expressing IL13Rα2. We found that IL13Rα2 was expressed by around 1% of liver resident memory T cells but not on circulating T cells. In support of their innate-like T cell character, the IL13Rα2 + T cells had higher expression of PLZF compared to IL13Rα2 - T cells and possessed the capacity to produce IL-22. However, only a minority of human liver sulfatide-reactive type II NKT cells expressed IL13Rα2. Collectively, these findings suggest that IL13Rα2 identifies tissue-resident intrahepatic T cells with innate characteristics and the capacity to produce IL-22. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Broadening CD4+ and CD8+ T Cell Responses against Hepatitis C Virus by Vaccination with NS3 Overlapping Peptide Panels in Cross-Priming Liposomes

PubMed Central

Filskov, Jonathan; Mikkelsen, Marianne; Hansen, Paul R.; Christensen, Jan P.; Thomsen, Allan R.; Andersen, Peter; Agger, Else Marie

2017-01-01

ABSTRACT Despite the introduction of effective drugs to treat patients with chronic hepatitis C virus (HCV) infection, a vaccine would be the only means to substantially reduce the worldwide disease burden. An incomplete understanding of how HCV interacts with its human host and evades immune surveillance has hampered vaccine development. It is generally accepted that in infected individuals, a narrow repertoire of exhausted T cells is a hallmark of persistent infection, whereas broad, vigorous CD4+ and CD8+ T cell responses are associated with control of acute hepatitis C. We employed a vaccine approach based on a mixture of peptides (pepmix) spanning the entire sequence of HCV nonstructural protein 3 (NS3) in cross-priming cationic liposomes (CAF09) to facilitate a versatile presentation of all possible T cell epitopes, regardless of the HLA background of the vaccine recipient. Here, we demonstrate that vaccination of mice with NS3 pepmix broadens the repertoire of epitope-specific T cells compared to the corresponding recombinant protein (rNS3). Moreover, vaccination with rNS3 induced only CD4+ T cells, whereas the NS3 pepmix induced a far more vigorous CD4+ T cell response and was as potent a CD8+ T cell inducer as an adenovirus-vectored vaccine expressing NS3. Importantly, the cellular responses are dominated by multifunctional T cells, such as gamma interferon-positive (IFN-γ+) tumor necrosis factor alpha-positive (TNF-α+) coproducers, and displayed cytotoxic capacity in mice. In conclusion, we present a novel vaccine approach against HCV, inducing a broadened T cell response targeting both immunodominant and potential subdominant epitopes, which may be key elements to counter T cell exhaustion and prevent chronicity. IMPORTANCE With at least 700,000 annual deaths, development of a vaccine against hepatitis C virus (HCV) has high priority, but the tremendous ability of the virus to dodge the human immune system poses great challenges. Furthermore, many chronic infections, including HCV infection, have a remarkable ability to drive initially strong CD4+ and CD8+ T cell responses against dominant epitopes toward an exhausted, dysfunctional state. Thus, new and innovative vaccine approaches to control HCV should be evaluated. Here, we report on a novel peptide-based nanoparticle vaccine strategy (NS3 pepmix) aimed at generating T cell immunity against potential subdominant T cell epitopes that are not efficiently targeted by vaccination with full-length recombinant protein (rNS3) or infection with HCV. As proof of concept, we found that NS3 pepmix excels in broadening the repertoire of epitope-specific, multifunctional, and cytotoxic CD4+ and CD8+ T cells compared to vaccination with rNS3, which generated only CD4+ T cell responses. PMID:28446674

Blocking the recruitment of naive CD4+ T cells reverses immunosuppression in breast cancer

PubMed Central

Su, Shicheng; Liao, Jianyou; Liu, Jiang; Huang, Di; He, Chonghua; Chen, Fei; Yang, LinBing; Wu, Wei; Chen, Jianing; Lin, Ling; Zeng, Yunjie; Ouyang, Nengtai; Cui, Xiuying; Yao, Herui; Su, Fengxi; Huang, Jian-dong; Lieberman, Judy; Liu, Qiang; Song, Erwei

2017-01-01

The origin of tumor-infiltrating Tregs, critical mediators of tumor immunosuppression, is unclear. Here, we show that tumor-infiltrating naive CD4+ T cells and Tregs in human breast cancer have overlapping TCR repertoires, while hardly overlap with circulating Tregs, suggesting that intratumoral Tregs mainly develop from naive T cells in situ rather than from recruited Tregs. Furthermore, the abundance of naive CD4+ T cells and Tregs is closely correlated, both indicating poor prognosis for breast cancer patients. Naive CD4+ T cells adhere to tumor slices in proportion to the abundance of CCL18-producing macrophages. Moreover, adoptively transferred human naive CD4+ T cells infiltrate human breast cancer orthotopic xenografts in a CCL18-dependent manner. In human breast cancer xenografts in humanized mice, blocking the recruitment of naive CD4+ T cells into tumor by knocking down the expression of PITPNM3, a CCL18 receptor, significantly reduces intratumoral Tregs and inhibits tumor progression. These findings suggest that breast tumor-infiltrating Tregs arise from chemotaxis of circulating naive CD4+ T cells that differentiate into Tregs in situ. Inhibiting naive CD4+ T cell recruitment into tumors by interfering with PITPNM3 recognition of CCL18 may be an attractive strategy for anticancer immunotherapy. PMID:28290464

Telomere Dynamics in Immune Senescence and Exhaustion Triggered by Chronic Viral Infection.

PubMed

Bellon, Marcia; Nicot, Christophe

2017-10-05

The progressive loss of immunological memory during aging correlates with a reduced proliferative capacity and shortened telomeres of T cells. Growing evidence suggests that this phenotype is recapitulated during chronic viral infection. The antigenic volume imposed by persistent and latent viruses exposes the immune system to unique challenges that lead to host T-cell exhaustion, characterized by impaired T-cell functions. These dysfunctional memory T cells lack telomerase, the protein capable of extending and stabilizing chromosome ends, imposing constraints on telomere dynamics. A deleterious consequence of this excessive telomere shortening is the premature induction of replicative senescence of viral-specific CD8+ memory T cells. While senescent cells are unable to expand, they can survive for extended periods of time and are more resistant to apoptotic signals. This review takes a closer look at T-cell exhaustion in chronic viruses known to cause human disease: Epstein-Barr virus (EBV), Hepatitis B/C/D virus (HBV/HCV/HDV), human herpesvirus 8 (HHV-8), human immunodeficiency virus (HIV), human T-cell leukemia virus type I (HTLV-I), human papillomavirus (HPV), herpes simplex virus-1/2(HSV-1/2), and Varicella-Zoster virus (VZV). Current literature linking T-cell exhaustion with critical telomere lengths and immune senescence are discussed. The concept that enduring antigen stimulation leads to T-cell exhaustion that favors telomere attrition and a cell fate marked by enhanced T-cell senescence appears to be a common endpoint to chronic viral infections.

Identification of p53 unbound to T-antigen in human cells transformed by simian virus 40 T-antigen.

PubMed

O'Neill, F J; Hu, Y; Chen, T; Carney, H

1997-02-27

In several clones of SV40-transformed human cells, we investigated the relative amounts of large T-Antigen (T-Ag) and p53 proteins, both unbound and associated within complexes, with the goal of identifying changes associated with transformation and immortalization. Cells were transformed by wild type (wt) T-Ag, a functionally temperature sensitive T-Ag (tsA58) and other T-Ag variants. Western analysis showed that while most of the T-Ag was ultimately bound by p53, most of the p53 remained unbound to T-Ag. Unbound p53 remained in the supernatant after a T-Ag immunoprecipitation and p53 was present in two to fourfold excess of T-Ag. In one transformant there was five to tenfold more p53 than T-Ag. p53 was present in transformants in amounts at least 200-fold greater than in untransformed human cells. In wt and variant T-Ag transformants, including those generated with tsA58 T-Ag, large amounts of unbound p53 were present in both pre-crisis and immortal cells and when the cells were grown at permissive or non-permissive temperatures. We also found that in transformants produced by tsA58, an SV40/JCV chimeric T-Ag and other variants, T-Ag appeared to form a complex with p53 slowly perhaps because one or both proteins matured slowly. The presence in transformed human cells of large amounts of unbound p53 and in excess of T-Ag suggests that sequestration of p53 by T-Ag, resulting from complex formation, is required neither for morphological transformation nor immortalization of human cells. Rather, these results support the proposal that high levels of p53, the T-Ag/p53 complexes, or other biochemical event(s), lead to transformation and immortalization of human cells by T-Ag.

Human β-cell Precursors Mature Into Functional Insulin-producing Cells in an Immunoisolation Device: Implications for Diabetes Cell Therapies

PubMed Central

Lee, Seung-Hee; Hao, Ergeng; Savinov, Alexei Y.; Geron, Ifat; Strongin, Alex Y.; Itkin-Ansari, Pamela

2009-01-01

Background Islet transplantation is limited by the need for chronic immunosuppression and the paucity of donor tissue. As new sources of human β-cells are developed (e.g., stem cell-derived tissue), transplanting them in a durable device could obviate the need for immunosuppression, while also protecting the patient from any risk of tumorigenicity. Here, we studied (1) the survival and function of encapsulated human β-cells and their progenitors and (2) the engraftment of encapsulated murine β-cells in allo- and autoimmune settings. Methods Human islets and human fetal pancreatic islet-like cell clusters were encapsulated in polytetrafluorethylene devices (TheraCyte) and transplanted into immunodeficient mice. Graft survival and function was measured by immunohistochemistry, circulating human C-peptide levels, and blood glucose levels. Bioluminescent imaging was used to monitor encapsulated neonatal murine islets. Results Encapsulated human islet-like cell clusters survived, replicated, and acquired a level of glucose responsive insulin secretion sufficient to ameliorate hyperglycemia in diabetic mice. Bioluminescent imaging of encapsulated murine neonatal islets revealed a dynamic process of cell death followed by regrowth, resulting in robust long-term allograft survival. Further, in the non-obese diabetic (NOD) mouse model of type I diabetes, encapsulated primary β-cells ameliorated diabetes without stimulating a detectable T-cell response. Conclusions We demonstrate for the first time that human β-cells function is compatible with encapsulation in a durable, immunoprotective device. Moreover, our study suggests that encapsulation of β-cells before terminal differentiation will be a successful approach for new cell-based therapies for diabetes, such as those derived from stem cells. PMID:19352116

Therapeutic manipulation of natural killer (NK) T cells in autoimmunity: are we close to reality?

PubMed Central

Simoni, Y; Diana, J; Ghazarian, L; Beaudoin, L; Lehuen, A

2013-01-01

T cells reactive to lipids and restricted by major histocompatibility complex (MHC) class I-like molecules represent more than 15% of all lymphocytes in human blood. This heterogeneous population of innate cells includes the invariant natural killer T cells (iNK T), type II NK T cells, CD1a,b,c-restricted T cells and mucosal-associated invariant T (MAIT) cells. These populations are implicated in cancer, infection and autoimmunity. In this review, we focus on the role of these cells in autoimmunity. We summarize data obtained in humans and preclinical models of autoimmune diseases such as primary biliary cirrhosis, type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, psoriasis and atherosclerosis. We also discuss the promise of NK T cell manipulations: restoration of function, specific activation, depletion and the relevance of these treatments to human autoimmune diseases. PMID:23199318

PDZ domain-binding motif of Tax sustains T-cell proliferation in HTLV-1-infected humanized mice.

PubMed

Pérès, Eléonore; Blin, Juliana; Ricci, Emiliano P; Artesi, Maria; Hahaut, Vincent; Van den Broeke, Anne; Corbin, Antoine; Gazzolo, Louis; Ratner, Lee; Jalinot, Pierre; Duc Dodon, Madeleine

2018-03-01

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive malignant proliferation of activated CD4+ T lymphocytes. The viral Tax oncoprotein is critically involved in both HTLV-1-replication and T-cell proliferation, a prerequisite to the development of ATLL. In this study, we investigated the in vivo contribution of the Tax PDZ domain-binding motif (PBM) to the lymphoproliferative process. To that aim, we examined T-cell proliferation in humanized mice (hu-mice) carrying a human hemato-lymphoid system infected with either a wild type (WT) or a Tax PBM-deleted (ΔPBM) provirus. We observed that the frequency of CD4+ activated T-cells in the peripheral blood and in the spleen was significantly higher in WT than in ΔPBM hu-mice. Likewise, human T-cells collected from WT hu-mice and cultivated in vitro in presence of interleukin-2 were proliferating at a higher level than those from ΔPBM animals. We next examined the association of Tax with the Scribble PDZ protein, a prominent regulator of T-cell polarity, in human T-cells analyzed either after ex vivo isolation or after in vitro culture. We confirmed the interaction of Tax with Scribble only in T-cells from the WT hu-mice. This association correlated with the presence of both proteins in aggregates at the leading edge of the cells and with the formation of long actin filopods. Finally, data from a comparative genome-wide transcriptomic analysis suggested that the PBM-PDZ association is implicated in the expression of genes regulating proliferation, apoptosis and cytoskeletal organization. Collectively, our findings suggest that the Tax PBM is an auxiliary motif that contributes to the sustained growth of HTLV-1 infected T-cells in vivo and in vitro and is essential to T-cell immortalization.

PDZ domain-binding motif of Tax sustains T-cell proliferation in HTLV-1-infected humanized mice

PubMed Central

Artesi, Maria; Jalinot, Pierre

2018-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive malignant proliferation of activated CD4+ T lymphocytes. The viral Tax oncoprotein is critically involved in both HTLV-1-replication and T-cell proliferation, a prerequisite to the development of ATLL. In this study, we investigated the in vivo contribution of the Tax PDZ domain-binding motif (PBM) to the lymphoproliferative process. To that aim, we examined T-cell proliferation in humanized mice (hu-mice) carrying a human hemato-lymphoid system infected with either a wild type (WT) or a Tax PBM-deleted (ΔPBM) provirus. We observed that the frequency of CD4+ activated T-cells in the peripheral blood and in the spleen was significantly higher in WT than in ΔPBM hu-mice. Likewise, human T-cells collected from WT hu-mice and cultivated in vitro in presence of interleukin-2 were proliferating at a higher level than those from ΔPBM animals. We next examined the association of Tax with the Scribble PDZ protein, a prominent regulator of T-cell polarity, in human T-cells analyzed either after ex vivo isolation or after in vitro culture. We confirmed the interaction of Tax with Scribble only in T-cells from the WT hu-mice. This association correlated with the presence of both proteins in aggregates at the leading edge of the cells and with the formation of long actin filopods. Finally, data from a comparative genome-wide transcriptomic analysis suggested that the PBM-PDZ association is implicated in the expression of genes regulating proliferation, apoptosis and cytoskeletal organization. Collectively, our findings suggest that the Tax PBM is an auxiliary motif that contributes to the sustained growth of HTLV-1 infected T-cells in vivo and in vitro and is essential to T-cell immortalization. PMID:29566098

Primer sets for cloning the human repertoire of T cell Receptor Variable regions

PubMed Central

Boria, Ilenia; Cotella, Diego; Dianzani, Irma; Santoro, Claudio; Sblattero, Daniele

2008-01-01

Background Amplification and cloning of naïve T cell Receptor (TR) repertoires or antigen-specific TR is crucial to shape immune response and to develop immuno-based therapies. TR variable (V) regions are encoded by several genes that recombine during T cell development. The cloning of expressed genes as large diverse libraries from natural sources relies upon the availability of primers able to amplify as many V genes as possible. Results Here, we present a list of primers computationally designed on all functional TR V and J genes listed in the IMGT®, the ImMunoGeneTics information system®. The list consists of unambiguous or degenerate primers suitable to theoretically amplify and clone the entire TR repertoire. We show that it is possible to selectively amplify and clone expressed TR V genes in one single RT-PCR step and from as little as 1000 cells. Conclusion This new primer set will facilitate the creation of more diverse TR libraries than has been possible using currently available primer sets. PMID:18759974

Objective scoring of transformed foci in BALB/c 3T3 cell transformation assay by statistical image descriptors.

PubMed

Urani, C; Corvi, R; Callegaro, G; Stefanini, F M

2013-09-01

In vitro cell transformation assays (CTAs) have been shown to model important stages of in vivo carcinogenesis and have the potential to predict carcinogenicity in humans. Advantages of CTAs are their ability of revealing both genotoxic and non-genotoxic carcinogens while reducing both experimental costs and the number of animals used. The endpoint of the CTA is foci formation, and requires classification under light microscopy based on morphology. Thus current limitations for the wide adoption of the assay partially depend on a fair degree of subjectivity in foci scoring. An objective evaluation may be obtained after separating foci from background monolayer in the digital image, and quantifying values of statistical descriptors which are selected to capture eye-scored morphological features. The aim of this study was to develop statistical descriptors to be applied to transformed foci of BALB/c 3T3, which cover foci size, multilayering and invasive cell growth into the background monolayer. Proposed descriptors were applied to a database of 407 foci images to explore the numerical features, and to illustrate open problems and potential solutions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Positive Selection in CD8+ T-Cell Epitopes of Influenza Virus Nucleoprotein Revealed by a Comparative Analysis of Human and Swine Viral Lineages.

PubMed

Machkovech, Heather M; Bedford, Trevor; Suchard, Marc A; Bloom, Jesse D

2015-11-01

Numerous experimental studies have demonstrated that CD8(+) T cells contribute to immunity against influenza by limiting viral replication. It is therefore surprising that rigorous statistical tests have failed to find evidence of positive selection in the epitopes targeted by CD8(+) T cells. Here we use a novel computational approach to test for selection in CD8(+) T-cell epitopes. We define all epitopes in the nucleoprotein (NP) and matrix protein (M1) with experimentally identified human CD8(+) T-cell responses and then compare the evolution of these epitopes in parallel lineages of human and swine influenza viruses that have been diverging since roughly 1918. We find a significant enrichment of substitutions that alter human CD8(+) T-cell epitopes in NP of human versus swine influenza virus, consistent with the idea that these epitopes are under positive selection. Furthermore, we show that epitope-altering substitutions in human influenza virus NP are enriched on the trunk versus the branches of the phylogenetic tree, indicating that viruses that acquire these mutations have a selective advantage. However, even in human influenza virus NP, sites in T-cell epitopes evolve more slowly than do nonepitope sites, presumably because these epitopes are under stronger inherent functional constraint. Overall, our work demonstrates that there is clear selection from CD8(+) T cells in human influenza virus NP and illustrates how comparative analyses of viral lineages from different hosts can identify positive selection that is otherwise obscured by strong functional constraint. There is a strong interest in correlates of anti-influenza immunity that are protective against diverse virus strains. CD8(+) T cells provide such broad immunity, since they target conserved viral proteins. An important question is whether T-cell immunity is sufficiently strong to drive influenza virus evolution. Although many studies have shown that T cells limit viral replication in animal models and are associated with decreased symptoms in humans, no studies have proven with statistical significance that influenza virus evolves under positive selection to escape T cells. Here we use comparisons of human and swine influenza viruses to rigorously demonstrate that human influenza virus evolves under pressure to fix mutations in the nucleoprotein that promote escape from T cells. We further show that viruses with these mutations have a selective advantage since they are preferentially located on the "trunk" of the phylogenetic tree. Overall, our results show that CD8(+) T cells targeting nucleoprotein play an important role in shaping influenza virus evolution. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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 non-therapeutic dimerizer to activate the suicide gene resulted in the elimination of only 76.4±2.0% gene modified cells in vitro, we found that co-administration of the dimerizer with either the BCL-2 inhibitor ABT-199, the pan-BCL inhibitor ABT-737, or mafosfamide, resulted in an additive effect up to complete cell elimination. Conclusions This strategy could be investigated for the safety of CAR T-cell applications, and targeting CD33 could be used as a ‘bridge” therapy for patients coming to allogeneic hematopoietic stem cell transplant, as anti-leukemia activity from infusing CAR.CD33 T-cells has been demonstrated in an ongoing clinical trial. Albeit never performed in the clinical setting, our future plan is to investigate the utility of iC9-CAR.CD33 T-cells as part of the conditioning therapy for an allogeneic hematopoietic stem cell transplant for acute myeloid leukemia, together with other myelosuppressive agents, whilst the activation of the inducible Caspase9 suicide gene would grant elimination of the infused gene modified T-cells prior to stem cell infusion to reduce the risk of engraftment failure as the CD33 is also expressed on a proportion of the donor stem cell graft. PMID:27907031

Refining human T-cell immunotherapy of cytomegalovirus disease: a mouse model with 'humanized' antigen presentation as a new preclinical study tool.

PubMed

Lemmermann, Niels A W; Reddehase, Matthias J

2016-12-01

With the cover headline 'T cells on the attack,' the journal Science celebrated individualized cancer immunotherapy by adoptive transfer of T cells as the 'Breakthrough of the Year' 2013 (J. Couzin-Frankel in Science 342:1432-1433, 2013). It is less well recognized and appreciated that individualized T cell immunotherapy of cytomegalovirus (CMV) infection is approaching clinical application for preventing CMV organ manifestations, interstitial CMV pneumonia in particular. This coincident medical development is particularly interesting as reactivated CMV infection is a major viral complication in the state of transient immunodeficiency after the therapy of hematopoietic malignancies by hematopoietic cell transplantation (HCT). It may thus be attractive to combine T cell immunotherapy of 'minimal residual disease/leukemia (MRD)' and CMV-specific T cell immunotherapy to combat both risks in HCT recipients simultaneously, and ideally with T cells derived from the respective HLA-matched HCT donor. Although clinical trials of human CMV-specific T cell immunotherapy were promising in that the incidence of virus reactivation and disease was found to be reduced with statistical significance, animal models are still instrumental for providing 'proof of concept' by directly documenting the prevention of viral multiple-organ histopathology and organ failure under controlled conditions of the absence versus presence of the therapy, which obviously is not feasible in an individual human patient. Further, animal models can make predictions regarding parameters that determine the efficacy of T cell immunotherapy for improved study design in clinical investigations, and they allow for manipulating host and virus genetics. The latter is of particular value as it opens the possibility for epitope specificity controls that are inherently missing in clinical trials. Here, we review a recently developed new mouse model that is more approximated to human CMV-specific T cell immunotherapy by 'humanizing' antigen presentation using antigenically chimeric CMV and HLA-transgenic mice to allow for an in vivo testing of the antiviral function of human CMV-specific T cells. As an important new message, this model predicts that T cell immunotherapy is most efficient if CD4 T cells are equipped with a transduced TCR directed against an epitope presented by MHC/HLA class-I for local delivery of 'cognate' help to CD8 effector T cells at infected MHC/HLA class-II-negative host tissue cells.

Peripheral blood-derived virus-specific memory stem T cells mature to functional effector memory subsets with self-renewal potency.

PubMed

Schmueck-Henneresse, Michael; Sharaf, Radwa; Vogt, Katrin; Weist, Benjamin J D; Landwehr-Kenzel, Sybille; Fuehrer, Henrike; Jurisch, Anke; Babel, Nina; Rooney, Cliona M; Reinke, Petra; Volk, Hans-Dieter

2015-06-01

Memory T cells expressing stem cell-like properties have been described recently. The capacity of self-renewal and differentiation into various memory/effector subsets make them attractive for adoptive T cell therapy to combat severe virus infections and tumors. The very few reports on human memory stem T cells (T(SCM)) are restricted to analyses on polyclonal T cells, but extensive data on Ag-specific T(SCM )are missing. This might be due to their very low frequency limiting their enrichment and characterization. In this article, we provide functional and phenotypic data on human viral-specific T(SCM), defined as CD8(+)CD45RA(+)CCR7(+)CD127(+)CD95(+). Whereas <1% of total T cells express the T(SCM) phenotype, human CMV-specific T(SCM) can be detected at frequencies similar to those seen in other subsets, resulting in ∼ 1 /10,000 human CMV-specific T(SCM). A new virus-specific expansion protocol of sort-purified T(SCM) reveals both upregulation of various T cell subset markers and preservation of their stem cell phenotype in a significant proportion, indicating both self-renewal and differentiation potency of virus-specific T cells sharing their TCR repertoire. Furthermore, we describe a simplified culture protocol that allows fast expansion of virus-specific T(SCM) starting from a mixed naive T/T(SCM) pool of PBLs. Due to the clinical-grade compatibility, this might be the basis for novel cell therapeutic options in life-threatening courses of viral and tumor disease. Copyright © 2015 by The American Association of Immunologists, Inc.

CD8 T-Cell Expansion and Inflammation Linked to CMV Coinfection in ART-treated HIV Infection

PubMed Central

Freeman, Michael L.; Mudd, Joseph C.; Shive, Carey L.; Younes, Souheil-Antoine; Panigrahi, Soumya; Sieg, Scott F.; Lee, Sulggi A.; Hunt, Peter W.; Calabrese, Leonard H.; Gianella, Sara; Rodriguez, Benigno; Lederman, Michael M.

2016-01-01

Background. Persistent CD8 T-cell expansion, low CD4/CD8 T-cell ratios, and heightened inflammation persist in antiretroviral therapy (ART)-treated human immunodeficiency virus (HIV) infection and are associated with increased risk of morbid outcomes. We explored the role of cytomegalovirus (CMV) infection in CD8 lymphocytosis and inflammation in ART-treated HIV infection. Methods. Absolute CD4 and CD8 T-cell counts were abstracted from clinical records and compared among 32 HIV-infected CMV-seronegative subjects, 126 age, CD4 and gender-matched HIV-infected CMV-seropositive subjects, and among 21 HIV-uninfected controls (9 CMV-negative, 12 CMV-positive). Plasma inflammatory indices were measured in a subset by ELISA. Results. Median CD8 counts/µL were higher in HIV-positive/CMV-positive patients (795) than in HIV-positive/CMV-negative subjects (522, P = .006) or in healthy controls (451, P = .0007), whereas CD8 T-cell counts were similar to controls' levels in HIV-positive/CMV-negative subjects. Higher plasma levels of IP-10 (P = .0011), TNF-RII (P = .0002), and D-dimer (P = .0444) were also found in coinfected patients than in HIV-positive/CMV-negative subjects. Conclusions. CMV infection is associated with higher CD8 T-cell counts, resultant lower CD4/CD8 ratios, and increased systemic inflammation in ART-treated HIV infection. CMV infection may contribute to risk for morbid outcomes in treated HIV infection. PMID:26400999

HIV Type 1 Disease Progression to AIDS and Death in a Rural Ugandan Cohort Is Primarily Dependent on Viral Load Despite Variable Subtype and T-Cell Immune Activation Levels

PubMed Central

Eller, Michael A.; Opollo, Marc S.; Liu, Michelle; Redd, Andrew D.; Eller, Leigh Anne; Kityo, Cissy; Kayiwa, Joshua; Laeyendecker, Oliver; Wawer, Maria J.; Milazzo, Mark; Kiwanuka, Noah; Gray, Ronald H.; Serwadda, David; Sewankambo, Nelson K.; Quinn, Thomas C.; Michael, Nelson L.; Wabwire-Mangen, Fred; Sandberg, Johan K.; Robb, Merlin L.

2015-01-01

Background. Untreated human immunodeficiency virus type 1 (HIV) infection is associated with persistent immune activation, which is an independent driver of disease progression in European and United States cohorts. In Uganda, HIV-1 subtypes A and D and recombinant AD viruses predominate and exhibit differential rates of disease progression. Methods. HIV-1 seroconverters (n = 156) from rural Uganda were evaluated to assess the effects of T-cell activation, viral load, and viral subtype on disease progression during clinical follow-up. Results. The frequency of activated T cells was increased in HIV-1–infected Ugandans, compared with community matched uninfected individuals, but did not differ significantly between viral subtypes. Higher HIV-1 load, subtype D, older age, and high T-cell activation levels were associated with faster disease progression to AIDS or death. In a multivariate Cox regression analysis, HIV-1 load was the strongest predictor of progression, with subtype also contributing. T-cell activation did not emerge an independent predictor of disease progression from this particular cohort. Conclusions. These findings suggest that the independent contribution of T-cell activation on morbidity and mortality observed in European and North American cohorts may not be directly translated to the HIV epidemic in East Africa. In this setting, HIV-1 load appears to be the primary determinant of disease progression. PMID:25404522

Acid sphingomyelinase mediates human CD4+ T-cell signaling: potential roles in T-cell responses and diseases

PubMed Central

Bai, Aiping; Guo, Yuan

2017-01-01

Acid sphingomyelinase (ASM) is a lipid hydrolase. By generating ceramide, ASM had been reported to have an important role in regulating immune cell functions inclusive of macrophages, NK cells, and CD8+ T cells, whereas the role of ASM bioactivity in regulation of human CD4+ T-cell functions remained uncertain. Recent studies have provided novel findings in this field. Upon stimulation of CD3 and/or CD28, ASM-dependent ceramide signaling mediates intracellular downstream signal cascades of CD3 and CD28, and regulates CD4+ T-cell activation and proliferation. Meanwhile, CD39 and CD161 have direct interactions with ASM, which mediates downstream signals inclusive of STAT3 and mTOR and thus defines human Th17 cells. Intriguingly, ASM mediates Th1 responses, but negatively regulates Treg functions. In this review, we summarized the pivotal roles of ASM in regulation of human CD4+ T-cell activation and responses. ASM/sphingolipid signaling may be a novel target for the therapy of human autoimmune diseases. PMID:28749465

Towards immunotherapy with redirected T cells in a large animal model: Ex vivo activation, expansion, and genetic modification of canine T cells

PubMed Central

Mata, Melinda; Vera, Juan; Gerken, Claudia; Rooney, Cliona M.; Miller, Tasha; Pfent, Catherine; Wang, Lisa L.; Wilson-Robles, Heather M.; Gottschalk, Stephen

2014-01-01

Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) has shown promising anti-tumor activity in early phase clinical studies, especially for hematological malignancies. However, most preclinical models do not reliably mimic human disease. We reasoned that developing an adoptive T-cell therapy approach for spontaneous osteosarcoma (OS) occurring in dogs would more closely reproduce the condition in human cancer. To generate CAR-expressing canine T cells we developed expansion and transduction protocols that allow for the generation of sufficient numbers of CAR-expressing canine T cells for future clinical studies in dogs within 2 weeks of ex vivo culture. To evaluate the functionality of CAR-expressing canine T cells we targeted HER2-positive OS. We demonstrate that canine OS is positive for HER2, and that canine T cells expressing a HER2-specific CAR with human-derived transmembrane and CD28.ζ signaling domains recognize and kill HER2-positive canine OS cell lines in an antigen-dependent manner. To reduce the potential immunogenicity of the CAR we evaluated a CAR with canine-derived transmembrane and signaling domains, and found no functional difference between human and canine CARs. Hence, we have successfully developed a strategy to generate CAR-expressing canine T cells for future preclinical studies in dogs. Testing T-cell therapies in an immunocompetent, outbred animal model may improve our ability to predict their safety and efficacy prior to conducting studies in humans. PMID:25198528

Population differences in immune marker profiles associated with human T-lymphotropic virus type I infection in Japan and Jamaica.

PubMed

Birmann, Brenda M; Breen, Elizabeth C; Stuver, Sherri; Cranston, Beverly; Martínez-Maza, Otoniel; Falk, Kerstin I; Okayama, Akihiko; Hanchard, Barrie; Mueller, Nancy; Hisada, Michie

2009-02-01

The natural history of human T-lymphotropic virus type I (HTLV-I) has been shown to differ markedly by geographic area. The differences include contrasting patterns of risk of adult T-cell lymphoma (ATL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which may be due in part to differences in host immune response to infection. To characterize variations in host immunity across populations, we compared serologic immune marker patterns in HTLV-I-endemic populations in Japan and Jamaica. We matched 204 participants with archived blood from the Miyazaki Cohort Study (Japan) and the Food Handlers Study (Jamaica)-i.e., 51 HTLV-I-positive ("carriers") and 51 HTLV-I-negative individuals ("noncarriers") from each population-by age, sex and blood collection year. We compared plasma concentrations of markers of T-cell-mediated (antigen-specific) and nonspecific immunity using regression models and correlation coefficients. Compared to Jamaican HTLV-I noncarriers, Japanese noncarriers had higher covariate-adjusted mean levels of T-cell activation markers, including antibody to Epstein-Barr virus nuclear antigen-1 (reciprocal titer 27 vs. 71, respectively, p=0.005), soluble interleukin-2 receptor-alpha (477 vs. 623 pg/mL, p=0.0008) and soluble CD30 (34 vs. 46 U/mL, p=0.0001) and lower levels of C-reactive protein (1.1 vs. 0.43 microg/mL, p=0.0004). HTLV-I infection was associated with activated T-cell immunity in Jamaicans but with diminished T-cell immunity in Japanese persons. The observed population differences in background and HTLV-I-related host immunity correspond closely to the divergent natural histories of infection observed among HTLV-I carriers in Japan and Jamaica and corroborate a role for host immune status in the contrasting patterns of ATL and HAM/TSP risk. Copyright (c) 2008 Wiley-Liss, Inc.

CP-25 Attenuates the Activation of CD4+ T Cells Stimulated with Immunoglobulin D in Human.

PubMed

Wu, Yu-Jing; Chen, Heng-Shi; Chen, Wen-Sheng; Dong, Jin; Dong, Xiao-Jie; Dai, Xing; Huang, Qiong; Wei, Wei

2018-01-01

Researchers have shown that the level of immunoglobulin D (IgD) is often elevated in patients with autoimmune diseases. The possible roles of IgD on the function of human T cell activation are still unclear. Paeoniflorin-6'- O -benzene sulfonate (code: CP-25), the chemistry structural modifications of paeoniflorin, was a novel drug of anti-inflammation and immunomodulation. The aims of this study were to determine if human CD4 + T cells could be activated by IgD via the IgD receptor (IgDR)-Lck pathway and whether the novel compound CP-25 could affect the activation of T cells by regulating Lck. Human CD4 + T cells were purified from peripheral blood mononuclear cells using microbeads. T cell viability and proliferation were detected by Cell Counting Kit-8 and CFSE Cell Proliferation Kit. Cytokines secreted by T cells were assessed with the Quantibody Human Inflammation Array. The binding affinity and expression of IgDR on T cells were detected by flow cytometry, and protein expression of IgDR, Lck, and P-Lck were analyzed by western blot. IgD was shown to bind to IgDR on CD4 + T cells in a concentration-dependent manner and stimulate the activation and proliferation of these cells by enhancing phosphorylation of the activating tyrosine residue of Lck (Tyr 394 ). CP-25 inhibited the IgD-stimulated activation and proliferation of CD4 + T cells, as well as the production of inflammatory cytokines; it was thus suggested that this process might be related to the downregulation of Lck (Tyr 394 ) phosphorylation. These results demonstrate that IgD amplifies the activation of CD4 + T cells, which could be mediated by Lck phosphorylation. Further, CP-25, via its ability to modulate Lck, is a novel potential therapeutic agent for the treatment of human autoimmune diseases.

Evidence that Singlet Oxygen-induced Human T Helper Cell Apoptosis Is the Basic Mechanism of Ultraviolet-A Radiation Phototherapy

PubMed Central

Morita, Akimichi; Werfel, Thomas; Stege, Helger; Ahrens, Constanze; Karmann, Karin; Grewe, Markus; Grether-Beck, Susanne; Ruzicka, Thomas; Kapp, Alexander; Klotz, Lars-Oliver; Sies, Helmut; Krutmann, Jean

1997-01-01

Ultraviolet A (UVA) irradiation is effectively used to treat patients with atopic dermatitis and other T cell mediated, inflammatory skin diseases. In the present study, successful phototherapy of atopic dermatitis was found to result from UVA radiation-induced apoptosis in skin-infiltrating T helper cells, leading to T cell depletion from eczematous skin. In vitro, UVA radiation-induced human T helper cell apoptosis was mediated through the FAS/FAS-ligand system, which was activated in irradiated T cells as a consequence of singlet oxygen generation. These studies demonstrate that singlet oxygen is a potent trigger for the induction of human T cell apoptosis. They also identify singlet oxygen generation as a fundamental mechanism of action operative in phototherapy. PMID:9362536

Identification of a novel proinflammatory human skin-homing Vγ9Vδ2 T cell subset with a potential role in psoriasis.

PubMed

Laggner, Ute; Di Meglio, Paola; Perera, Gayathri K; Hundhausen, Christian; Lacy, Katie E; Ali, Niwa; Smith, Catherine H; Hayday, Adrian C; Nickoloff, Brian J; Nestle, Frank O

2011-09-01

γδ T cells mediate rapid tissue responses in murine skin and participate in cutaneous immune regulation including protection against cancer. The role of human γδ cells in cutaneous homeostasis and pathology is characterized poorly. In this study, we show in vivo evidence that human blood contains a distinct subset of proinflammatory cutaneous lymphocyte Ag and CCR6-positive Vγ9Vδ2 T cells, which is rapidly recruited into perturbed human skin. Vγ9Vδ2 T cells produced an array of proinflammatory mediators including IL-17A and activated keratinocytes in a TNF-α- and IFN-γ-dependent manner. Examination of the common inflammatory skin disease psoriasis revealed a striking reduction of circulating Vγ9Vδ2 T cells in psoriasis patients compared with healthy controls and atopic dermatitis patients. Decreased numbers of circulating Vγ9Vδ2 T cells normalized after successful treatment with psoriasis-targeted therapy. Taken together with the increased presence of Vγ9Vδ2 T cells in psoriatic skin, these data indicate redistribution of Vγ9Vδ2 T cells from the blood to the skin compartment in psoriasis. In summary, we report a novel human proinflammatory γδ T cell involved in skin immune surveillance with immediate response characteristics and with potential clinical relevance in inflammatory skin disease.

HLA-B*57 and IFNL4-Related Polymorphisms Are Associated With Protection Against HIV-1 Disease Progression in Controllers

PubMed Central

Dominguez-Molina, Beatriz; Tarancon-Diez, Laura; Hua, Stephane; Abad-Molina, Cristina; Rodriguez-Gallego, Esther; Machmach, Kawthar; Vidal, Francesc; Tural, Cristina; Moreno, Santiago; Goñi, María José; Ramírez de Arellano, Elena; del Val, Margarita; Gonzalez-Escribano, María Francisca; Del Romero, Jorge; Rodriguez, Carmen; Capa, Laura; Viciana, Pompeyo; Alcamí, José; Yu, Xu G.; Walker, Bruce D.; Leal, Manuel; Lichterfeld, Mathias

2017-01-01

Abstract Background. Human immunodeficiency virus type 1 (HIV-1) controllers maintain HIV-1 viremia at low levels (normally <2000 HIV-RNA copies/mL) without antiretroviral treatment. However, some HIV-1 controllers have evidence of immunologic progression with marked CD4+ T-cell decline. We investigated host genetic factors associated with protection against CD4+ T-cell loss in HIV-1 controllers. Methods. We analyzed the association of interferon-lambda 4 (IFNL4)–related polymorphisms and human leukocyte antigen (HLA)-B haplotypes within long-term nonprogressor HIV-1 controllers (LTNP-Cs; defined by maintaining CD4+ T-cells counts >500 cells/mm3 for more than 7 years after HIV-1 diagnosis) vs non-LTNP-Cs who developed CD4+ T-cell counts <500 cells/mm3. Both a Spanish study cohort (n = 140) and an international validation cohort (n = 914) were examined. Additionally, in a subgroup of individuals, HIV-1–specific T-cell responses and soluble cytokines were analyzed. Results. HLA-B*57 was independently associated with the LTNP-C phenotype (odds ratio [OR], 3.056 [1.029–9.069]; P = .044 and OR, 1.924 [1.252–2.957]; P = .003) while IFNL4 genotypes represented independent factors for becoming non-LTNP-C (TT/TT, ss469415590; OR, 0.401 [0.171–0.942]; P = .036 or A/A, rs12980275; OR, 0.637 [0.434–0.934]; P = .021) in the Spanish and validation cohorts, respectively, after adjusting for sex, age at HIV-1 diagnosis, IFNL4-related polymorphisms, and different HLA-B haplotypes. LTNP-Cs showed lower plasma induced protein 10 (P = .019) and higher IFN-γ (P = .02) levels than the HIV-1 controllers with diminished CD4+ T-cell numbers. Moreover, LTNP-Cs exhibited higher quantities of interleukin (IL)2+CD57- and IFN-γ +CD57- HIV-1–specific CD8+ T cells (P = .002 and .041, respectively) than non-LTNP-Cs. Conclusions. We defined genetic markers able to segregate stable HIV-1 controllers from those who experience CD4+ T-cell decline. These findings allow for identification of HIV-1 controllers at risk for immunologic progression and provide avenues for personalized therapeutic interventions and precision medicine for optimizing clinical care of these individuals. PMID:27986689

CD8+ T-cell mediated anti-malaria protection induced by malaria vaccines; assessment of hepatic CD8+ T cells by SCBC assay.

PubMed

Zhou, Jing; Kaiser, Alaina; Ng, Colin; Karcher, Rachel; McConnell, Tim; Paczkowski, Patrick; Fernandez, Cristina; Zhang, Min; Mackay, Sean; Tsuji, Moriya

2017-07-03

Malaria is a severe infectious disease with relatively high mortality, thus having been a scourge of humanity. There are a few candidate malaria vaccines that have shown a protective efficacy in humans against malaria. One of the candidate human malaria vaccines, which is based on human malaria sporozoites and called PfSPZ Vaccine, has been shown to protect a significant proportion of vaccine recipients from getting malaria. PfSPZ Vaccine elicits a potent response of hepatic CD8+ T cells that are specific for malaria antigens in non-human primates. To further characterize hepatic CD8+ T cells induced by the sporozoite-based malaria vaccine in a mouse model, we have used a cutting-edge Single-cell Barcode (SCBC) assay, a recently emerged approach/method for investigating the nature of T-cells responses during infection or cancer. Using the SCBC technology, we have identified a population of hepatic CD8+ T cells that are polyfunctional at a single cell level only in a group of vaccinated mice upon malaria challenge. The cytokines/chemokines secreted by these polyfunctional CD8+ T-cell subsets include MIP-1α, RANTES, IFN-γ, and/or IL-17A, which have shown to be associated with protective T-cell responses against certain pathogens. Therefore, a successful induction of such polyfunctional hepatic CD8+ T cells may be a key to the development of effective human malaria vaccine. In addition, the SCBC technology could provide a new level of diagnostic that will allow for a more accurate determination of vaccine efficacy.

In vivo expression of the HBZ gene of HTLV-1 correlates with proviral load, inflammatory markers and disease severity in HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP)

PubMed Central

Saito, Mineki; Matsuzaki, Toshio; Satou, Yorifumi; Yasunaga, Jun-ichirou; Saito, Kousuke; Arimura, Kimiyoshi; Matsuoka, Masao; Ohara, Yoshiro

2009-01-01

Background Recently, human T-cell leukemia virus type 1 (HTLV-1) basic leucine zipper factor (HBZ), encoded from a minus strand mRNA was discovered and was suggested to play an important role in adult T cell leukemia (ATL) development. However, there have been no reports on the role of HBZ in patients with HTLV-1 associated inflammatory diseases. Results We quantified the HBZ and tax mRNA expression levels in peripheral blood from 56 HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients, 10 ATL patients, 38 healthy asymptomatic carriers (HCs) and 20 normal uninfected controls, as well as human leukemic T-cell lines and HTLV-1-infected T-cell lines, and the data were correlated with clinical parameters. The spliced HBZ gene was transcribed in all HTLV-1-infected individuals examined, whereas tax mRNA was not transcribed in significant numbers of subjects in the same groups. Although the amount of HBZ mRNA expression was highest in ATL, medium in HAM/TSP, and lowest in HCs, with statistical significance, neither tax nor the HBZ mRNA expression per HTLV-1-infected cell differed significantly between each clinical group. The HTLV-1 HBZ, but not tax mRNA load, positively correlated with disease severity and with neopterin concentration in the cerebrospinal fluid of HAM/TSP patients. Furthermore, HBZ mRNA expression per HTLV-1-infected cell was decreased after successful immunomodulatory treatment for HAM/TSP. Conclusion These findings suggest that in vivo expression of HBZ plays a role in HAM/TSP pathogenesis. PMID:19228429

Efficient Transduction of Human and Rhesus Macaque Primary T Cells by a Modified Human Immunodeficiency Virus Type 1-Based Lentiviral Vector.

PubMed

He, Huan; Xue, Jing; Wang, Weiming; Liu, Lihong; Ye, Chaobaihui; Cong, Zhe; Kimata, Jason T; Qin, Chuan; Zhou, Paul

2017-03-01

Human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors efficiently transduce genes to human, but not rhesus, primary T cells and hematopoietic stem cells (HSCs). The poor transduction of HIV-1 vectors to rhesus cells is mainly due to species-specific restriction factors such as rhesus TRIM5α. Previously, several strategies to modify HIV-1 vectors were developed to overcome rhesus TRIM5α restriction. While the modified HIV-1 vectors efficiently transduce rhesus HSCs, they remain suboptimal for rhesus primary T cells. Recently, HIV-1 variants that encode combinations of LNEIE mutations in capsid (CA) protein and SIVmac239 Vif were found to replicate efficiently in rhesus primary T cells. Thus, the present study tested whether HIV-1 vectors packaged by a packaging construct containing these CA substitutions could efficiently transduce both human and rhesus primary CD4 T cells. To accomplish this, LNEIE mutations were made in the packaging construct CEMΔ8.9, and recombinant HIV-1 vectors packaged by Δ8.9 WT or Δ8.9 LNEIE were generated. Transduction rates, CA stability, and vector integration in CEMss-CCR5 and CEMss-CCR5-rhTRIM5α/green fluorescent protein cells, as well as transduction rates in human and rhesus primary CD4 T cells by Δ8.9 WT or Δ8.9 LNEIE-packaged HIV-1 vectors, were compared. Finally, the influence of rhesus TRIM5α variations in transduction rates to primary CD4 T cells from a cohort of 37 Chinese rhesus macaques was studied. While it maintains efficient transduction for human T-cell line and primary CD4 T cells, Δ8.9 LNEIE-packaged HIV-1 vector overcomes rhesus TRIM5α-mediated CA degradation, resulting in significantly higher transduction efficiency of rhesus primary CD4 T cells than Δ8.9 WT-packaged HIV-1 vector. Rhesus TRIM5α variations strongly influence transduction efficiency of rhesus primary CD4 T cells by both Δ8.9 WT or Δ8.9 LNEIE-packaged HIV-1 vectors. Thus, it is concluded that Δ8.9 LNEIE-packaged HIV-1 vector overcomes rhesus TRIM5α restriction and efficiently transduces both human and rhesus primary T cells.

Efficient Transduction of Human and Rhesus Macaque Primary T Cells by a Modified Human Immunodeficiency Virus Type 1–Based Lentiviral Vector

PubMed Central

He, Huan; Xue, Jing; Wang, Weiming; Liu, Lihong; Ye, Chaobaihui; Cong, Zhe; Kimata, Jason T.; Qin, Chuan; Zhou, Paul

2017-01-01

Human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors efficiently transduce genes to human, but not rhesus, primary T cells and hematopoietic stem cells (HSCs). The poor transduction of HIV-1 vectors to rhesus cells is mainly due to species-specific restriction factors such as rhesus TRIM5α. Previously, several strategies to modify HIV-1 vectors were developed to overcome rhesus TRIM5α restriction. While the modified HIV-1 vectors efficiently transduce rhesus HSCs, they remain suboptimal for rhesus primary T cells. Recently, HIV-1 variants that encode combinations of LNEIE mutations in capsid (CA) protein and SIVmac239 Vif were found to replicate efficiently in rhesus primary T cells. Thus, the present study tested whether HIV-1 vectors packaged by a packaging construct containing these CA substitutions could efficiently transduce both human and rhesus primary CD4 T cells. To accomplish this, LNEIE mutations were made in the packaging construct CEMΔ8.9, and recombinant HIV-1 vectors packaged by Δ8.9 WT or Δ8.9 LNEIE were generated. Transduction rates, CA stability, and vector integration in CEMss-CCR5 and CEMss-CCR5-rhTRIM5α/green fluorescent protein cells, as well as transduction rates in human and rhesus primary CD4 T cells by Δ8.9 WT or Δ8.9 LNEIE-packaged HIV-1 vectors, were compared. Finally, the influence of rhesus TRIM5α variations in transduction rates to primary CD4 T cells from a cohort of 37 Chinese rhesus macaques was studied. While it maintains efficient transduction for human T-cell line and primary CD4 T cells, Δ8.9 LNEIE-packaged HIV-1 vector overcomes rhesus TRIM5α-mediated CA degradation, resulting in significantly higher transduction efficiency of rhesus primary CD4 T cells than Δ8.9 WT-packaged HIV-1 vector. Rhesus TRIM5α variations strongly influence transduction efficiency of rhesus primary CD4 T cells by both Δ8.9 WT or Δ8.9 LNEIE-packaged HIV-1 vectors. Thus, it is concluded that Δ8.9 LNEIE-packaged HIV-1 vector overcomes rhesus TRIM5α restriction and efficiently transduces both human and rhesus primary T cells. PMID:28042947

[60]Fullerene derivative modulates adenosine and metabotropic glutamate receptors gene expression: a possible protective effect against hypoxia

PubMed Central

2014-01-01

Background Glutamate, the main excitatory neurotransmitter, is involved in learning and memory processes but at higher concentration results excitotoxic causing degeneration and neuronal death. Adenosine is a nucleoside that exhibit neuroprotective effects by modulating of glutamate release. Hypoxic and related oxidative conditions, in which adenosine and metabotropic glutamate receptors are involved, have been demonstrated to contribute to neurodegenerative processes occurring in certain human pathologies. Results Human neuroblastoma cells (SH-SY5Y) were used to evaluate the long time (24, 48 and 72 hours) effects of a [60]fullerene hydrosoluble derivative (t3ss) as potential inhibitor of hypoxic insult. Low oxygen concentration (5% O2) caused cell death, which was avoided by t3ss exposure in a concentration dependent manner. In addition, gene expression analysis by real time PCR of adenosine A1, A2A and A2B and metabotropic glutamate 1 and 5 receptors revealed that t3ss significantly increased A1 and mGlu1 expression in hypoxic conditions. Moreover, t3ss prevented the hypoxia-induced increase in A2A mRNA expression. Conclusions As t3ss causes overexpression of adenosine A1 and metabotropic glutamate receptors which have been shown to be neuroprotective, our results point to a radical scavenger protective effect of t3ss through the enhancement of these neuroprotective receptors expression. Therefore, the utility of these nanoparticles as therapeutic target to avoid degeneration and cell death of neurodegenerative diseases is suggested. PMID:25123848

The human Vδ2+ T-cell compartment comprises distinct innate-like Vγ9+ and adaptive Vγ9- subsets.

PubMed

Davey, Martin S; Willcox, Carrie R; Hunter, Stuart; Kasatskaya, Sofya A; Remmerswaal, Ester B M; Salim, Mahboob; Mohammed, Fiyaz; Bemelman, Frederike J; Chudakov, Dmitriy M; Oo, Ye H; Willcox, Benjamin E

2018-05-02

Vδ2 + T cells form the predominant human γδ T-cell population in peripheral blood and mediate T-cell receptor (TCR)-dependent anti-microbial and anti-tumour immunity. Here we show that the Vδ2 + compartment comprises both innate-like and adaptive subsets. Vγ9 + Vδ2 + T cells display semi-invariant TCR repertoires, featuring public Vγ9 TCR sequences equivalent in cord and adult blood. By contrast, we also identify a separate, Vγ9 - Vδ2 + T-cell subset that typically has a CD27 hi CCR7 + CD28 + IL-7Rα + naive-like phenotype and a diverse TCR repertoire, however in response to viral infection, undergoes clonal expansion and differentiation to a CD27 lo CD45RA + CX 3 CR1 + granzymeA/B + effector phenotype. Consistent with a function in solid tissue immunosurveillance, we detect human intrahepatic Vγ9 - Vδ2 + T cells featuring dominant clonal expansions and an effector phenotype. These findings redefine human γδ T-cell subsets by delineating the Vδ2 + T-cell compartment into innate-like (Vγ9 + ) and adaptive (Vγ9 - ) subsets, which have distinct functions in microbial immunosurveillance.

Type 1 Responses of Human Vγ9Vδ2 T Cells to Influenza A Viruses▿

PubMed Central

Qin, Gang; Liu, Yinping; Zheng, Jian; Ng, Iris H. Y.; Xiang, Zheng; Lam, Kwok-Tai; Mao, Huawei; Li, Hong; Peiris, J. S. Malik; Lau, Yu-Lung; Tu, Wenwei

2011-01-01

γδ T cells are essential constituents of antimicrobial and antitumor defenses. We have recently reported that phosphoantigen isopentenyl pyrophosphate (IPP)-expanded human Vγ9Vδ2 T cells participated in anti-influenza virus immunity by efficiently killing both human and avian influenza virus-infected monocyte-derived macrophages (MDMs) in vitro. However, little is known about the noncytolytic responses and trafficking program of γδ T cells to influenza virus. In this study, we found that Vγ9Vδ2 T cells expressed both type 1 cytokines and chemokine receptors during influenza virus infection, and IPP-expanded cells had a higher capacity to produce gamma interferon (IFN-γ). Besides their potent cytolytic activity against pandemic H1N1 virus-infected cells, IPP-activated γδ T cells also had noncytolytic inhibitory effects on seasonal and pandemic H1N1 viruses via IFN-γ but had no such effects on avian H5N1 or H9N2 virus. Avian H5N1 and H9N2 viruses induced significantly higher CCL3, CCL4, and CCL5 production in Vγ9Vδ2 T cells than human seasonal H1N1 virus. CCR5 mediated the migration of Vγ9Vδ2 T cells toward influenza virus-infected cells. Our findings suggest a novel therapeutic strategy of using phosphoantigens to boost the antiviral activities of human Vγ9Vδ2 T cells against influenza virus infection. PMID:21752902

Humanized Mouse Model of Skin Inflammation Is Characterized by Disturbed Keratinocyte Differentiation and Influx of IL-17A Producing T Cells

PubMed Central

de Oliveira, Vivian L.; Keijsers, Romy R. M. C.; van de Kerkhof, Peter C. M.; Seyger, Marieke M. B.; Fasse, Esther; Svensson, Lars; Latta, Markus; Norsgaard, Hanne; Labuda, Tord; Hupkens, Pieter; van Erp, Piet E. J.; Joosten, Irma; Koenen, Hans J. P. M.

2012-01-01

Humanized mouse models offer a challenging possibility to study human cell function in vivo. In the huPBL-SCID-huSkin allograft model human skin is transplanted onto immunodeficient mice and allowed to heal. Thereafter allogeneic human peripheral blood mononuclear cells are infused intra peritoneally to induce T cell mediated inflammation and microvessel destruction of the human skin. This model has great potential for in vivo study of human immune cells in (skin) inflammatory processes and for preclinical screening of systemically administered immunomodulating agents. Here we studied the inflammatory skin response of human keratinocytes and human T cells and the concomitant systemic human T cell response. As new findings in the inflamed human skin of the huPBL-SCID-huSkin model we here identified: 1. Parameters of dermal pathology that enable precise quantification of the local skin inflammatory response exemplified by acanthosis, increased expression of human β-defensin-2, Elafin, K16, Ki67 and reduced expression of K10 by microscopy and immunohistochemistry. 2. Induction of human cytokines and chemokines using quantitative real-time PCR. 3. Influx of inflammation associated IL-17A-producing human CD4+ and CD8+ T cells as well as immunoregulatory CD4+Foxp3+ cells using immunohistochemistry and -fluorescence, suggesting that active immune regulation is taking place locally in the inflamed skin. 4. Systemic responses that revealed activated and proliferating human CD4+ and CD8+ T cells that acquired homing marker expression of CD62L and CLA. Finally, we demonstrated the value of the newly identified parameters by showing significant changes upon systemic treatment with the T cell inhibitory agents cyclosporine-A and rapamycin. In summary, here we equipped the huPBL-SCID-huSkin humanized mouse model with relevant tools not only to quantify the inflammatory dermal response, but also to monitor the peripheral immune status. This combined approach will gain our understanding of the dermal immunopathology in humans and benefit the development of novel therapeutics for controlling inflammatory skin diseases. PMID:23094018

Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

PubMed Central

Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

1992-01-01

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes. Images PMID:1533935

TIM-3 Does Not Act as a Receptor for Galectin-9

PubMed Central

Leitner, Judith; Rieger, Armin; Pickl, Winfried F.; Zlabinger, Gerhard; Grabmeier-Pfistershammer, Katharina; Steinberger, Peter

2013-01-01

T cell immunoglobulin and mucin protein 3 (TIM-3) is a type I cell surface protein that was originally identified as a marker for murine T helper type 1 cells. TIM-3 was found to negatively regulate murine T cell responses and galectin-9 was described as a binding partner that mediates T cell inhibitory effects of TIM-3. Moreover, it was reported that like PD-1 the classical exhaustion marker, TIM-3 is up-regulated in exhausted murine and human T cells and TIM-3 blockade was described to restore the function of these T cells. Here we show that the activation of human T cells is not affected by the presence of galectin-9 or antibodies to TIM-3. Furthermore, extensive studies on the interaction of galectin-9 with human and murine TIM-3 did not yield evidence for specific binding between these molecules. Moreover, profound differences were observed when analysing the expression of TIM-3 and PD-1 on T cells of HIV-1-infected individuals: TIM-3 was expressed on fewer cells and also at much lower levels. Furthermore, whereas PD-1 was preferentially expressed on CD45RA−CD8 T cells, the majority of TIM-3-expressing CD8 T cells were CD45RA+. Importantly, we found that TIM-3 antibodies were ineffective in increasing anti-HIV-1 T cell responses in vitro, whereas PD-L antibodies potently reverted the dysfunctional state of exhausted CD8 T cells. Taken together, our results are not in support of an interaction between TIM-3 and galectin-9 and yield no evidence for a functional role of TIM-3 in human T cell activation. Moreover, our data indicate that PD-1, but not TIM-3, is a promising target to ameliorate T cell exhaustion. PMID:23555261

IL-17+ regulatory T cells in the microenvironments of chronic inflammation and cancer.

PubMed

Kryczek, Ilona; Wu, Ke; Zhao, Ende; Wei, Shuang; Vatan, Linhua; Szeliga, Wojciech; Huang, Emina; Greenson, Joel; Chang, Alfred; Roliński, Jacek; Radwan, Piotr; Fang, Jingyuan; Wang, Guobin; Zou, Weiping

2011-04-01

Foxp3(+)CD4(+) regulatory T (Treg) cells inhibit immune responses and temper inflammation. IL-17(+)CD4(+) T (Th17) cells mediate inflammation of autoimmune diseases. A small population of IL-17(+)Foxp3(+)CD4(+) T cells has been observed in peripheral blood in healthy human beings. However, the biology of IL-17(+)Foxp3(+)CD4(+) T cells remains poorly understood in humans. We investigated their phenotype, cytokine profile, generation, and pathological relevance in patients with ulcerative colitis. We observed that high levels of IL-17(+)Foxp3(+)CD4(+) T cells were selectively accumulated in the colitic microenvironment and associated colon carcinoma. The phenotype and cytokine profile of IL-17(+)Foxp3(+)CD4(+) T cells was overlapping with Th17 and Treg cells. Myeloid APCs, IL-2, and TGF-β are essential for their induction from memory CCR6(+) T cells or Treg cells. IL-17(+)Foxp3(+)CD4(+) T cells functionally suppressed T cell activation and stimulated inflammatory cytokine production in the colitic tissues. Our data indicate that IL-17(+)Foxp3(+) cells may be "inflammatory" Treg cells in the pathological microenvironments. These cells may contribute to the pathogenesis of ulcerative colitis through inducing inflammatory cytokines and inhibiting local T cell immunity, and in turn may mechanistically link human chronic inflammation to tumor development. Our data therefore challenge commonly held beliefs of the anti-inflammatory role of Treg cells and suggest a more complex Treg cell biology, at least in the context of human chronic inflammation and associated carcinoma.

Human CD3+ T-Cells with the Anti-ERBB2 Chimeric Antigen Receptor Exhibit Efficient Targeting and Induce Apoptosis in ERBB2 Overexpressing Breast Cancer Cells

PubMed Central

Munisvaradass, Rusheni; Kumar, Suresh; Govindasamy, Chandramohan; Alnumair, Khalid S.; Mok, Pooi Ling

2017-01-01

Breast cancer is a common malignancy among women. The innate and adaptive immune responses failed to be activated owing to immune modulation in the tumour microenvironment. Decades of scientific study links the overexpression of human epidermal growth factor receptor 2 (ERBB2) antigen with aggressive tumours. The Chimeric Antigen Receptor (CAR) coding for specific tumour-associated antigens could initiate intrinsic T-cell signalling, inducing T-cell activation, and cytotoxic activity without the need for major histocompatibility complex recognition. This renders CAR as a potentially universal immunotherapeutic option. Herein, we aimed to establish CAR in CD3+ T-cells, isolated from human peripheral blood mononucleated cells that could subsequently target and induce apoptosis in the ERBB2 overexpressing human breast cancer cell line, SKBR3. Constructed CAR was inserted into a lentiviral plasmid containing a green fluorescent protein tag and produced as lentiviral particles that were used to transduce activated T-cells. Transduced CAR-T cells were then primed with SKBR3 cells to evaluate their functionality. Results showed increased apoptosis in SKBR3 cells co-cultured with CAR-T cells compared to the control (non–transduced T-cells). This study demonstrates that CAR introduction helps overcome the innate limitations of native T-cells leading to cancer cell apoptosis. We recommend future studies should focus on in vivo cytotoxicity of CAR-T cells against ERBB2 expressing tumours. PMID:28885562

Human CD3+ T-Cells with the Anti-ERBB2 Chimeric Antigen Receptor Exhibit Efficient Targeting and Induce Apoptosis in ERBB2 Overexpressing Breast Cancer Cells.

PubMed

Munisvaradass, Rusheni; Kumar, Suresh; Govindasamy, Chandramohan; Alnumair, Khalid S; Mok, Pooi Ling

2017-09-08

Breast cancer is a common malignancy among women. The innate and adaptive immune responses failed to be activated owing to immune modulation in the tumour microenvironment. Decades of scientific study links the overexpression of human epidermal growth factor receptor 2 (ERBB2) antigen with aggressive tumours. The Chimeric Antigen Receptor (CAR) coding for specific tumour-associated antigens could initiate intrinsic T-cell signalling, inducing T-cell activation, and cytotoxic activity without the need for major histocompatibility complex recognition. This renders CAR as a potentially universal immunotherapeutic option. Herein, we aimed to establish CAR in CD3+ T-cells, isolated from human peripheral blood mononucleated cells that could subsequently target and induce apoptosis in the ERBB2 overexpressing human breast cancer cell line, SKBR3. Constructed CAR was inserted into a lentiviral plasmid containing a green fluorescent protein tag and produced as lentiviral particles that were used to transduce activated T-cells. Transduced CAR-T cells were then primed with SKBR3 cells to evaluate their functionality. Results showed increased apoptosis in SKBR3 cells co-cultured with CAR-T cells compared to the control (non-transduced T-cells). This study demonstrates that CAR introduction helps overcome the innate limitations of native T-cells leading to cancer cell apoptosis. We recommend future studies should focus on in vivo cytotoxicity of CAR-T cells against ERBB2 expressing tumours.

Emodin induces human T cell apoptosis in vitro by ROS-mediated endoplasmic reticulum stress and mitochondrial dysfunction

PubMed Central

Qu, Kai; Shen, Nai-ying; Xu, Xin-sen; Su, Hai-bo; Wei, Ji-chao; Tai, Ming-hui; Meng, Fan-di; Zhou, Lei; Zhang, Yue-lang; Liu, Chang

2013-01-01

Aim: To elucidate the molecular mechanisms underlying the immunosuppressive effects of emodin isolated from Rheum palmatum L. Methods: Human T cells were isolated from the peripheral venous blood of 10 healthy adult donors. Cell viability was analyzed with MTT assay. AO/EB and Annexin V/PI staining and DNA damage assay were used to detect cell apoptosis. Fluorescence staining was used to detect the levels of ROS, the mitochondrial membrane potential and intracellular Ca2+. Colorimetry was used to detect the levels of MDA and total SOD and GSH/GSSG ratio. The expression and activity of caspase-3, -4, and -9 were detected with Western blotting and a fluorometric assay. Western blotting was also used to detect the expression of Bcl-2, Bax, cytochrome C, and endoplasmic reticulum (ER) markers. Results: Emodin (1, 10, and 100 μmol/L) inhibited the growth of human T cells and induced apoptosis in dose- and time dependent manners. Emodin triggered ER stress and significantly elevated intracellular free Ca2+ in human T cells. It also disrupted mitochondrial membrane potential, and increased cytosolic level of cytochrome C, and the levels of activated cleavage fragments of caspase-3, -4, and -9 in human T cells. Furthermore, emodin significantly increased the levels of ROS and MDA, inhibited both SOD level and GSH/GSSG ratio in human T cells, whereas co-incubation with the ROS scavenger N-acetylcysteine (NAC, 20 μmol/L) almost completely blocked emodin-induced ER stress and mitochondrial dysfunction in human T cells, and decreased the caspase cascade-mediated apoptosis. Conclusion: Emodin exerts immunosuppressive actions at least partly by inducing apoptosis of human T cells, which is triggered by ROS-mediated ER stress and mitochondrial dysfunction. PMID:23811723

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.

[gammadelta T cells stimulated by zoledronate kill osteosarcoma cells].

PubMed

Jiang, Hui; Xu, Qiang; Yang, Chao; Cao, Zhen-Guo; Li, Zhao-Xu; Ye, Zhao-Ming

2010-12-01

To investigate the cytotoxicity of human γδT cells from PBMCs stimulated by zoledronate against osteosarcoma cell line HOS in vitro and in vivo and evaluate the relavent pathways. The peripheral blood mononuclear cells (PBMCs)of healthy donors were stimulated by single dose zoledronate and cultured in the present of IL-2 for two weeks, analysising the percentage of γδT cells on a FACSCalibur cytometer.Study the cytotoxicity of γδT cells against the osteosarcoma line HOS using LDH release assay kit. Pre-treatment of γδT cells with anti-human γδTCR antibody, anti-human NKG2D antibody and concanamycin A to bolck the relavent pathways for evaluating the mechenisms of its cytotoxicity. In vivo, BALB/c mice were inoculated subcutaneously osteosarcoma cell HOS for developing hypodermal tumors. And they were randomized into two groups: unteated group, γδT cell therapy group. Tumor volume and weight of the two groups were compared. After two weeks of culture, γδT cells from zoledronate-stimulated PBMCs could reach (95±3)%. When the E:T as 6:1, 12:1, 25:1, 50:1, the percentage of osteosarcoma cell HOS killed by γδT cells was 26.8%, 31.5%, 37.8%, 40.9%, respectively.When anti-huma γδTCR antibody, anti-human NKG2D antibody and concanamycin A blocked the relavent pathways, the percentage was 32.3%, 4.7%, 16.7% ( E:T as 25:1), respectively. In vivo, the tumor inhibition rate of the group of γδT cell therapy was 42.78%. γδT cells derived from PBMCs stimulated by zoledronate can acquired pure γδT cells. And they show strong cytoxicity against osteosarcoma cell line HOS in vitro and in vivo.

T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones.

PubMed

Theaker, Sarah M; Rius, Cristina; Greenshields-Watson, Alexander; Lloyd, Angharad; Trimby, Andrew; Fuller, Anna; Miles, John J; Cole, David K; Peakman, Mark; Sewell, Andrew K; Dolton, Garry

2016-03-01

Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8(+) or CD4(+) polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6 weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein-Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

IL-10 suppresses Th17 cells and promotes regulatory T cells in the CD4+ T cell population of rheumatoid arthritis patients.

PubMed

Heo, Yu-Jung; Joo, Young-Bin; Oh, Hye-Jwa; Park, Mi-Kyung; Heo, Yang-Mi; Cho, Mi-La; Kwok, Seung-Ki; Ju, Ji-Hyeon; Park, Kyung-Su; Cho, Seok Goo; Park, Sung-Hwan; Kim, Ho-Youn; Min, Jun-Ki

2010-01-04

Interleukin-17-producing CD4(+) T cells (Th17 cells) are the dominant pathogenic cellular component in autoimmune inflammatory diseases, including autoimmune arthritis. IL-10 promotes the generation of Foxp3(+) regulatory T cells via the IL-10 receptor signal. The objective of this study was to examine whether IL-10, which acts as an anti-inflammatory cytokine, has a suppressive effect on the activation of human Th17 cells. Expression of IL-17 and IL-10 was examined immunohistochemically in tissue obtained from rheumatoid arthritis patients. Human peripheral blood CD4(+) T cells were isolated and cultured under various stimulatory conditions. Th17 cells and regulatory T (Treg) cells were detected by flow cytometry. The gene expression of related cytokines and transcription factors were assessed by ELISA and RT-PCR. IL-17 was overexpressed in rheumatoid arthritis patients. IL-10 treatment significantly decreased the numbers of IL-17-producing and RORc-expressing cells among human CD4(+) T cells that had been activated in vitro by Th17-differentiating conditions in autoimmune arthritis patients. IL-10 induced Foxp3(+) regulatory T cells in the human CD4(+) T cell population. Our results demonstrate that IL-17 is overexpressed in autoimmune disease patients and that IL-10 suppresses IL-17 expression. IL-10 may be useful in the treatment of autoimmune diseases.

Effector/memory CD8+ T cells synergize with co-stimulation competent macrophages to trigger autoimmune peripheral neuropathy.

PubMed

Yang, Mu; Shi, Xiang Qun; Peyret, Corentin; Oladiran, Oladayo; Wu, Sonia; Chambon, Julien; Fournier, Sylvie; Zhang, Ji

2018-04-05

Autoimmune peripheral neuropathy (APN) such as Guillain Barre Syndrome (GBS) is a debilitating illness and sometimes life threatening. The molecular and cellular mechanisms remain elusive but exposure to environmental factors including viral/bacterial infection and injury is highly associated with disease incidence. We demonstrated previously that both male and female B7.2 (CD86) transgenic L31 and L31/CD4KO mice develop spontaneous APN. Here we further reveal that CD8 + T cells in these mice exhibit an effector/memory phenotype, which bears a resemblance to the CD8 + T cell response following persistent cytomegalovirus (CMV) infection in humans and mice, whilst CMV has been considered as one of the most relevant pathogens in APN development. These activated, peripheral myelin Ag specific CD8 + T cells are required for the disease initiation. While an injury to a peripheral nerve results in Wallerian degeneration in control littermates, the same injury accelerates the development of APN in other non-injured nerves of L31 mice which have a predisposed inflammatory background consisting of effector/memory CD8 + T (CD8 + T EM ) cells. However, CD8 + T EM cells alone are not sufficient. A certain threshold of B7.2 expression on nerve macrophages is an additional requisite. Our findings reveal that indeed, the synergism between CD8 + T EM cells and co-stimulation competent macrophages is crucial in inducing autoimmune-mediated peripheral neuropathy. The identification of decisive molecular/cellular players connecting environmental triggers and the occurrence of APN provides opportunities to prevent disease onset, reduce relapses and develop new therapeutic strategies. Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.

Development of operational models of receptor activation including constitutive receptor activity and their use to determine the efficacy of the chemokine CCL17 at the CC chemokine receptor CCR4.

PubMed

Slack, R J; Hall, D A

2012-07-01

BACKGROUND AND PURPOSE The operational model provides a key conceptual framework for the analysis of pharmacological data. However, this model does not include constitutive receptor activity, a frequent phenomenon in modern pharmacology, particularly in recombinant systems. Here, we developed extensions of the operational model which include constitutive activity and applied them to effects of agonists at the chemokine receptor CCR4. EXPERIMENTAL APPROACH The effects of agonists of CCR4 on [(35) S]GTPγS binding to recombinant cell membranes and on the filamentous (F-) actin content of human CD4(+) CCR4(+) T cells were determined. The basal [(35) S]GTPγS binding was changed by varying the GDP concentration whilst the basal F-actin contents of the higher expressing T cell populations were elevated, suggesting constitutive activity of CCR4. Both sets of data were analysed using the mathematical models. RESULTS The affinity of CCL17 (also known as TARC) derived from analysis of the T cell data (pK(a) = 9.61 ± 0.17) was consistent with radioligand binding experiments (9.50 ± 0.11) while that from the [(35) S]GTPγS binding experiments was lower (8.27 ± 0.09). Its intrinsic efficacy differed between the two systems (110 in T cells vs. 11). CONCLUSIONS AND IMPLICATIONS The presence of constitutive receptor activity allows the absolute intrinsic efficacy of agonists to be determined without a contribution from the signal transduction system. Intrinsic efficacy estimated in this way is consistent with Furchgott's definition of this property. CCL17 may have a higher intrinsic efficacy at CCR4 in human T cells than that expressed recombinantly in CHO cells. © 2012 GSK Services Unlimited. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Human influenza viruses and CD8(+) T cell responses.

PubMed

Grant, Emma J; Quiñones-Parra, Sergio M; Clemens, E Bridie; Kedzierska, Katherine

2016-02-01

Influenza A viruses (IAVs) cause significant morbidity and mortality worldwide, despite new strain-specific vaccines being available annually. As IAV-specific CD8(+) T cells promote viral control in the absence of neutralizing antibodies, and can mediate cross-reactive immunity toward distinct IAVs to drive rapid recovery from both mild and severe influenza disease, there is great interest in developing a universal T cell vaccine. However, despite detailed studies in mouse models of influenza virus infection, there is still a paucity of data on human epitope-specific CD8(+) T cell responses to IAVs. This review focuses on our current understanding of human CD8(+) T cell immunity against distinct IAVs and discusses the possibility of achieving a CD8(+) T cell mediated-vaccine that protects against multiple, distinct IAV strains across diverse human populations. We also review the importance of CD8(+) T cell immunity in individuals highly susceptible to severe influenza infection, including those hospitalised with influenza, the elderly and Indigenous populations. Copyright © 2016 Elsevier B.V. All rights reserved.

Regulatory T cells in human disease and their potential for therapeutic manipulation

PubMed Central

Taams, Leonie S; Palmer, Donald B; Akbar, Arne N; Robinson, Douglas S; Brown, Zarin; Hawrylowicz, Catherine M

2006-01-01

Regulatory T cells are proposed to play a central role in the maintenance of immunological tolerance in the periphery, and studies in many animal models demonstrate their capacity to inhibit inflammatory pathologies in vivo. At a recent meeting [Clinical Application of Regulatory T Cells, 7–8 April 2005, Horsham, UK, organized by the authors of this review, in collaboration with the British Society for Immunology and Novartis] evidence was discussed that certain human autoimmune, infectious and allergic diseases are associated with impaired regulatory T-cell function. In contrast, evidence from several human cancer studies and some infections indicates that regulatory T cells may impair the development of protective immunity. Importantly, certain therapies, both those that act non-specifically to reduce inflammation and antigen-specific immunotherapies, may induce or enhance regulatory T-cell function. The purpose of this review was to summarize current knowledge on regulatory T-cell function in human disease, and to assess critically how this can be tailored to suit the therapeutic manipulation of immunity. PMID:16630018

Human renal adipose tissue induces the invasion and progression of renal cell carcinoma.

PubMed

Campo-Verde-Arbocco, Fiorella; López-Laur, José D; Romeo, Leonardo R; Giorlando, Noelia; Bruna, Flavia A; Contador, David E; López-Fontana, Gastón; Santiano, Flavia E; Sasso, Corina V; Zyla, Leila E; López-Fontana, Constanza M; Calvo, Juan C; Carón, Rubén W; Creydt, Virginia Pistone

2017-11-07

We evaluated the effects of conditioned media (CMs) of human adipose tissue from renal cell carcinoma located near the tumor (hRATnT) or farther away from the tumor (hRATfT), on proliferation, adhesion and migration of tumor (786-O and ACHN) and non-tumor (HK-2) human renal epithelial cell lines. Human adipose tissues were obtained from patients with renal cell carcinoma (RCC) and CMs from hRATnT and hRATfT incubation. Proliferation, adhesion and migration were quantified in 786-O, ACHN and HK-2 cell lines incubated with hRATnT-, hRATfT- or control-CMs. We evaluated versican, adiponectin and leptin expression in CMs from hRATnT and hRATfT. We evaluated AdipoR1/2, ObR, pERK, pAkt y pPI3K expression on cell lines incubated with CMs. No differences in proliferation of cell lines was found after 24 h of treatment with CMs. All cell lines showed a significant decrease in cell adhesion and increase in cell migration after incubation with hRATnT-CMs vs. hRATfT- or control-CMs. hRATnT-CMs showed increased levels of versican and leptin, compared to hRATfT-CMs. AdipoR2 in 786-O and ACHN cells decreased significantly after incubation with hRATfT- and hRATnT-CMs vs. control-CMs. We observed a decrease in the expression of pAkt in HK-2, 786-O and ACHN incubated with hRATnT-CMs. This result could partially explain the observed changes in migration and cell adhesion. We conclude that hRATnT released factors, such as leptin and versican, could enhance the invasive potential of renal epithelial cell lines and could modulate the progression of the disease.

ILK Induces Cardiomyogenesis in the Human Heart

PubMed Central

Traister, Alexandra; Aafaqi, Shabana; Masse, Stephane; Dai, Xiaojing; Li, Mark; Hinek, Aleksander; Nanthakumar, Kumaraswamy; Hannigan, Gregory; Coles, John G.

2012-01-01

Background Integrin-linked kinase (ILK) is a widely conserved serine/threonine kinase that regulates diverse signal transduction pathways implicated in cardiac hypertrophy and contractility. In this study we explored whether experimental overexpression of ILK would up-regulate morphogenesis in the human fetal heart. Methodology/Principal Findings Primary cultures of human fetal myocardial cells (19–22 weeks gestation) yielded scattered aggregates of cardioblasts positive for the early cardiac lineage marker nk×2.5 and containing nascent sarcomeres. Cardiac cells in colonies uniformly expressed the gap junction protein connexin 43 (C×43) and displayed a spectrum of differentiation with only a subset of cells exhibiting the late cardiomyogenic marker troponin T (cTnT) and evidence of electrical excitability. Adenovirus-mediated overexpression of ILK potently increased the number of new aggregates of primitive cardioblasts (p<0.001). The number of cardioblast colonies was significantly decreased (p<0.05) when ILK expression was knocked down with ILK targeted siRNA. Interestingly, overexpression of the activation resistant ILK mutant (ILKR211A) resulted in much greater increase in the number of new cell aggregates as compared to overexpression of wild-type ILK (ILKWT). The cardiomyogenic effects of ILKR211A and ILKWT were accompanied by concurrent activation of β-catenin (p<0.001) and increase expression of progenitor cell marker islet-1, which was also observed in lysates of transgenic mice with cardiac-specific over-expression of ILKR211A and ILKWT. Finally, endogenous ILK expression was shown to increase in concert with those of cardiomyogenic markers during directed cardiomyogenic differentiation in human embryonic stem cells (hESCs). Conclusions/Significance In the human fetal heart ILK activation is instructive to the specification of mesodermal precursor cells towards a cardiomyogenic lineage. Induction of cardiomyogenesis by ILK overexpression bypasses the requirement of proximal PI3K activation for transduction of growth factor- and β1-integrin-mediated differentiation signals. Altogether, our data indicate that ILK represents a novel regulatory checkpoint during human cardiomyogenesis. PMID:22666394

Molecular Imprint of Exposure to Naturally Occurring Genetic Variants of Human Cytomegalovirus on the T cell Repertoire

NASA Astrophysics Data System (ADS)

Smith, Corey; Gras, Stephanie; Brennan, Rebekah M.; Bird, Nicola L.; Valkenburg, Sophie A.; Twist, Kelly-Anne; Burrows, Jacqueline M.; Miles, John J.; Chambers, Daniel; Bell, Scott; Campbell, Scott; Kedzierska, Katherine; Burrows, Scott R.; Rossjohn, Jamie; Khanna, Rajiv

2014-02-01

Exposure to naturally occurring variants of herpesviruses in clinical settings can have a dramatic impact on anti-viral immunity. Here we have evaluated the molecular imprint of variant peptide-MHC complexes on the T-cell repertoire during human cytomegalovirus (CMV) infection and demonstrate that primary co-infection with genetic variants of CMV was coincident with development of strain-specific T-cell immunity followed by emergence of cross-reactive virus-specific T-cells. Cross-reactive CMV-specific T cells exhibited a highly conserved public T cell repertoire, while T cells directed towards specific genetic variants displayed oligoclonal repertoires, unique to each individual. T cell recognition foot-print and pMHC-I structural analyses revealed that the cross-reactive T cells accommodate alterations in the pMHC complex with a broader foot-print focussing on the core of the peptide epitope. These findings provide novel molecular insight into how infection with naturally occurring genetic variants of persistent human herpesviruses imprints on the evolution of the anti-viral T-cell repertoire.

Nucleotide composition analysis of tRNA from leukemia patient cell samples and human cell lines.

PubMed Central

Agris, P F

1975-01-01

A technique developed for analysis of less than microgram quantities of tRNA has been applied to the study of human leukemia. Leucocytes from peripheal blood and bone marrow samples of six, untreated leukemia patients and cells of five different established human cell lines were maintained for 18 hours in media containing (32P)-phosphate. Incorporation of radioactive phosphate into the cells from the patient samples was slightly less than that of the cell lines. Likewise, incorporation of (32P)-phosphate into the tRNA of the patient samples (approximately 5 x 106 DPM/mug tRNA) was also less then that incorporated into the tRNA of the cell lines. The major and minor nucleotide compositions of the unfractionated tRNA preparations from each patient sample and each cell line were determined and compared. Similarities and differences in the major and minor nucleotide compositions of the tRNA preparations are discussed with reference to types of leukemia and the importance of patient sample analysis versus analysis of cultured human cells. PMID:1057159

A promising sword of tomorrow: Human γδ T cell strategies reconcile allo-HSCT complications.

PubMed

Hu, Yongxian; Cui, Qu; Luo, Chao; Luo, Yi; Shi, Jimin; Huang, He

2016-05-01

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is potentially a curative therapeutic option for hematological malignancies. In clinical practice, transplantation associated complications greatly affected the final therapeutical outcomes. Currently, primary disease relapse, graft-versus-host disease (GVHD) and infections remain the three leading causes of a high morbidity and mortality in allo-HSCT patients. Various strategies have been investigated in the past several decades including human γδ T cell-based therapeutical regimens. In different microenvironments, human γδ T cells assume features reminiscent of classical Th1, Th2, Th17, NKT and regulatory T cells, showing diverse biological functions. The cytotoxic γδ T cells could be utilized to target relapsed malignancies, and recently regulatory γδ T cells are defined as a novel implement for GVHD management. In addition, human γδ Τ cells facilitate control of post-transplantation infections and participate in tissue regeneration and wound healing processes. These features potentiate γδ T cells a versatile therapeutical agent to target transplantation associated complications. This review focuses on insights of applicable potentials of human γδ T cells reconciling complications associated with allo-HSCT. We believe an improved understanding of pertinent γδ T cell functions would be further exploited in the design of innovative immunotherapeutic approaches in allo-HSCT, to reduce mortality and morbidity, as well as improve quality of life for patients after transplantation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Modulation of Autoimmune T-Cell Memory by Stem Cell Educator Therapy: Phase 1/2 Clinical Trial

PubMed Central

Delgado, Elias; Perez-Basterrechea, Marcos; Suarez-Alvarez, Beatriz; Zhou, Huimin; Revuelta, Eva Martinez; Garcia-Gala, Jose Maria; Perez, Silvia; Alvarez-Viejo, Maria; Menendez, Edelmiro; Lopez-Larrea, Carlos; Tang, Ruifeng; Zhu, Zhenlong; Hu, Wei; Moss, Thomas; Guindi, Edward; Otero, Jesus; Zhao, Yong

2015-01-01

Background Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that causes a deficit of pancreatic islet β cells. The complexities of overcoming autoimmunity in T1D have contributed to the challenges the research community faces when devising successful treatments with conventional immune therapies. Overcoming autoimmune T cell memory represents one of the key hurdles. Methods In this open-label, phase 1/phase 2 study, Caucasian T1D patients (N = 15) received two treatments with the Stem Cell Educator (SCE) therapy, an approach that uses human multipotent cord blood-derived multipotent stem cells (CB-SCs). SCE therapy involves a closed-loop system that briefly treats the patient's lymphocytes with CB-SCs in vitro and returns the “educated” lymphocytes (but not the CB-SCs) into the patient's blood circulation. This study is registered with ClinicalTrials.gov, NCT01350219. Findings Clinical data demonstrated that SCE therapy was well tolerated in all subjects. The percentage of naïve CD4+ T cells was significantly increased at 26 weeks and maintained through the final follow-up at 56 weeks. The percentage of CD4+ central memory T cells (TCM) was markedly and constantly increased at 18 weeks. Both CD4+ effector memory T cells (TEM) and CD8+ TEM cells were considerably decreased at 18 weeks and 26 weeks respectively. Additional clinical data demonstrated the modulation of C–C chemokine receptor 7 (CCR7) expressions on naïve T, TCM, and TEM cells. Following two treatments with SCE therapy, islet β-cell function was improved and maintained in individuals with residual β-cell function, but not in those without residual β-cell function. Interpretation Current clinical data demonstrated the safety and efficacy of SCE therapy in immune modulation. SCE therapy provides lasting reversal of autoimmune memory that could improve islet β-cell function in Caucasian subjects. Funding Obra Social “La Caixa”, Instituto de Salud Carlos III, Red de Investigación Renal, European Union FEDER Funds, Principado de Asturias, FICYT, and Hackensack University Medical Center Foundation. PMID:26844283

Suppression of in vitro murine T cell proliferation by human adipose tissue-derived mesenchymal stem cells is dependent mainly on cyclooxygenase-2 expression

PubMed Central

Kim, Jin-Hee; Lee, Yong-Taek; Hong, Jun Man

2013-01-01

Mesenchymal stem cells (MSCs) of human origin have been frequently applied to experimental animal models to evaluate their immunomodulatory functions. MSCs are known to be activated by cytokines from T cells, predominantly by interferon-γ (IFN-γ), in conjunction with other cytokines such as tumor necrosis factor-α (TNF-α) and interlukin-1β. Because IFN-γ is not cross-reactive between human and mouse species, the manner in which human MSCs administered in experimental animals are activated and stimulated to function has been questioned. In the present study, we established MSCs from human adipose tissue. They successfully suppressed the proliferation of not only human peripheral blood mononuclear cells but also mouse splenic T cells. When these human MSCs were stimulated with a culture supernatant of mouse T cells or recombinant murine TNF-α, they expressed cyclooxygenase-2 (COX-2), but not indoleamine 2,3-dioxygenase. The dominant role of COX-2 in suppressing mouse T cell proliferation was validated by the addition of COX-2 inhibitor in the co-culture, wherein the suppressed proliferation was almost completely recovered. In conclusion, human MSCs in a murine environment were activated, at least in part, by TNF-α and mainly used COX-2 as a tool for the suppression of in vitro T cell proliferation. These results should be considered when interpreting results for human MSCs in experimental animals. PMID:24386599

An inducible caspase 9 safety switch for T-cell therapy

PubMed Central

Straathof, Karin C.; Pulè, Martin A.; Yotnda, Patricia; Dotti, Gianpietro; Vanin, Elio F.; Brenner, Malcolm K.; Heslop, Helen E.; Spencer, David M.; Rooney, Cliona M.

2005-01-01

The efficacy of adoptive T-cell therapy as treatment for malignancies may be enhanced by genetic modification of infused cells. However, oncogenic events due to vector/transgene integration, and toxicities due to the infused cells themselves, have tempered enthusiasm. A safe and efficient means of removing aberrant cells in vivo would ameliorate these concerns. We describe a “safety switch” that can be stably and efficiently expressed in human T cells without impairing phenotype, function, or antigen specificity. This reagent is based on a modified human caspase 9 fused to a human FK506 binding protein (FKBP) to allow conditional dimerization using a small molecule pharmaceutical. A single 10-nM dose of synthetic dimerizer drug induces apoptosis in 99% of transduced cells selected for high transgene expression in vitro and in vivo. This system has several advantages over currently available suicide genes. First, it consists of human gene products with low potential immunogenicity. Second, administration of dimerizer drug has no effects other than the selective elimination of transduced T cells. Third, inducible caspase 9 maintains function in T cells overexpressing antiapoptotic molecules. These characteristics favor incorporation of inducible caspase 9 as a safety feature in human T-cell therapies. PMID:15728125

Enhanced Induction of T Cell Immunity Using Dendritic Cells Pulsed with HIV Tat and HCMV-pp65 Fusion Protein In Vitro

PubMed Central

Park, Jung-Sun; Park, Soo-Young; Cho, Hyun-Il; Sohn, Hyun-Jung

2011-01-01

Background Cytotoxic T lymphocytes (CTLs) appear to play an important role in the control and prevention of human cytomegalovirus (HCMV) infection. The pp65 antigen is a structural protein, which has been defined as a potential target for effective immunity against HCMV infection. Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (Tat) into protein facilitates rapid, efficient entry into cells. Methods To establish a strategy for the generation of HCMV-specific CTLs in vitro, recombinant truncated N- and C-terminal pp65 protein (pp65 N&C) and N- and C-terminal pp65 protein fused with Tat (Tat/pp65 N&C) was produced in E.coli system. Peripheral blood mononuclear cells were stimulated with dendritic cells (DCs) pulsed with pp65 N&C or Tat/pp65 N&C protein and immune responses induced was examined using IFN-γ ELISPOT assay, cytotoxicity assay and tetramer staining. Results DCs pulsed with Tat/pp65N&C protein could induce higher T-cell responses in vitro compared with pp65N&C. Moreover, the DCs pulsed with Tat/pp65 N&C could stimulate both of CD8+ and CD4+ T-cell responses. The T cells induced by DCs pulsed with Tat/pp65 N&C showed higher cytotoxicity than that of pp65-pulsed DCs against autologous lymphoblastoid B-cell line (LCL) expressing the HCMV-pp65 antigen. Conclusion Our results suggest that DCs pulsed with Tat/pp65 N&C protein effectively induced pp65-specific CTL in vitro. Tat fusion recombinant protein may be useful for the development of adoptive T-cell immunotherapy and DC-based vaccines. PMID:21860612

CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

PubMed Central

Alonso-Camino, Vanesa; Sánchez-Martín, David; Compte, Marta; Nuñez-Prado, Natalia; Diaz, Rosa M; Vile, Richard; Alvarez-Vallina, Luis

2013-01-01

A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells. PMID:23695536

Optimized depletion of chimeric antigen receptor T cells in murine xenograft models of human acute myeloid leukemia

PubMed Central

Kenderian, Saad S.; Shen, Feng; Ruella, Marco; Shestova, Olga; Kozlowski, Miroslaw; Li, Yong; Schrank-Hacker, April; Morrissette, Jennifer J. D.; Carroll, Martin; June, Carl H.; Grupp, Stephan A.; Gill, Saar

2017-01-01

We and others previously reported potent antileukemia efficacy of CD123-redirected chimeric antigen receptor (CAR) T cells in preclinical human acute myeloid leukemia (AML) models at the cost of severe hematologic toxicity. This observation raises concern for potential myeloablation in patients with AML treated with CD123-redirected CAR T cells and mandates novel approaches for toxicity mitigation. We hypothesized that CAR T-cell depletion with optimal timing after AML eradication would preserve leukemia remission and allow subsequent hematopoietic stem cell transplantation. To test this hypothesis, we compared 3 CAR T-cell termination strategies: (1) transiently active anti-CD123 messenger RNA–electroporated CART (RNA-CART123); (2) T-cell ablation with alemtuzumab after treatment with lentivirally transduced anti–CD123-4-1BB-CD3ζ T cells (CART123); and (3) T-cell ablation with rituximab after treatment with CD20-coexpressing CART123 (CART123-CD20). All approaches led to rapid leukemia elimination in murine xenograft models of human AML. Subsequent antibody-mediated depletion of CART123 or CART123-CD20 did not impair leukemia remission. Time-course studies demonstrated that durable leukemia remission required CAR T-cell persistence for 4 weeks prior to ablation. Upon CAR T-cell termination, we further demonstrated successful hematopoietic engraftment with a normal human donor to model allogeneic stem cell rescue. Results from these studies will facilitate development of T-cell depletion strategies to augment the feasibility of CAR T-cell therapy for patients with AML. PMID:28246194

The bovine model for elucidating the role of γδ T cells in controlling infectious diseases of importance to cattle and humans.

PubMed

Baldwin, Cynthia L; Telfer, Janice C

2015-07-01

There are several instances of co-investigation and related discoveries and achievements in bovine and human immunology; perhaps most interesting is the development of the BCG vaccine, the tuberculin skin test and the more recent interferon-gamma test that were developed first in cattle to prevent and diagnosis bovine tuberculosis and then applied to humans. There are also a number of immune-physiological traits that ruminant share with humans including the development of their immune systems in utero which increases the utility of cattle as a model for human immunology. These are reviewed here with a particular focus on the use of cattle to unravel γδ T cell biology. Based on the sheer number of γδ T cells in this γδ T cell high species, it is reasonable to expect γδ T cells to play an important role in protective immune responses. For that reason alone cattle may provide good models for elucidating at least some of the roles γδ T cells play in protective immunity in all species. This includes fundamental research on γδ T cells as well as the responses of ruminant γδ T cells to a variety of infectious disease situations including to protozoan and bacterial pathogens. The role that pattern recognition receptors (PRR) play in the activation of γδ T cells may be unique relative to αβ T cells. Here we focus on that of the γδ T cell specific family of molecules known as WC1 or T19 in ruminants, which are part of the CD163 scavenger receptor cysteine rich (SRCR) family that includes SCART1 and SCART2 expressed on murine γδ T cells. We review the evidence for WC1 being a PRR as well as an activating co-receptor and the role that γδ T cells bearing these receptors play in immunity to leptospirosis and tuberculosis. This includes the generation of memory responses to vaccines, thereby continuing the tradition of co-discovery between cattle and humans. Copyright © 2014 Elsevier Ltd. All rights reserved.

The lysosomotropic drug LeuLeu-OMe induces lysosome disruption and autophagy-independent cell death in Trypanosoma brucei

PubMed Central

Koh, Hazel X.; Aye, Htay M.; Tan, Kevin S. W.; He, Cynthia Y.

2015-01-01

Background: Trypanosoma brucei is a blood-borne, protozoan parasite that causes African sleeping sickness in humans and nagana in animals. The current chemotherapy relies on only a handful of drugs that display undesirable toxicity, poor efficacy and drug-resistance. In this study, we explored the use of lysosomotropic drugs to induce bloodstream form T. brucei cell death via lysosome destabilization. Methods: We measured drug concentrations that inhibit cell proliferation by 50% (IC_{50) for several compounds, chosen based on their lysosomotropic effects previously reported in Plasmodium falciparum. The lysosomal effects and cell death induced by L-leucyl-L-leucyl methyl ester (LeuLeu-OMe) were further analyzed by flow cytometry and immunofluorescence analyses of different lysosomal markers. The effect of autophagy in LeuLeu-OMe-induced lysosome destabilization and cytotoxicity was also investigated in control and autophagy-deficient cells. Results: LeuLeu-OMe was selected for detailed analyses due to its strong inhibitory profile against T. brucei with minimal toxicity to human cell lines in vitro. Time-dependent immunofluorescence studies confirmed an effect of LeuLeu-OMe on the lysosome. LeuLeu-OMe-induced cytotoxicity was also found to be dependent on the acidic pH of the lysosome. Although an increase in autophagosomes was observed upon LeuLeu-OMe treatment, autophagy was not required for the cell death induced by LeuLeu-OMe. Necrosis appeared to be the main cause of cell death upon LeuLeu-OMe treatment. Conclusions: LeuLeu-OMe is a lysosomotropic agent capable of destabilizing lysosomes and causing necrotic cell death in bloodstream form of T. brucei. PMID:28357304}

Human Keratinocytes Are Vanilloid Resistant

PubMed Central

Pecze, László; Szabó, Kornélia; Széll, Márta; Jósvay, Katalin; Kaszás, Krisztián; Kúsz, Erzsébet; Letoha, Tamás; Prorok, János; Koncz, István; Tóth, András; Kemény, Lajos; Vizler, Csaba; Oláh, Zoltán

2008-01-01

Background Use of capsaicin or resiniferatoxin (RTX) as analgesics is an attractive therapeutic option. RTX opens the cation channel inflammatory pain/vanilloid receptor type 1 (TRPV1) permanently and selectively removes nociceptive neurons by Ca2+-cytotoxicity. Paradoxically, not only nociceptors, but non-neuronal cells, including keratinocytes express full length TRPV1 mRNA, while patient dogs and experimental animals that underwent topical treatment or anatomically targeted molecular surgery have shown neither obvious behavioral, nor pathological side effects. Methods To address this paradox, we assessed the vanilloid sensitivity of the HaCaT human keratinocyte cell line and primary keratinocytes from skin biopsies. Results Although both cell types express TRPV1 mRNA, neither responded to vanilloids with Ca2+-cytotoxicity. Only ectopic overproduction of TRPV1 rendered HaCaT cells sensitive to low doses (1–50 nM) of vanilloids. The TRPV1-mediated and non-receptor specific Ca2+-cytotoxity ([RTX]>15 µM) could clearly be distinguished, thus keratinocytes were indeed resistant to vanilloid-induced, TRPV1-mediated Ca2+-entry. Having a wider therapeutic window than capsaicin, RTX was effective in subnanomolar range, but even micromolar concentrations could not kill human keratinocytes. Keratinocytes showed orders of magnitudes lower TRPV1 mRNA level than sensory ganglions, the bona fide therapeutic targets in human pain management. In addition to TRPV1, TRPV1b, a dominant negative splice variant was also noted in keratinocytes. Conclusion TRPV1B expression, together with low TRPV1 expression, may explain the vanilloid paradox: even genuinely TRPV1 mRNA positive cells can be spared with therapeutic (up to micromolar) doses of RTX. This additional safety information might be useful for planning future human clinical trials. PMID:18852901

Of mice and men: how animal models advance our understanding of T-cell function in RA.

PubMed

Kobezda, Tamás; Ghassemi-Nejad, Sheida; Mikecz, Katalin; Glant, Tibor T; Szekanecz, Zoltán

2014-03-01

The involvement of autoreactive T cells in the pathogenesis of rheumatoid arthritis (RA) as well as in autoimmune animal models of arthritis has been well established; however, unanswered questions, such as the role of joint-homing T cells, remain. Animal models of arthritis are superb experimental tools in demonstrating how T cells trigger joint inflammation, and thus can help to further our knowledge of disease mechanisms and potential therapies. In this Review, we discuss the similarities and differences in T-cell subsets and functions between RA and mouse arthritis models. For example, various T-cell subsets are involved in both human and mouse arthritis, but differences might exist in the cytokine regulation and plasticity of these cells. With regard to joint-homing T cells, an abundance of synovial T cells is present in humans compared with mice. On the other hand, local expansion of type 17 T-helper (TH17) cells is observed in some animal models, but not in RA. Finally, whereas T-cell depletion therapy essentially failed in RA, antibody targeting of T cells can work, at least preventatively, in most arthritis models. Clearly, additional human and animal studies are needed to fill the gap in our understanding of the specific contribution of T-cell subsets to arthritis in mice and men.

The IDO1 selective inhibitor epacadostat enhances dendritic cell immunogenicity and lytic ability of tumor antigen-specific T cells.

PubMed

Jochems, Caroline; Fantini, Massimo; Fernando, Romaine I; Kwilas, Anna R; Donahue, Renee N; Lepone, Lauren M; Grenga, Italia; Kim, Young-Seung; Brechbiel, Martin W; Gulley, James L; Madan, Ravi A; Heery, Christopher R; Hodge, James W; Newton, Robert; Schlom, Jeffrey; Tsang, Kwong Y

2016-06-21

Epacadostat is a novel inhibitor of indoleamine-2,3-dioxygenase-1 (IDO1) that suppresses systemic tryptophan catabolism and is currently being evaluated in ongoing clinical trials. We investigated the effects of epacadostat on (a) human dendritic cells (DCs) with respect to maturation and ability to activate human tumor antigen-specific cytotoxic T-cell (CTL) lines, and subsequent T-cell lysis of tumor cells, (b) human regulatory T cells (Tregs), and (c) human peripheral blood mononuclear cells (PBMCs) in vitro. Simultaneous treatment with epacadostat and IFN-γ plus lipopolysaccharide (LPS) did not change the phenotype of matured human DCs, and as expected decreased the tryptophan breakdown and kynurenine production. Peptide-specific T-cell lines stimulated with DCs pulsed with peptide produced significantly more IFN-γ, TNFα, GM-CSF and IL-8 if the DCs were treated with epacadostat. These T cells also displayed higher levels of tumor cell lysis on a per cell basis. Epacadostat also significantly decreased Treg proliferation induced by IDO production from IFN-γ plus LPS matured human DCs, although the Treg phenotype did not change. Multicolor flow cytometry was performed on human PBMCs treated with epacadostat; analysis of 123 discrete immune cell subsets revealed no changes in major immune cell types, an increase in activated CD83+ conventional DCs, and a decrease in immature activated Tim3+ NK cells. These studies show for the first time several effects of epacadostat on human DCs, and subsequent effects on CTL and Tregs, and provide a rationale as to how epacadostat could potentially increase the efficacy of immunotherapeutics, including cancer vaccines.

Human T cells engineered to express a programmed death 1/28 costimulatory retargeting molecule display enhanced antitumor activity.

PubMed

Ankri, Chen; Shamalov, Katerina; Horovitz-Fried, Miryam; Mauer, Shmuel; Cohen, Cyrille J

2013-10-15

Adoptive transfer of T cells genetically modified to express cancer-specific receptors can mediate impressive tumor regression in terminally ill patients. However, T cell function and persistence over time could be hampered by the activation of inhibitory costimulatory pathways, such as programmed death 1 (PD1)/programmed death ligand 1, leading to T cell exhaustion and providing tumor cells with an escape mechanism from immunosurveillance. In addition, the lack of positive costimulation at the tumor site can further dampen T cell response. Thus, as T cell genetic engineering has become clinically relevant, we aimed at enhancing T cell antitumor activity by genetically diverting T cell-negative costimulatory signals into positive ones using chimeric costimulatory retargeting molecules and which are composed of the PD1 extracellular domain fused to the signaling domains of positive costimulatory molecules such as CD28 and 4-1BB. After characterizing the optimal PD1 chimera, we designed and optimized a tripartite retroviral vector that enables the simultaneous expression of this chimeric molecule in conjunction with a cancer-specific TCR. Human T cells, transduced to express a PD1/28 chimeric molecule, exhibited enhanced cytokine secretion and upregulation of activation markers upon coculture with tumor cells. These engineered cells also proliferated better compared with control cells. Finally, we tested the function of these cells in two xenograft models of human melanoma tumors and show that PD1/28-engineered human T cells demonstrated superior antitumor function. Overall, we propose that engineering T cells with a costimulatory retargeting molecule can enhance their function, which bears important implications for the improvement of T cell immunotherapy.

Human Atopic Dermatitis Skin-derived T Cells can Induce a Reaction in Mouse Keratinocytes in vivo.

PubMed

Martel, B C; Blom, L; Dyring-Andersen, B; Skov, L; Thestrup-Pedersen, K; Skov, S; Skak, K; Poulsen, L K

2015-08-01

In atopic dermatitis (AD), the inflammatory response between skin-infiltrating T cells and keratinocytes is fundamental to the development of chronic lesional eczema. The aim of this study was to investigate whether skin-derived T cells from AD patients could induce an inflammatory response in mice through keratinocyte activation and consequently cause the development of eczematous lesions. Punch biopsies of the lesional skin from AD patients were used to establish skin-derived T cell cultures, which were transferred to NOD.Cg-Prkd(scid) Il2rg(tm1Sug) /JicTac (NOG) mice. We found that the subcutaneous injection of the human AD skin-derived T cells resulted in the migration of the human T cells from subcutis to the papillary dermis followed by the development of erythema and oedema in the mouse skin. Furthermore, the human T cells induced a transient proliferative response in the mouse keratinocytes shown as increased numbers of Ki-67(+) keratinocytes and increased epidermal thickness. Out of six established AD skin-derived T cell cultures, two were superior at inducing a skin reaction in the mice, and these cultures were found to contain >10% CCR10(+) T cells compared to <2% for the other cultures. In comparison, blood-derived in vitro-differentiated Th2 cells only induced a weak response in a few of the mice. Thus, we conclude that human AD skin-derived T cells can induce a reaction in the mouse skin through the induction of a proliferative response in the mouse keratinocytes. © 2015 The Foundation for the Scandinavian Journal of Immunology.

CXCL16 and CXCR6 are upregulated in psoriasis and mediate cutaneous recruitment of human CD8+ T cells.

PubMed

Günther, Claudia; Carballido-Perrig, Nicole; Kaesler, Susanne; Carballido, José M; Biedermann, Tilo

2012-03-01

Psoriatic skin lesions are characterized by an inflammatory infiltrate, consisting of dendritic cells, monocytes, and both CD4(+) and CD8(+) T lymphocytes. Although the chemokines involved in the migration of CD4(+) T cells into psoriatic skin are well characterized, those regulating CD8(+) T-cell recruitment are less understood. We found that the percentages of peripheral blood CD8(+) T cells expressing CXCR6 were higher in psoriatic patients than in healthy or atopic individuals. In addition, CXCR6 expression in psoriatic patients was more abundant in the CD8(+) than in the CD4(+) T-cell compartment. CXCR6 mRNA expression was also stronger in skin CD8(+) T cells than in the corresponding blood-derived counterparts. Immunofluorescence analysis revealed profound upregulation of the CXCR6 ligand CXCL16 by monocytes, keratinocytes, and dendritic cells in psoriatic skin compared with healthy or atopic dermatitis skin. In line with this, CXCR6(+) CD8(+) T cells also were most prevalent in psoriatic skin. Furthermore, CXCL16 induced Ca(2+) influx and chemotactic migration of psoriatic skin-derived CD8(+) T cells in vitro. Most importantly, CXCL16 potently recruited human CD8(+) T cells to human skin grafts previously transplanted onto SCID mice in vivo. These investigations indicate that CXCL16-CXCR6 interactions mediate homing of CD8(+) T cells into human skin, and thereby contribute to psoriasis pathogenesis.

The pyrimidin analogue cyclopentenyl cytosine induces alloantigen-specific non-responsiveness of human T lymphocytes

PubMed Central

Nikolaeva, N; Bemelman, F J; Yong, S-L; Verschuur, A; van Lier, R A W; ten Berge, I J M

2008-01-01

Cyclopentenyl cytosine (CPEC) has been shown to induce apoptosis in human T lymphoblastic cell lines and T cells from leukaemia patients. In this study we have addressed the question of whether CPEC is able to decrease proliferation and effector functions of human alloresponsive T lymphocytes and induce T cell anergy. The proliferative capacity of human peripheral blood mononuclear cells in response to allogeneic stimulation was measured by 5,6-carboxy-succinimidyl-diacetate-fluorescein-ester staining. Flow cytometric analysis was performed using surface CD4, CD8, CD25, CD103 and intracellular perforin, granzyme A, granzyme B, caspase-3 and forkhead box P3 (FoxP3) markers. The in vivo immunosuppressive capacity was tested in a murine skin graft model. Addition of CPEC at a concentration of 20 nM strongly decreased the expansion and cytotoxicity of alloreactive T cells. Specific restimulation in the absence of CPEC showed that the cells became anergic. The drug induced caspase-dependent apoptosis of alloreactive T lymphocytes. Finally, CPEC increased the percentage of CD25high FoxP3+ CD4+ and CD103+ CD8+ T cells, and potentiated the effect of rapamycin in increasing the numbers of alloreactive regulatory T cells. Treatment with CPEC of CBA/CA mice transplanted with B10/Br skin grafts significantly prolonged graft survival. We conclude that CPEC inhibits proliferation and cytotoxicity of human alloreactive T cells and induces alloantigen non-responsiveness in vitro. PMID:18062797

CD4+CD62L+ Central Memory T Cells Can Be Converted to Foxp3+ T Cells

PubMed Central

Zhang, Xiaolong; Chang Li, Xian; Xiao, Xiang; Sun, Rui; Tian, Zhigang; Wei, Haiming

2013-01-01

The peripheral Foxp3+ Treg pool consists of naturally arising Treg (nTreg) and adaptive Treg cells (iTreg). It is well known that naive CD4+ T cells can be readily converted to Foxp3+ iTreg in vitro, and memory CD4+ T cells are resistant to conversion. In this study, we investigated the induction of Foxp3+ T cells from various CD4+ T-cell subsets in human peripheral blood. Though naive CD4+ T cells were readily converted to Foxp3+ T cells with TGF-β and IL-2 treatment in vitro, such Foxp3+ T cells did not express the memory marker CD45RO as do Foxp3+ T cells induced in the peripheral blood of Hepatitis B Virus (HBV) patients. Interestingly, a subset of human memory CD4+ T cells, defined as CD62L+ central memory T cells, could be induced by TGF-β to differentiate into Foxp3+ T cells. It is well known that Foxp3+ T cells derived from human CD4+CD25- T cells in vitro are lack suppressive functions. Our data about the suppressive functions of CD4+CD62L+ central memory T cell-derived Foxp3+ T cells support this conception, and an epigenetic analysis of these cells showed a similar methylation pattern in the FOXP3 Treg-specific demethylated region as the naive CD4+ T cell-derived Foxp3+ T cells. But further research showed that mouse CD4+ central memory T cells also could be induced to differentiate into Foxp3+ T cells, such Foxp3+ T cells could suppress the proliferation of effector T cells. Thus, our study identified CD4+CD62L+ central memory T cells as a novel potential source of iTreg. PMID:24155942

Chimeric antigen receptor T cells secreting anti-PD-L1 antibodies more effectively regress renal cell carcinoma in a humanized mouse model.

PubMed

Suarez, Eloah Rabello; Chang, De Kuan; Sun, Jiusong; Sui, Jianhua; Freeman, Gordon J; Signoretti, Sabina; Zhu, Quan; Marasco, Wayne A

2016-06-07

Advances in the treatment of metastatic clear cell renal cell carcinoma (ccRCC) have led to improved progression-free survival of many patients; however the therapies are toxic, rarely achieve durable long-term complete responses and are not curative. Herein we used a single bicistronic lentiviral vector to develop a new combination immunotherapy that consists of human anti-carbonic anhydrase IX (CAIX)-targeted chimeric antigen receptor (CAR) T cells engineered to secrete human anti-programmed death ligand 1 (PD-L1) antibodies at the tumor site. The local antibody delivery led to marked immune checkpoint blockade. Tumor growth diminished 5 times and tumor weight reduced 50-80% when compared with the anti-CAIX CAR T cells alone in a humanized mice model of ccRCC. The expression of PD-L1 and Ki67 in the tumors decreased and an increase in granzyme B levels was found in CAR T cells. The anti-PD-L1 IgG1 isotype, which is capable of mediating ADCC, was also able to recruit human NK cells to the tumor site in vivo. These armed second-generation CAR T cells empowered to secrete human anti-PD-L1 antibodies in the ccRCC milieu to combat T cell exhaustion is an innovation in this field that should provide renewed potential for CAR T cell immunotherapy of solid tumors where limited efficacy is currently seen.

Phenotype of NK-Like CD8(+) T Cells with Innate Features in Humans and Their Relevance in Cancer Diseases

PubMed Central

Barbarin, Alice; Cayssials, Emilie; Jacomet, Florence; Nunez, Nicolas Gonzalo; Basbous, Sara; Lefèvre, Lucie; Abdallah, Myriam; Piccirilli, Nathalie; Morin, Benjamin; Lavoue, Vincent; Catros, Véronique; Piaggio, Eliane; Herbelin, André; Gombert, Jean-Marc

2017-01-01

Unconventional T cells are defined by their capacity to respond to signals other than the well-known complex of peptides and major histocompatibility complex proteins. Among the burgeoning family of unconventional T cells, innate-like CD8(+) T cells in the mouse were discovered in the early 2000s. This subset of CD8(+) T cells bears a memory phenotype without having encountered a foreign antigen and can respond to innate-like IL-12 + IL-18 stimulation. Although the concept of innate memory CD8(+) T cells is now well established in mice, whether an equivalent memory NK-like T-cell population exists in humans remains under debate. We recently reported that CD8(+) T cells responding to innate-like IL-12 + IL-18 stimulation and co-expressing the transcription factor Eomesodermin (Eomes) and KIR/NKG2A membrane receptors with a memory/EMRA phenotype may represent a new, functionally distinct innate T cell subset in humans. In this review, after a summary on the known innate CD8(+) T-cell features in the mouse, we propose Eomes together with KIR/NKG2A and CD49d as a signature to standardize the identification of this innate CD8(+) T-cell subset in humans. Next, we discuss IL-4 and IL-15 involvement in the generation of innate CD8(+) T cells and particularly its possible dependency on the promyelocytic leukemia zinc-finger factor expressing iNKT cells, an innate T cell subset well documented for its susceptibility to tumor immune subversion. After that, focusing on cancer diseases, we provide new insights into the potential role of these innate CD8(+) T cells in a physiopathological context in humans. Based on empirical data obtained in cases of chronic myeloid leukemia, a myeloproliferative syndrome controlled by the immune system, and in solid tumors, we observe both the possible contribution of innate CD8(+) T cells to cancer disease control and their susceptibility to tumor immune subversion. Finally, we note that during tumor progression, innate CD8(+) T lymphocytes could be controlled by immune checkpoints. This study significantly contributes to understanding of the role of NK-like CD8(+) T cells and raises the question of the possible involvement of an iNKT/innate CD8(+) T cell axis in cancer. PMID:28396661

Evaluation of the impact of chitosan/DNA nanoparticles on the differentiation of human naive CD4+ T cells

NASA Astrophysics Data System (ADS)

Liu, Lanxia; Bai, Yuanyuan; Zhu, Dunwan; Song, Liping; Wang, Hai; Dong, Xia; Zhang, Hailing; Leng, Xigang

2011-06-01

Chitosan (CS) is one of the most widely studied polymers in non-viral gene delivery since it is a cationic polysaccharide that forms nanoparticles with DNA and hence protects the DNA against digestion by DNase. However, the impact of CS/DNA nanoparticle on the immune system still remains poorly understood. Previous investigations did not found CS/DNA nanoparticles had any significant impact on the function of human and murine macrophages. To date, little is known about the interaction between CS/DNA nanoparticles and naive CD4+ T cells. This study was designed to investigate whether CS/DNA nanoparticles affect the initial differentiation direction of human naive CD4+ T cells. The indirect impact of CS/DNA nanoparticles on naive CD4+ T cell differentiation was investigated by incubating the nanoparticles with human macrophage THP-1 cells in one chamber of a transwell co-incubation system, with the enriched human naive CD4+ T cells being placed in the other chamber of the transwell. The nanoparticles were also co-incubated with the naive CD4+ T cells to explore their direct impact on naive CD4+ T cell differentiation by measuring the release of IL-4 and IFN-γ from the cells. It was demonstrated that CS/DNA nanoparticles induced slightly elevated production of IL-12 by THP-1 cells, possibly owing to the presence of CpG motifs in the plasmid. However, this macrophage stimulating activity was much less significant as compared with lipopolysaccharide and did not impact on the differentiation of the naive CD4+ T cells. It was also demonstrated that, when directly exposed to the naive CD4+ T cells, the nanoparticles induced neither the activation of the naive CD4+ T cells in the absence of recombinant cytokines (recombinant human IL-4 or IFN-γ) that induce naive CD4+ T cell polarization, nor any changes in the differentiation direction of naive CD4+ T cells in the presence of the corresponding cytokines.

Intestinal IFN-γ-producing type 1 regulatory T cells coexpress CCR5 and programmed cell death protein 1 and downregulate IL-10 in the inflamed guts of patients with inflammatory bowel disease.

PubMed

Alfen, Johanna Sophie; Larghi, Paola; Facciotti, Federica; Gagliani, Nicola; Bosotti, Roberto; Paroni, Moira; Maglie, Stefano; Gruarin, Paola; Vasco, Chiara Maria; Ranzani, Valeria; Frusteri, Cristina; Iseppon, Andrea; Moro, Monica; Crosti, Maria Cristina; Gatti, Stefano; Pagani, Massimiliano; Caprioli, Flavio; Abrignani, Sergio; Flavell, Richard A; Geginat, Jens

2018-01-31

IL-10 is an anti-inflammatory cytokine required for intestinal immune homeostasis. It mediates suppression of T-cell responses by type 1 regulatory T (T R 1) cells but is also produced by CD25 + regulatory T (Treg) cells. We aimed to identify and characterize human intestinal T R 1 cells and to investigate whether they are a relevant cellular source of IL-10 in patients with inflammatory bowel diseases (IBDs). CD4 + T cells isolated from the intestinal lamina propria of human subjects and mice were analyzed for phenotype, cytokine production, and suppressive capacities. Intracellular IL-10 expression by CD4 + T-cell subsets in the inflamed guts of patients with IBD (Crohn disease or ulcerative colitis) was compared with that in cells from noninflamed control subjects. Finally, the effects of proinflammatory cytokines on T-cell IL-10 expression were analyzed, and IL-1β and IL-23 responsiveness was assessed. Intestinal T R 1 cells could be identified by coexpression of CCR5 and programmed cell death protein 1 (PD-1) in human subjects and mice. CCR5 + PD-1 + T R 1 cells expressed IFN-γ and efficiently suppressed T-cell proliferation and transfer colitis. Intestinal IFN-γ + T R 1 cells, but not IL-7 receptor-positive T H cells or CD25 + Treg cells, showed lower IL-10 expression in patients with IBDs. T R 1 cells were responsive to IL-23, and IFN-γ + T R 1 cells downregulated IL-10 with IL-1β and IL-23. Conversely, CD25 + Treg cells expressed higher levels of IL-1 receptor but showed stable IL-10 expression. We provide the first ex vivo characterization of human intestinal T R 1 cells. Selective downregulation of IL-10 by IFN-γ + T R 1 cells in response to proinflammatory cytokines is likely to drive excessive intestinal inflammation in patients with IBDs. Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Identification of a novel pro-inflammatory human skin-homing Vγ9Vδ2 T cell subset with a potential role in psoriasis

PubMed Central

LAGGNER, Ute; DI MEGLIO, Paola; PERERA, Gayathri K.; HUNDHAUSEN, Christian; LACY, Katie E.; ALI, Niwa; SMITH, Catherine H.; HAYDAY, Adrian C.; NICKOLOFF, Brian J.; NESTLE, Frank O.

2011-01-01

γδ T cells mediate rapid tissue responses in murine skin and participate in cutaneous immune regulation including protection against cancer. The role of human γδ cells in cutaneous homeostasis and pathology is poorly characterized. In this study we show in vivo evidence that human blood contains a distinct subset of pro-inflammatory cutaneous lymphocyte antigen (CLA) and C-C chemokine receptor (CCR) 6 positive Vγ9Vδ2 T cells, which is rapidly recruited into perturbed human skin. Vγ9Vδ2 T cells produced an array of pro-inflammatory mediators including IL-17A and activated keratinocytes in a TNF-α and IFN-γ dependent manner. Examination of the common inflammatory skin disease psoriasis revealed a striking reduction of circulating Vγ9Vδ2 T cells in psoriasis patients compared to healthy controls and atopic dermatitis patients. Decreased numbers of circulating Vγ9Vδ2 T cells normalized after successful treatment with psoriasis-targeted therapy. Together with the increased presence of Vγ9Vδ2 T cells in psoriatic skin, this data indicates redistribution of Vγ9Vδ2 T cells from the blood to the skin compartment in psoriasis. In summary, we report a novel human pro-inflammatory γδ T cell involved in skin immune surveillance with immediate response characteristics and with potential clinical relevance in inflammatory skin disease. PMID:21813772

Deep Surveying of the Transcriptional and Alternative Splicing Signatures for Decidual CD8+ T Cells at the First Trimester of Human Healthy Pregnancy.

PubMed

Zeng, Weihong; Liu, Xinmei; Liu, Zhicui; Zheng, Ying; Yu, Tiantian; Fu, Shaliu; Li, Xiao; Zhang, Jing; Zhang, Siming; Ma, Xiaoling; Liu, Xiao-Rui; Qin, Xiaoli; Khanniche, Asma; Zhang, Yan; Tian, Fuju; Lin, Yi

2018-01-01

Decidual CD8 + (dCD8) T cells have been proposed to play important roles in immune protection against the invading pathogens and in tolerance toward the growing semi-allogeneic fetus during early pregnancy. However, their phenotypic and functional characteristics remain poorly defined. Here, we performed the first analysis of the transcriptional and alternative splicing (AS) signatures for human first-trimester dCD8 T cells using high-throughput mRNA sequencing. Our data revealed that dCD8 T cells have distinct transcriptional and AS landscapes when compared with their autologous peripheral blood CD8 + (pCD8) T counterparts. Furthermore, human dCD8 T cells were observed to contain CD8-Treg and effector-memory T-cell subsets, and display enhanced functionality in terms of degranulation and cytokine production on a per-cell basis. Additionally, we have identified the novel splice junctions that use a high ratio of the non-canonical splicing motif GC-AG and found that AS is not a major contributor to the gene expression-level changes between paired pCD8 and dCD8 T cells. Together, our findings not only provide a comprehensive framework of the transcriptional and AS landscapes but also reveal the functional feature of human dCD8 T cells, which are of great importance in understanding the biology of these cells and the physiology of human healthy pregnancy.

T-dependence of human B lymphocyte proliferative response to mitogens.

PubMed

Brochier, J; Samarut, C; Gueho, J P; Revillard, J P

1976-01-01

Human peripheral blood and tonsil lymphocytes were fractionated on anti-Ig-coated Sephadex columns or by centrifugation after rosetting with native sheep erythrocytes. Both methods allowed the recovery of B and T-enriched populations the purity of which was checked by fluorescein-labelled anti-Ig serum, E and EAC rosette formation, and heterologous antisera specific for B or T lymphocytes. The proliferative response of T cells to PHA, Con A, PWM, and ALS was not found different from that of unfractionated cells, whereas no response of the B cells could be observed to these mitogens providing that no contaminating T cells were present. Addition of T lymphocytes to these unresponsive B cells allowed them to respond to phytomitogens, but not to ALS. X-irradiated T cells could, to some extent, replace the diving T lymphocytes; no T-replacing factor could be found in cell-free supernatants from T cells, whether or not they had been activated by mitrogens. This model of B-T cooperation appears useful for studying the differentiation and maturation of human B lymphocytes.

The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies.

PubMed

Tsukahara, T; Iwase, N; Kawakami, K; Iwasaki, M; Yamamoto, C; Ohmine, K; Uchibori, R; Teruya, T; Ido, H; Saga, Y; Urabe, M; Mizukami, H; Kume, A; Nakamura, M; Brentjens, R; Ozawa, K

2015-02-01

Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3(+) T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

Induction of suppression through human T cell interactions.

PubMed

Lydyard, P M; Hayward, A R

1980-02-01

Concanavalin A (Con A) activated T cells, devoid of cells bearing Fc receptors for IgG (T - TG) help human B lymphocytes to differentiate into plasma cells (PC) in response to pokeweed mitogen (PWM). PC differentiation is reduced when adult T cells are added to such cultures. The radiosensitivity of suppression and the radioresistance of help enabled us to show that adult T cells include a suppressor-precursor which is activated by irradiated Con A-precultured T cells. Newborn T cells which include active suppressors, are both poor stimulators of suppressor-precursors and poor helpers of B cells. Our results suggest that at least two cells may mediate Con A-induced suppression, one which suppresses directly and is radiosensitive and another which is radioresistant and stimulates suppressor-precursors in a target population of T cells.

Curtailed T-cell activation curbs effector differentiation and generates CD8+ T cells with a naturally-occurring memory stem cell phenotype.

PubMed

Zanon, Veronica; Pilipow, Karolina; Scamardella, Eloise; De Paoli, Federica; De Simone, Gabriele; Price, David A; Martinez Usatorre, Amaia; Romero, Pedro; Mavilio, Domenico; Roberto, Alessandra; Lugli, Enrico

2017-09-01

Human T memory stem (T SCM ) cells with superior persistence capacity and effector functions are emerging as important players in the maintenance of long-lived T-cell memory and are thus considered an attractive population to be used in adoptive transfer-based immunotherapy of cancer. However, the molecular signals regulating their generation remain poorly defined. Here we show that curtailed T-cell receptor stimulation curbs human effector CD8 + T-cell differentiation and allows the generation of CD45RO - CD45RA + CCR7 + CD27 + CD95 + -phenotype cells from highly purified naïve T-cell precursors, resembling naturally-occurring human T SCM . These cells proliferate extensively in vitro and in vivo, express low amounts of effector-associated genes and transcription factors and undergo considerable self-renewal in response to IL-15 while retaining effector differentiation potential. Such a phenotype is associated with a lower number of mitochondria compared to highly-activated effector T cells committed to terminal differentiation. These results shed light on the molecular signals that are required to generate long-lived memory T cells with potential application in adoptive cell transfer immunotherapy. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co.KGaA, Weinheim.

MERTK as negative regulator of human T cell activation

PubMed Central

Cabezón, Raquel; Carrera-Silva, E. Antonio; Flórez-Grau, Georgina; Errasti, Andrea E.; Calderón-Gómez, Elisabeth; Lozano, Juan José; España, Carolina; Ricart, Elena; Panés, Julián; Rothlin, Carla Vanina; Benítez-Ribas, Daniel

2015-01-01

The aim of this study was to test the hypothesis whether MERTK, which is up-regulated in human DCs treated with immunosuppressive agents, is directly involved in modulating T cell activation. MERTK is a member of the TAM family and contributes to regulating innate immune response to ACs by inhibiting DC activation in animal models. However, whether MERTK interacts directly with T cells has not been addressed. Here, we show that MERTK is highly expressed on dex-induced human tol-DCs and participates in their tolerogenic effect. Neutralization of MERTK in allogenic MLR, as well as autologous DC–T cell cultures, leads to increased T cell proliferation and IFN-γ production. Additionally, we identify a previously unrecognized noncell-autonomous regulatory function of MERTK expressed on DCs. Mer-Fc protein, used to mimic MERTK on DCs, suppresses naïve and antigen-specific memory T cell activation. This mechanism is mediated by the neutralization of the MERTK ligand PROS1. We find that MERTK and PROS1 are expressed in human T cells upon TCR activation and drive an autocrine proproliferative mechanism. Collectively, these results suggest that MERTK on DCs controls T cell activation and expansion through the competition for PROS1 interaction with MERTK in the T cells. In conclusion, this report identified MERTK as a potent suppressor of T cell response. PMID:25624460

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

NASA Astrophysics Data System (ADS)

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

1992-07-01

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

Mucosal-associated invariant T cells from induced pluripotent stem cells: A novel approach for modeling human diseases

PubMed Central

Sugimoto, Chie; Fujita, Hiroyoshi; Wakao, Hiroshi

2016-01-01

Mice have frequently been used to model human diseases involving immune dysregulation such as autoimmune and inflammatory diseases. These models help elucidate the mechanisms underlying the disease and in the development of novel therapies. However, if mice are deficient in certain cells and/or effectors associated with human diseases, how can their functions be investigated in this species? Mucosal-associated invariant T (MAIT) cells, a novel innate-like T cell family member, are a good example. MAIT cells are abundant in humans but scarce in laboratory mice. MAIT cells harbor an invariant T cell receptor and recognize nonpeptidic antigens vitamin B2 metabolites from bacteria and yeasts. Recent studies have shown that MAIT cells play a pivotal role in human diseases such as bacterial infections and autoimmune and inflammatory diseases. MAIT cells possess granulysin, a human-specific effector molecule, but granulysin and its homologue are absent in mice. Furthermore, MAIT cells show poor proliferation in vitro. To overcome these problems and further our knowledge of MAIT cells, we have established a method to expand MAIT cells via induced pluripotent stem cells (iPSCs). In this review, we describe recent advances in the field of MAIT cell research and our approach for human disease modeling with iPSC-derived MAIT cells. PMID:27114747

Hypercholesterolemia induces T cell expansion in humanized immune mice.

PubMed

Proto, Jonathan D; Doran, Amanda C; Subramanian, Manikandan; Wang, Hui; Zhang, Mingyou; Sozen, Erdi; Rymond, Christina C; Kuriakose, George; D'Agati, Vivette; Winchester, Robert; Sykes, Megan; Yang, Yong-Guang; Tabas, Ira

2018-06-01

Emerging data suggest that hypercholesterolemia has stimulatory effects on adaptive immunity and that these effects can promote atherosclerosis and perhaps other inflammatory diseases. However, research in this area has relied primarily on inbred strains of mice whose adaptive immune system can differ substantially from that of humans. Moreover, the genetically induced hypercholesterolemia in these models typically results in plasma cholesterol levels that are much higher than those in most humans. To overcome these obstacles, we studied human immune system-reconstituted mice (hu-mice) rendered hypercholesterolemic by treatment with adeno-associated virus 8-proprotein convertase subtilisin/kexin type 9 (AAV8-PCSK9) and a high-fat/high-cholesterol Western-type diet (WD). These mice had a high percentage of human T cells and moderate hypercholesterolemia. Compared with hu-mice that had lower plasma cholesterol, the PCSK9-WD mice developed a T cell-mediated inflammatory response in the lung and liver. Human CD4+ and CD8+ T cells bearing an effector memory phenotype were significantly elevated in the blood, spleen, and lungs of PCSK9-WD hu-mice, whereas splenic and circulating regulatory T cells were reduced. These data show that moderately high plasma cholesterol can disrupt human T cell homeostasis in vivo. This process may not only exacerbate atherosclerosis, but also contribute to T cell-mediated inflammatory diseases in the hypercholesterolemia setting.

Virus Infection Stages and Distinct Th1 or Th17/Th22 T-Cell Responses in Malaria/SHIV Coinfection Correlate with Different Outcomes of Disease

PubMed Central

Ryan-Payseur, Bridgett; Ali, Zahida; Huang, Dan; Chen, Crystal Y.; Yan, Lin; Wang, Richard C.; Collins, William E.; Wang, Yunqi

2011-01-01

Background. Malaria and AIDS represent 2 leading causes of death from infectious diseases worldwide, and their high geographic overlap means coinfection is prevalent. It remains unknown whether distinct immune responses during coinfection with malaria and human immunodeficiency virus (HIV) affect clinical outcomes. Methods. We tested this hypothesis by employing macaque models of coinfection with malaria and simian-human immunodeficiency virus (SHIV). Results. Plasmodium fragile malaria coinfection of acutely SHIV-infected macaques induced hyperimmune activation and remarkable expansion of CD4+ and CD8+ T effector cells de novo producing interferon γ or tumor necrosis factor α. Malaria-driven cellular hyperactivation/expansion and high-level Th1-cytokines enhanced SHIV disease characterized by increasing CD4+ T-cell depletion, profound lymphoid depletion or destruction, and even necrosis in lymph nodes and spleens. Importantly, malaria/SHIV-mediated depletion, destruction, and necrosis in lymphoid tissues led to bursting parasite replication and fatal virus-associated malaria. Surprisingly, chronically SHIV-infected macaques without AIDS employed different defense mechanisms during malaria coinfection, and mounted unique ∼200-fold expansion of interleukin 17+/interleukin 22+ T effectors with profound Th1 suppression. Such remarkable expansion of Th17/Th22 cells and inhibition of Th1 response coincided with development of immunity against fatal virus-associated malaria without accelerating SHIV disease. Conclusions. These novel findings suggest that virus infection status and selected Th1 or Th17/Th22 responses after malaria/AIDS-virus coinfection correlate with distinct outcomes of virus infection and malaria. PMID:21921207

Tacrolimus potently inhibits human osteoclastogenesis induced by IL-17 from human monocytes alone and suppresses human Th17 differentiation.

PubMed

Yago, Toru; Nanke, Yuki; Kawamoto, Manabu; Yamanaka, Hisashi; Kotake, Shigeru

2012-08-01

Tacrolimus (FK506, Prograf®) is an orally available, T cell specific and anti-inflammatory agent that has been proposed as a therapeutic drug in rheumatoid arthritis (RA) patients. It has been known that T cells have a critical role in the pathogenesis of RA. Recent studies suggest that Th17 cells, which mainly produce IL-17, are involved in many autoimmune inflammatory disease including RA. The present study was undertaken to assess the effect of tacrolimus on IL-17-induced human osteoclastogenesis and human Th17 differentiation. Human CD14(+) monocytes were cultured in the presence of macrophage-colony stimulating factor (M-CSF) and IL-17. From day 4, tacrolimus was added to these cultures. Osteoclasts were immunohistologically stained for vitronectin receptor 10days later. IL-17 production from activated T cells stimulated with IL-23 was measured by enzyme-linked immunosorbent assay (ELISA). Th17 differentiation from naïve T cells was assayed by flow cytometry. Tacrolimus potently inhibited IL-17-induced osteoclastogenesis from human monocytes and osteoclast activation. Addition of tacrolimus also reduced production of IL-17 in human activated T cells stimulated with IL-23. Interestingly, the population of human IL-17(+)IFN-γ(-) CD4 T cells or IL-17(+)TNF-α(+) CD4 T cells were decreased by adding of tacrolimus. The present study demonstrates that the inhibitory effect of tacrolimus on IL-17-induced osteoclastogenesis from human monocytes. Tacrolimus also inhibited expression of IL-17 or TNF-α by reducing the proportion of Th17, suggesting that therapeutic effect on Th17-associated disease such as RA, inflammatory bowel disease, multiple sclerosis, psoriasis, or allograft rejection. Copyright © 2012 Elsevier Ltd. All rights reserved.

Expansion of CD4+CD25+FOXP3+ regulatory T cells in infants of mothers with type 1 diabetes

PubMed Central

Luopajärvi, Kristiina; Nieminen, Janne K; Ilonen, Jorma; Åkerblom, Hans K; Knip, Mikael; Vaarala, Outi

2014-01-01

Background Reduced risk for type 1 diabetes (T1D) has been reported in the offspring of mothers with T1D when compared with children of affected fathers. Objective To evaluate the hypothesis that exposure of the offspring to maternal insulin therapy induces regulatory mechanisms in utero, we compared the FOXP3 expressing regulatory T cells in cord blood (CB) of infants born to mothers with or without T1D. Subjects and Methods Cord blood mononuclear cells (CBMCs) from 20 infants with maternal T1D and from 20 infants with an unaffected mother were analyzed for the numbers of CD4+CD25+FOXP3+ cells ex vivo and after in vitro stimulation with human insulin by flow cytometry. The mRNA expression of FOXP3, NFATc2, STIM1, interleukin (IL)-10, and transforming growth factor (TGF)-β was measured by real-time reverse transcription polymerase chain reaction. Results The percentage of FOXP3+ cells in CD4+CD25high cells was higher in the CB of the infants with maternal T1D when compared with the infants of unaffected mothers (p = 0.023). After in vitro insulin stimulation an increase in the percentage of FOXP3+ cells in CD4+CD25high cells (p = 0.0002) as well as upregulation of FOXP3, NFATc2, STIM1, IL-10, and TGF-β transcripts in CBMCs (p < 0.013 for all; Wilcoxon test) was observed only in the offspring of mothers with T1D, in whom the disease-related PTPN22 allele was associated with reduced STIM1 and NFATc2 response in insulin-stimulated CBMCs (p = 0.007 and p = 0.014). Conclusions We suggest that maternal insulin treatment induces expansion of regulatory T cells in the fetus, which might contribute to the lower risk of diabetes in children with maternal vs. paternal diabetes. PMID:22332874

Characterization of New Zealand White Rabbit Gut-Associated Lymphoid Tissues and Use as Viral Oncology Animal Model.

PubMed

Haines, Robyn A; Urbiztondo, Rebeccah A; Haynes, Rashade A H; Simpson, Elaine; Niewiesk, Stefan; Lairmore, Michael D

2016-01-01

Rabbits have served as a valuable animal model for the pathogenesis of various human diseases, including those related to agents that gain entry through the gastrointestinal tract such as human T cell leukemia virus type 1. However, limited information is available regarding the spatial distribution and phenotypic characterization of major rabbit leukocyte populations in mucosa-associated lymphoid tissues. Herein, we describe the spatial distribution and phenotypic characterization of leukocytes from gut-associated lymphoid tissues (GALT) from 12-week-old New Zealand White rabbits. Our data indicate that rabbits have similar distribution of leukocyte subsets as humans, both in the GALT inductive and effector sites and in mesenteric lymph nodes, spleen, and peripheral blood. GALT inductive sites, including appendix, cecal tonsil, Peyer's patches, and ileocecal plaque, had variable B cell/T cell ratios (ranging from 4.0 to 0.8) with a predominance of CD4 T cells within the T cell population in all four tissues. Intraepithelial and lamina propria compartments contained mostly T cells, with CD4 T cells predominating in the lamina propria compartment and CD8 T cells predominating in the intraepithelial compartment. Mesenteric lymph node, peripheral blood, and splenic samples contained approximately equal percentages of B cells and T cells, with a high proportion of CD4 T cells compared with CD8 T cells. Collectively, our data indicate that New Zealand White rabbits are comparable with humans throughout their GALT and support future studies that use the rabbit model to study human gut-associated disease or infectious agents that gain entry by the oral route. © The Author 2016. Published by Oxford University Press on behalf of the Institute for Laboratory Animal Research. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Generation of knock-in primary human T cells using Cas9 ribonucleoproteins

DOE PAGES

Schumann, Kathrin; Lin, Steven; Boyer, Eric; ...

2015-07-27

T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4 + T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs).more » Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ~40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 ( PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ~20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.« less

Generation of knock-in primary human T cells using Cas9 ribonucleoproteins

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

Schumann, Kathrin; Lin, Steven; Boyer, Eric

T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4 + T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs).more » Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ~40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 ( PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ~20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.« less

Role of interleukin 1 in antigen-specific T cell proliferation.

PubMed

Chu, E; Rosenwasser, L J; Dinarello, C A; Lareau, M; Geha, R S

1984-03-01

The role of interleukin 1 (IL 1) in human antigen-specific T cell proliferation was examined. Nylon wool-purified T cells proliferated in the presence of autologous monocytes (Mo.) pulsed for 18 h with tetanus toxoid (TT) antigen (Mo.TT). Irradiation of Mo.TT with ultraviolet (UV) light (72 J/m2) abolished their capacity to support T cell proliferation and drastically reduced their capacity to secrete IL 1 after stimulation with Staphylococcus albus. The defect in antigen presentation induced by UV irradiation of Mo.TT was reversed in a dose-dependent manner by the addition of two different preparations containing human interleukin 1 (IL 1). The first preparation consisted of supernatants of Mo. stimulated with Con A for 18 hr and in which Con A activity was blocked by alpha-D-methyl-mannoside (Mo.-Con A-Sup). The second preparation consisted of human IL 1 partially purified from supernatants of human peripheral blood mononuclear cells stimulated with S. albus. This IL 1 copurified with human leukocyte pyrogen (LP) and was termed IL 1/LP. Both IL 1-containing preparations enhanced the response of C57BL/6 mouse thymocytes to phytohemagglutinin. A rabbit antibody to human IL 1/LP inhibited the capacity of T cells to proliferate in response to Mo.TT and inhibited the capacity of Mo.-Con A-Sup to reconstitute the T cell response to UV-irradiated Mo.TT. IL 1/LP was not necessary for T cells to recognize the immunogenic moiety presented by Mo., because monolayers of UV-irradiated Mo.TT were equivalent to monolayers of unirradiated MO.TT in their capacity to adsorb TT-reactive T cells specifically. Furthermore, the addition of rabbit antibody to IL 1/LP did not interfere with the capacity of UV-irradiated Mo.TT to adsorb TT-reactive T cells. The results obtained in this study indicate that IL 1 is involved in optimal antigen-driven proliferation of human T lymphocytes.

T Cells Are Not Required for Pathogenesis in the Syrian Hamster Model of Hantavirus Pulmonary Syndrome ▿

PubMed Central

Hammerbeck, Christopher D.; Hooper, Jay W.

2011-01-01

Andes virus (ANDV) is associated with a lethal vascular leak syndrome in humans termed hantavirus pulmonary syndrome (HPS). In hamsters, ANDV causes a respiratory distress syndrome closely resembling human HPS. The mechanism for the massive vascular leakage associated with HPS is poorly understood; however, T cell immunopathology has been implicated on the basis of circumstantial and corollary evidence. Here, we show that following ANDV challenge, hamster T cell activation corresponds with the onset of disease. However, treatment with cyclophosphamide or specific T cell depletion does not impact the course of disease or alter the number of surviving animals, despite significant reductions in T cell number. These data demonstrate, for the first time, that T cells are not required for hantavirus pathogenesis in the hamster model of human HPS. Depletion of T cells from Syrian hamsters did not significantly influence early events in disease progression. Moreover, these data argue for a mechanism of hantavirus-induced vascular permeability that does not involve T cell immunopathology. PMID:21775442

T cells are not required for pathogenesis in the Syrian hamster model of hantavirus pulmonary syndrome.

PubMed

Hammerbeck, Christopher D; Hooper, Jay W

2011-10-01

Andes virus (ANDV) is associated with a lethal vascular leak syndrome in humans termed hantavirus pulmonary syndrome (HPS). In hamsters, ANDV causes a respiratory distress syndrome closely resembling human HPS. The mechanism for the massive vascular leakage associated with HPS is poorly understood; however, T cell immunopathology has been implicated on the basis of circumstantial and corollary evidence. Here, we show that following ANDV challenge, hamster T cell activation corresponds with the onset of disease. However, treatment with cyclophosphamide or specific T cell depletion does not impact the course of disease or alter the number of surviving animals, despite significant reductions in T cell number. These data demonstrate, for the first time, that T cells are not required for hantavirus pathogenesis in the hamster model of human HPS. Depletion of T cells from Syrian hamsters did not significantly influence early events in disease progression. Moreover, these data argue for a mechanism of hantavirus-induced vascular permeability that does not involve T cell immunopathology.

The molecular bases of δ/αβ T cell-mediated antigen recognition.

PubMed

Pellicci, Daniel G; Uldrich, Adam P; Le Nours, Jérôme; Ross, Fiona; Chabrol, Eric; Eckle, Sidonia B G; de Boer, Renate; Lim, Ricky T; McPherson, Kirsty; Besra, Gurdyal; Howell, Amy R; Moretta, Lorenzo; McCluskey, James; Heemskerk, Mirjam H M; Gras, Stephanie; Rossjohn, Jamie; Godfrey, Dale I

2014-12-15

αβ and γδ T cells are disparate T cell lineages that can respond to distinct antigens (Ags) via the use of the αβ and γδ T cell Ag receptors (TCRs), respectively. Here we characterize a population of human T cells, which we term δ/αβ T cells, expressing TCRs comprised of a TCR-δ variable gene (Vδ1) fused to joining α and constant α domains, paired with an array of TCR-β chains. We demonstrate that these cells, which represent ∼50% of all Vδ1(+) human T cells, can recognize peptide- and lipid-based Ags presented by human leukocyte antigen (HLA) and CD1d, respectively. Similar to type I natural killer T (NKT) cells, CD1d-lipid Ag-reactive δ/αβ T cells recognized α-galactosylceramide (α-GalCer); however, their fine specificity for other lipid Ags presented by CD1d, such as α-glucosylceramide, was distinct from type I NKT cells. Thus, δ/αβTCRs contribute new patterns of Ag specificity to the human immune system. Furthermore, we provide the molecular bases of how δ/αβTCRs bind to their targets, with the Vδ1-encoded region providing a major contribution to δ/αβTCR binding. Our findings highlight how components from αβ and γδTCR gene loci can recombine to confer Ag specificity, thus expanding our understanding of T cell biology and TCR diversity. © 2014 Pellicci et al.

Therapeutic effect of targeted Fas-expressing adenoviruses method combining γδ T cells in a mouse model of human ovarian carcinoma.

PubMed

Zeng, Dingyuan; Lin, Jiajing; He, Hongying; Tan, Guangping; Lan, Ying; Jiang, Fuyan; Sheng, Shuting

2018-02-01

The present study aimed to investigate the therapeutic effect and safety of targeted use of Fas-expressing adenoviruses combined with γδ T cell-mediated killing to treat human ovarian cancer xenografts in BALB/c mice. Shuttle plasmids containing control elements of human telomerase reverse transcriptase promoter and two-step transcriptional amplification system were constructed and packaged into adenovirus-5 vectors to generate expression of an exogenous Fas gene. A mouse xenograft model of human ovarian carcinoma was constructed. A total of 35 BALB/c mice were randomly divided into five groups, which were injected with PBS, γδ T cells, Fas-expressing adenoviruses, taxol, or Fas-expressing adenovirus and γδ T cells. The weight and volume of tumors in mice in each group was monitored. Tissue sections of the various tissues of mice in the Fas-expressing adenovirus and γδ T cells group was compared with those in the PBS group to evaluate the safety of Fas-expressing adenovirus and γδ T cells in the treatment of human ovarian cancer xenograft tumors. The results of the present study indicated that mice in all treatment groups were alive at the end of the treatment course. Tumor weight and volume was the highest in the PBS group, followed successively by the adenovirus group, the γδ T cell group, the adenovirus and γδ T cell group, and the taxol group. The weight and volume inhibition rate in adenovirus and γδ T cell group were significantly higher compared with in the PBS group (P<0.05). Pathological observation of tissue samples revealed that none of vital organs in the adenovirus and γδ T cell group developed any evident morphological changes during treatment, when compared with healthy controls. In conclusion, the combined therapy with Fas-expressing adenoviruses and γδ T cells is efficient and safe for the treatment of mouse human ovarian carcinoma xenografts.

CD8 apoptosis may be a predictor of T cell number normalization after immune reconstitution in HIV

PubMed Central

Lewis, Dorothy E; Gross, Kimber L; Diez, Martine M; Martinez, Maria L; Lukefahr, Helen N; Kozinetz, Claudia A; Arduino, Roberto C

2007-01-01

Background As part of the Houston Vanguard study, a subset of 10 patients randomized to receive IL-2 therapy were compared to 4 patients randomized to not receive IL-2, for markers of T cell activation and death during the first three cycles of IL-2. All patients were treated with combination antiretroviral therapy (ART) and were virally suppressed. The purpose of the study was to examine the role of CD8+ T cell death in responses to ART and IL-2 therapy. Methods Lymphocytes were examined at Day 0, 5 and 30 days during three cycles of IL-2 therapy. CD25, CD38, HLA-DR expression and annexin (cell death) were examined on CD4 and CD8 subpopulations. Follow up studies examined CD4 levels and CD4:CD8 reconstitution after 6 years using both univariant and multivariate analyses. Results Human lymphocytes responded to IL-2 therapy by upregulation of CD25 on CD4+ T cells, leading to an increase in CD4 cell counts. CD8+ T cells did not increase CD25 expression, but upregulated activation antigens (CD38 and DR) and had increased death. At baseline, 7 of the 14 patients had high CD8+ T cell apoptosis (mean 17.0% ± 6.0). We did an exploratory analysis of immune status after six years, and found that baseline CD8+ T cell apoptosis was correlated with CD4 cell count gain beginning two years post enrollment. Patients with low levels of CD8+ T cell apoptosis at baseline (mean 2.2% ± 2.1) had significantly higher CD4 cell counts and more normalized CD4:CD8 ratios than patients with high CD8+ T cell apoptosis (mean CD4 cell counts 1,209 ± 164 vs 754 ± 320 cells/mm3; CD4:CD8 ratios 1.55 vs. 0.70, respectively). Conclusion We postulate that CD8+ T cell apoptosis may reflect inherent activation status, which continues in some patients even though viral replication is suppressed which influences the ability of CD4+ T cells to rebound. Levels of CD8+ T cell apoptosis may therefore be an independent predictor of immune status, which should be shown in a prospective study. PMID:17263884

T Cell Proliferation and Colitis Are Initiated by Defined Intestinal Microbes.

PubMed

Chiaranunt, Pailin; Tometich, Justin T; Ji, Junyi; Hand, Timothy W

2018-07-01

Inflammatory bowel disease has been associated with the dysregulation of T cells specific to Ags derived from the intestinal microbiota. How microbiota-specific T cells are regulated is not completely clear but is believed to be mediated by a combination of IgA, regulatory T cells, and type 3 innate lymphoid cells. To test the role of these regulatory components on microbiota-specific T cells, we bred CBir1 TCR transgenic (CBir1Tg) mice (specific to flagellin from common intestinal bacteria) onto a lymphopenic Rag1 -/- background. Surprisingly, T cells from CBir1Tg mice bred onto a Rag1 -/- background could not induce colitis and did not differentiate to become effectors under lymphopenic conditions, despite deficits in immunoregulatory factors, such as IgA, regulatory T cells, and type 3 innate lymphoid cells. In fact, upon transfer of conventional CBir1Tg T cells into lymphopenic mice, the vast majority of proliferating T cells responded to Ags other than CBir1 flagellin, including those found on other bacteria, such as Helicobacter spp. Thus, we discovered a caveat in the CBir1Tg model within our animal facility that illustrates the limitations of using TCR transgenics at mucosal surfaces, where multiple TCR specificities can respond to the plethora of foreign Ags. Our findings also indicate that T cell specificity to the microbiota alone is not sufficient to induce T cell activation and colitis. Instead, other interrelated factors, such as the composition and ecology of the intestinal microbiota and host access to Ag, are paramount in controlling the activation of microbiota-specific T cell clones. Copyright © 2018 by The American Association of Immunologists, Inc.

Bystander CD4+ T lymphocytes survive in HIV-infected human lymphoid tissue

NASA Technical Reports Server (NTRS)

Grivel, Jean-Charles; Biancotto, Angelique; Ito, Yoshinori; Lima, Rosangela G.; Margolis, Leonid B.

2003-01-01

HIV infection is associated with depletion of CD4(+) T cells. The mechanisms of this phenomenon remain to be understood. In particular, it remains controversial whether and to what extent uninfected ("bystander") CD4(+) T cells die in HIV-infected individuals. We address this question using a system of human lymphoid tissue ex vivo. Tissue blocks were inoculated with HIV-1. After productive infection was established, they were treated with the reverse transcriptase inhibitor nevirapine to protect from infection those CD4(+) T cells that had not yet been infected. These CD4(+) T cells residing in HIV-infected tissue are by definition bystanders. Our results demonstrate that after nevirapine application the number of bystander CD4(+) T cells is conserved. Thus, in the context of HIV-infected human lymphoid tissue, productive HIV infection kills infected cells but is not sufficient to cause the death of a significant number of uninfected CD4(+) T cells.

Nuclear calcium is required for human T cell activation

PubMed Central

Samstag, Yvonne

2016-01-01

Calcium signals in stimulated T cells are generally considered single entities that merely trigger immune responses, whereas costimulatory events specify the type of reaction. Here we show that the “T cell calcium signal” is a composite signal harboring two distinct components that antagonistically control genomic programs underlying the immune response. Using human T cells from healthy individuals, we establish nuclear calcium as a key signal in human T cell adaptogenomics that drives T cell activation and is required for signaling to cyclic adenosine monophosphate response element–binding protein and the induction of CD25, CD69, interleukin-2, and γ-interferon. In the absence of nuclear calcium signaling, cytosolic calcium activating nuclear factor of activated T cells translocation directed the genomic response toward enhanced expression of genes that negatively modulate T cell activation and are associated with a hyporesponsive state. Thus, nuclear calcium controls the T cell fate decision between a proliferative immune response and tolerance. Modulators of nuclear calcium–driven transcription may be used to develop a new type of pro-tolerance immunosuppressive therapy. PMID:27810914

Blocking immunosuppression by human Tregs in vivo with antibodies targeting integrin αVβ8.

PubMed

Stockis, Julie; Liénart, Stéphanie; Colau, Didier; Collignon, Amandine; Nishimura, Stephen L; Sheppard, Dean; Coulie, Pierre G; Lucas, Sophie

2017-11-21

Human regulatory T cells (Tregs) suppress other T cells by converting the latent, inactive form of TGF-β1 into active TGF-β1. In Tregs, TGF-β1 activation requires GARP, a transmembrane protein that binds and presents latent TGF-β1 on the surface of Tregs stimulated through their T cell receptor. However, GARP is not sufficient because transduction of GARP in non-Treg T cells does not induce active TGF-β1 production. RGD-binding integrins were shown to activate TGF-β1 in several non-T cell types. Here we show that αVβ8 dimers are present on stimulated human Tregs but not in other T cells, and that antibodies against αV or β8 subunits block TGF-β1 activation in vitro. We also show that αV and β8 interact with GARP/latent TGF-β1 complexes in human Tregs. Finally, a blocking antibody against β8 inhibited immunosuppression by human Tregs in a model of xenogeneic graft-vs.-host disease induced by the transfer of human T cells in immunodeficient mice. These results show that TGF-β1 activation on the surface of human Tregs implies an interaction between the integrin αVβ8 and GARP/latent TGF-β1 complexes. Immunosuppression by human Tregs can be inhibited by antibodies against GARP or against the integrin β8 subunit. Such antibodies may prove beneficial against cancer or chronic infections.

Chromosome translocations in T. scripta: the dose-rate effect and in vivo lymphocyte radiation response.

PubMed

Ulsh, B A; Whicker, F W; Congdon, J D; Bedford, J S; Hinton, T G

2001-01-01

Using a whole-chromosome FISH painting probe we previously developed for chromosome 1 of the yellow-bellied slider turtle (Trachemys scripta), we investigated the dose-rate effect for radiation-induced symmetrical translocations in T. scripta fibroblasts and lymphocytes. The dose rate below which no reduction in effect per unit dose is observed with further dose protraction was approximately 23 cGy h(-1). We estimated the whole-genome spontaneous background level of complete, apparently simple symmetrical translocations in T. scripta lymphocytes to be approximately 1.20 x 10(-3)/cell projected from aberrations occurring in chromosome 1. Similar spontaneous background levels reported for humans are some 6- to 25-fold higher, ranging from about 6 x 10(-3) to 3.4 x 10(-2) per cell. This relatively low background level for turtles would be a significant advantage for resolution of effects at low doses and dose rates. We also chronically irradiated turtles over a range of doses from 0-8 Gy delivered at approximately 5.5 cGy h(-1) and constructed a lymphocyte dose-response curve for complete, apparently simple symmetrical translocations suitable for use with animals chronically exposed to radiation in contaminated environments. The best-fitting calibration curve (not constrained through the zero dose estimate) was of the form Y(as) = c + aD + bD(2), where Y(as) was the number of apparently simple symmetrical translocations per cell, D was the dose (Gy), a = (0.0058 +/- 0.0009), b = (-0.00033 +/- 0.00011), and c = (0.0015 +/- 0.0013). With additional whole-chromosome probes to improve sensitivity, environmental biodosimetry using stable chromosome translocations could provide a practical and genetically relevant measurement end point for ecological risk assessments and biomonitoring programs.

Thyroid hormone enhanced human hepatoma cell motility involves brain-specific serine protease 4 activation via ERK signaling

PubMed Central

2014-01-01

Background The thyroid hormone, 3, 3′, 5-triiodo-L-thyronine (T3), has been shown to modulate cellular processes via interactions with thyroid hormone receptors (TRs), but the secretory proteins that are regulated to exert these effects remain to be characterized. Brain-specific serine protease 4 (BSSP4), a member of the human serine protease family, participates in extracellular matrix remodeling. However, the physiological role and underlying mechanism of T3-mediated regulation of BSSP4 in hepatocellular carcinogenesis are yet to be established. Methods The thyroid hormone response element was identified by reporter and chromatin immunoprecipitation assays. The cell motility was analyzed via transwell and SCID mice. The BSSP4 expression in clinical specimens was examined by Western blot and quantitative reverse transcription polymerase chain reaction. Results Upregulation of BSSP4 at mRNA and protein levels after T3 stimulation is a time- and dose-dependent manner in hepatoma cell lines. Additionally, the regulatory region of the BSSP4 promoter stimulated by T3 was identified at positions -609/-594. BSSP4 overexpression enhanced tumor cell migration and invasion, both in vitro and in vivo. Subsequently, BSSP4-induced migration occurs through the ERK 1/2-C/EBPβ-VEGF cascade, similar to that observed in HepG2-TRα1 and J7-TRα1 cells. BSSP4 was overexpressed in clinical hepatocellular carcinoma (HCC) patients, compared with normal subjects, and positively associated with TRα1 and VEGF to a significant extent. Importantly, a mild association between BSSP4 expression and distant metastasis was observed. Conclusions Our findings collectively support a potential role of T3 in cancer cell progression through regulation of the BSSP4 protease via the ERK 1/2-C/EBPβ-VEGF cascade. BSSP4 may thus be effectively utilized as a novel marker and anti-cancer therapeutic target in HCC. PMID:24980078

CD83 Antibody Inhibits Human B Cell Responses to Antigen as well as Dendritic Cell-Mediated CD4 T Cell Responses.

PubMed

Wong, Kuan Y; Baron, Rebecca; Seldon, Therese A; Jones, Martina L; Rice, Alison M; Munster, David J

2018-05-15

Anti-CD83 Ab capable of Ab-dependent cellular cytotoxicity can deplete activated CD83 + human dendritic cells, thereby inhibiting CD4 T cell-mediated acute graft-versus-host disease. As CD83 is also expressed on the surface of activated B lymphocytes, we hypothesized that anti-CD83 would also inhibit B cell responses to stimulation. We found that anti-CD83 inhibited total IgM and IgG production in vitro by allostimulated human PBMC. Also, Ag-specific Ab responses to immunization of SCID mice xenografted with human PBMC were inhibited by anti-CD83 treatment. This inhibition occurred without depletion of all human B cells because anti-CD83 lysed activated CD83 + B cells by Ab-dependent cellular cytotoxicity and spared resting (CD83 - ) B cells. In cultured human PBMC, anti-CD83 inhibited tetanus toxoid-stimulated B cell proliferation and concomitant dendritic cell-mediated CD4 T cell proliferation and expression of IFN-γ and IL-17A, with minimal losses of B cells (<20%). In contrast, the anti-CD20 mAb rituximab depleted >80% of B cells but had no effect on CD4 T cell proliferation and cytokine expression. By virtue of the ability of anti-CD83 to selectively deplete activated, but not resting, B cells and dendritic cells, with the latter reducing CD4 T cell responses, anti-CD83 may be clinically useful in autoimmunity and transplantation. Advantages might include inhibited expansion of autoantigen- or alloantigen-specific B cells and CD4 T cells, thus preventing further production of pathogenic Abs and inflammatory cytokines while preserving protective memory and regulatory cells. Copyright © 2018 by The American Association of Immunologists, Inc.

Genetically-modified pig mesenchymal stromal cells: xenoantigenicity and effect on human T-cell xenoresponses.

PubMed

Ezzelarab, Mohamed; Ezzelarab, Corin; Wilhite, Tyler; Kumar, Goutham; Hara, Hidetaka; Ayares, David; Cooper, David K C

2011-01-01

Mesenchymal stromal cells (MSC) are being investigated as immunomodulatory therapy in the field of transplantation, particularly islet transplantation. While MSC can regenerate across species barriers, the immunoregulatory influence of genetically modified pig MSC (pMSC) on the human and non-human primate T-cell responses has not been studied. Mesenchymal stromal cells from wild-type (WT), α1,3-galactosyltransferase gene knockout (GTKO) and GTKO pigs transgenic for the human complement-regulatory protein CD46 (GTKO/CD46) were isolated and tested for differentiation. Antibody binding and T-cell responses to WT and GTKO pMSC in comparison with GTKO pig aortic endothelial cells (pAEC) were investigated. The expression of swine leukocyte antigen (SLA) class II (SLA II) was tested. Costimulatory molecules CD80 and CD86 mRNA levels were measured. Human T-cell proliferation and the production of pro-inflammatory cytokines in response to GTKO and GTKO/CD46 pMSC in comparison with human MSC (hMSC) were evaluated. α1,3-galactosyltransferase gene knockout and GTKO/CD46 pMSC isolation and differentiation were achieved in vitro. Binding of human antibodies and T-cell responses were lower to GTKO than those to WT pMSC. Human and baboon (naïve and sensitized) antibody binding were significantly lower to GTKO pMSC than to GTKO pAEC. Before activation, <1% of GTKO pMSC expressed SLA II, compared with 2.5% of GTKO pAEC. After pig interferon-gamma (pIFN-γ) activation, 99% of GTKO pAEC upregulated SLA II expression, compared with 49% of GTKO pMSC. Only 3% of GTKO pMSC expressed CD80 compared with 80% of GTKO pAEC without activation. After pIFN-γ activation, GTKO pAEC upregulated CD86 mRNA level stronger than GTKO pMSC. The human CD4(+) T-cell response to GTKO pMSC was significantly weaker than that to GTKO pAEC, even after pIFN-γ activation. More than 99% of GTKO/CD46 pMSC expressed hCD46. Human peripheral blood mononuclear cells and CD4(+) T-cell responses to GTKO and GTKO/CD46 pMSC were comparable with those to hMSC, and all were significantly lower than to GTKO pAEC. GTKO/CD46 pMSC downregulated human T-cell proliferation as efficiently as hMSC. The level of proinflammatory cytokines IL-2, IFN-γ, TNF-α, and sCD40L correlated with the downregulation of T-cell proliferation by all types of MSC. Genetically modified pMSC is significantly less immunogenic than WT pMSC. GTKO/CD46 pMSC downregulates the human T-cell responses to pig antigens as efficiently as human MSC, which can be advantageous for therapeutic cell xenotransplantation. © 2011 John Wiley & Sons A/S.

A fully human chimeric antigen receptor with potent activity against cancer cells but reduced risk for off-tumor toxicity

PubMed Central

Song, De-Gang; Ye, Qunrui; Poussin, Mathilde; Liu, Lin; Figini, Mariangela; Powell, Daniel J.

2015-01-01

Chimeric antigen receptors (CARs) can redirect T cells against antigen-expressing tumors in an HLA-independent manner. To date, various CARs have been constructed using mouse single chain antibody variable fragments (scFvs) of high affinity that are immunogenic in humans and have the potential to mediate “on-target” toxicity. Here, we developed and evaluated a fully human CAR comprised of the human C4 folate receptor-alpha (αFR)-specific scFv coupled to intracellular T cell signaling domains. Human T cells transduced to express the C4 CAR specifically secreted proinflammatory cytokine and exerted cytolytic functions when cultured with αFR-expressing tumors in vitro. Adoptive transfer of C4 CAR T cells mediated the regression of large, established human ovarian cancer in a xenogeneic mouse model. Relative to a murine MOv19 scFv-based αFR CAR, C4 CAR T cells mediated comparable cytotoxic tumor activity in vitro and in vivo but had lower affinity for αFR protein and exhibited reduced recognition of normal cells expressing low levels of αFR. Thus, T cells expressing a fully human CAR of intermediate affinity can efficiently kill antigen-expressing tumors in vitro and in vivo and may overcome issues of transgene immunogenicity and “on-target off-tumor” toxicity that plague trials utilizing CARs containing mouse-derived, high affinity scFvs. PMID:26101914

Endothelial cell-derived microparticles induce plasmacytoid dendritic cell maturation: potential implications in inflammatory diseases

PubMed Central

Angelot, Fanny; Seillès, Estelle; Biichlé, Sabeha; Berda, Yael; Gaugler, Béatrice; Plumas, Joel; Chaperot, Laurence; Dignat-George, Françoise; Tiberghien, Pierre; Saas, Philippe; Garnache-Ottou, Francine

2009-01-01

Background Increased circulating endothelial microparticles, resulting from vascular endothelium dysfunction, and plasmacytoid dendritic cell activation are both encountered in common inflammatory disorders. The aim of our study was to determine whether interactions between endothelial microparticles and plasmacytoid dendritic cells could contribute to such pathologies. Design and Methods Microparticles generated from endothelial cell lines, platelets or activated T cells were incubated with human plasmacytoid dendritic cells sorted from healthy donor blood or with monocyte-derived dendritic cells. Dendritic cell maturation was evaluated by flow cytometry, cytokine secretion as well as naive T-cell activation and polarization. Labeled microparticles were also used to study cellular interactions. Results Endothelial microparticles induced plasmacytoid dendritic cell maturation. In contrast, conventional dendritic cells were resistant to endothelial microparticle-induced maturation. In addition to upregulation of co-stimulatory molecules, endothelial microparticle-matured plasmacytoid dendritic cells secreted inflammatory cytokines (interleukins 6 and 8, but no interferon-α) and also induced allogeneic naive CD4+ T cells to proliferate and to produce type 1 cytokines such as interferon-γ and tumor necrosis factor-α. Endothelial microparticle endocytosis by plasmacytoid dendritic cells appeared to be required for plasmacytoid dendritic cell maturation. Importantly, the ability of endothelial microparticles to induce plasmacytoid dendritic cells to mature was specific as microparticles derived from activated T cells or platelets (the major source of circulating microparticules in healthy subjects) did not induce such plasmacytoid dendritic cell maturation. Conclusions Our data show that endothelial microparticles specifically induce plasmacytoid dendritic cell maturation and production of inflammatory cytokines. This novel activation pathway may be implicated in various inflammatory disorders and endothelial microparticles could be an important immunmodulatory therapeutic target. PMID:19648164

Soluble GARP has potent antiinflammatory and immunomodulatory impact on human CD4⁺ T cells.

PubMed

Hahn, Susanne A; Stahl, Heiko F; Becker, Christian; Correll, Anita; Schneider, Franz-Joseph; Tuettenberg, Andrea; Jonuleit, Helmut

2013-08-15

Glycoprotein A repetitions predominant (GARP) is expressed on the surface of activated human regulatory T cells (Treg) and regulates the bioavailability of transforming growth factor-β (TGF-β). GARP has been assumed to require membrane anchoring. To investigate the function of GARP in more detail, we generated a soluble GARP protein (sGARP) and analyzed its impact on differentiation and activation of human CD4⁺ T cells. We demonstrate that sGARP efficiently represses proliferation and differentiation of naïve CD4⁺ T cells into T effector cells. Exposure to sGARP induces Foxp3, decreases proliferation and represses interleukin (IL)-2 and interferon-γ production, resulting in differentiation of naïve T cells into induced Treg. This is associated with Smad2/3 phosphorylation and partially inhibited by blockade of TGF-β signaling. Furthermore, in the presence of the proinflammatory cytokines IL-6 and IL-23, sGARP facilitates the differentiation of naïve T cells into Th17 cells. More important, in a preclinical humanized mouse model of xenogeneic graft-versus-host disease (GVHD), sGARP prevents T cell-mediated destructive inflammation by enhancing Treg and inhibiting T effector cell activity. These results demonstrate a crucial role of sGARP in modulation of peripheral tolerance and T effector cell function, opening the possibility to use sGARP as a potent immunomodulator of inflammatory diseases including transplant rejection, autoimmunity, and allergy.

The molecular bases of δ/αβ T cell–mediated antigen recognition

PubMed Central

Pellicci, Daniel G.; Uldrich, Adam P.; Le Nours, Jérôme; Ross, Fiona; Chabrol, Eric; Eckle, Sidonia B.G.; de Boer, Renate; Lim, Ricky T.; McPherson, Kirsty; Besra, Gurdyal; Howell, Amy R.; Moretta, Lorenzo; McCluskey, James; Heemskerk, Mirjam H.M.; Gras, Stephanie

2014-01-01

αβ and γδ T cells are disparate T cell lineages that can respond to distinct antigens (Ags) via the use of the αβ and γδ T cell Ag receptors (TCRs), respectively. Here we characterize a population of human T cells, which we term δ/αβ T cells, expressing TCRs comprised of a TCR-δ variable gene (Vδ1) fused to joining α and constant α domains, paired with an array of TCR-β chains. We demonstrate that these cells, which represent ∼50% of all Vδ1+ human T cells, can recognize peptide- and lipid-based Ags presented by human leukocyte antigen (HLA) and CD1d, respectively. Similar to type I natural killer T (NKT) cells, CD1d-lipid Ag-reactive δ/αβ T cells recognized α-galactosylceramide (α-GalCer); however, their fine specificity for other lipid Ags presented by CD1d, such as α-glucosylceramide, was distinct from type I NKT cells. Thus, δ/αβTCRs contribute new patterns of Ag specificity to the human immune system. Furthermore, we provide the molecular bases of how δ/αβTCRs bind to their targets, with the Vδ1-encoded region providing a major contribution to δ/αβTCR binding. Our findings highlight how components from αβ and γδTCR gene loci can recombine to confer Ag specificity, thus expanding our understanding of T cell biology and TCR diversity. PMID:25452463

Langerhans cells from human oral epithelium are more effective at stimulating allogeneic T cells in vitro than Langerhans cells from skin.

PubMed

Hasséus, B; Jontell, M; Bergenholtz, G; Dahlgren, U I

2004-06-01

This report is focused on the functional capacity of Langerhans cells (LC) in the epithelium of skin and oral mucosa, which both meet different antigenic challenges. The capacity of LC from human oral and skin epithelium to provide co-stimulatory signals to T cells in vitro was compared. LC in a crude suspension of oral epithelial cells had a significantly enhanced T cell co-stimulatory capacity compared to skin epithelial cells. This applied both to cultures with concanavalin A (con-A)-stimulated syngeneic T cells and to a mixed epithelial cell lymphocyte reaction involving allogeneic T cells. The co-stimulatory capacity of oral and skin epithelial cells was reduced by >70% if monoclonal antibodies against HLA-DR, -DP and -DQ were added to the cultures with allogeneic T cells, indicating the involvement of HLA class II expressing LC. Immunohistochemistry revealed that 6% of the epithelial cells were CD1a + LC in sections from both oral and skin epithelium. Interleukin (IL)-8 production was higher in cultures of oral epithelial cells and con-A stimulated T cells than in corresponding cultures with skin epithelial cells as accessory cells. The results suggest that LC in human oral epithelium are more efficient at stimulating T cells than those of skin.

Rescue of Tolerant CD8+ T Cells during Cancer Immunotherapy with IL2:Antibody Complexes.

PubMed

Klevorn, Lauryn E; Berrien-Elliott, Melissa M; Yuan, Jinyun; Kuehm, Lindsey M; Felock, Gregory D; Crowe, Sean A; Teague, Ryan M

2016-12-01

Interleukin-2 (IL2) was among the earliest reagents used for cancer immunotherapy due to its ability to support the survival and function of tumor-reactive T cells. However, treatment with IL2 is accompanied by off-target toxicity and low response rates in patients. In mouse models, these issues are largely overcome when IL2 is administered as a cytokine/antibody complex (IL2c). The complex has a longer serum half-life and can be designed for preferential cytokine delivery to specific cells of interest. Early studies showed IL2c could boost antitumor immunity in mice by activating tumor-reactive CD8 + T cells. But such functional T cells are often limited in the tumor microenvironment, where instead unresponsive tolerant T cells are eventually eliminated by apoptosis, representing a major obstacle to the success of cancer immunotherapy. We found that IL2c treatment rescued tumor-specific CD8 + T cells from a state of established tolerance, providing effective immunotherapy in tumor-bearing mice. Expression of the transcription factor T-bet was necessary to drive intratumoral IFNγ production and effector activity by T cells rescued with IL2c. Furthermore, IL2c promoted T-bet expression in human CD4 + and CD8 + T cells in humanized tumor-bearing mice, but also increased the frequency of Foxp3 + regulatory T cells. Our study reveals a novel role for IL2c as a powerful immunotherapeutic reagent capable of reversing tolerance in tumor-reactive T cells, and provides the first evidence that IL2c influences human T cells in vivo, highlighting the translational potential to modulate human antitumor immune responses. Cancer Immunol Res; 4(12); 1016-26. ©2016 AACR. ©2016 American Association for Cancer Research.

Analysis of close associations of uropod-associated proteins in human T-cells using the proximity ligation assay

PubMed Central

Baumann, Tommy; Affentranger, Sarah

2013-01-01

We have shown previously that the raft-associated proteins flotillin-1 and -2 are rapidly recruited to the uropods of chemoattractant-stimulated human neutrophils and T-cells and are involved in cell polarization. Other proteins such as the adhesion receptor PSGL-1, the actin-membrane linker proteins ezrin/radixin/moesin (ERM) and the signaling enzyme phosphatidylinositol-4-phosphate 5-kinase type Iγ90 (PIPKIγ90) also accumulate in the T-cell uropod. Using the in situ proximity ligation assay (PLA) we now have investigated putative close associations of these proteins in human freshly isolated T-cells before and after chemokine addition. The PLA allows in situ subcellular localization of close proximity of endogenous proteins at single-molecule resolution in fixed cells. It allows detection also of weaker and transient complexes that would not be revealed with co-immunoprecipitation approaches. We previously provided evidence for heterodimer formation of tagged flotillin-1 and -2 in T-cells before and after chemokine addition using fluorescence resonance energy transfer (FRET). We now confirm these findings using PLA for the endogenous flotillins in fixed human T-cells. Moreover, in agreement with the literature, our PLA findings confirm a close association of endogenous PSGL-1 and ERM proteins both in resting and chemokine-activated human T-cells. In addition, we provide novel evidence using the PLA for close associations of endogenous activated ERM proteins with PIPKIγ90 and of endogenous flotillins with PSGL-1 in human T-cells, before and after chemokine addition. Our findings suggest that preformed clusters of these proteins coalesce in the uropod upon cell stimulation. PMID:24167781

Therapeutic blockade of LIGHT interaction with HVEM and LTβR attenuates in vivo cytotoxic allogeneic responses

PubMed Central

del Rio, Maria-Luisa; Fernandez-Renedo, Carlos; Scheu, Stefanie; Pfeffer, Klaus; Shintani, Yasushi; Kronenberg, Mitchell; Chaloin, Olivier; Schneider, Pascal; Rodriguez-Barbosa, Jose-Ignacio

2016-01-01

Background TNF/TNFR superfamily members conform a group of molecular interaction pathways of essential relevance during the process of T cell activation and differentiation towards effector cells and particularly for the maintenance phase of the immune response. Specific blockade of these interacting pathways, such as CD40/CD40L, contributes to modulate the deleterious outcome of allogeneic immune responses. We postulated that antagonizing the interaction of LIGHT expression on activated T cells with its receptors, HVEM and LTβR may decrease T cell-mediated allogeneic responses. Methods A flow cytometry competition assay was designed to identify anti-LIGHT monoclonal antibodies capable to prevent the interaction of mouse LIGHT with its receptors expressed on transfected cells. An antibody with the desired specificity was evaluated in a short-term in vivo allogeneic cytotoxic assay and tested for its ability to detect endogenous mouse LIGHT. Results We provide evidence for the first time that in mice, as previously described in humans, LIGHT protein is rapidly and transiently expressed after T cell activation, and this expression was stronger on CD8 T cells than on CD4 T cells. Two anti-LIGHT antibodies prevented interactions of mouse LIGHT with its two known receptors HVEM and LTβR. In vivo administration of anti-LIGHT antibody (clone 10F12) ameliorated host anti-donor short-term cytotoxic response in WT B6 mice, although to a lesser extent than that observed in LIGHT-deficient mice. Conclusions The therapeutic targeting of LIGHT may contribute to achieve a better control of cytotoxic responses refractory to current immunosuppressive drugs in transplantation. PMID:25226173

CXCL4 is a novel inducer of human Th17 cells and correlates with IL‐17 and IL‐22 in psoriatic arthritis

PubMed Central

Affandi, Alsya J.; Silva‐Cardoso, Sandra C.; Garcia, Samuel; Leijten, Emmerik F. A.; van Kempen, Tessa S.; Marut, Wioleta; van Roon, Joel A. G.

2018-01-01

Abstract CXCL4 regulates multiple facets of the immune response and is highly upregulated in various Th17‐associated rheumatic diseases. However, whether CXCL4 plays a direct role in the induction of IL‐17 production by human CD4+ T cells is currently unclear. Here, we demonstrated that CXCL4 induced human CD4+ T cells to secrete IL‐17 that co‐expressed IFN‐γ and IL‐22, and differentiated naïve CD4+ T cells to become Th17‐cytokine producing cells. In a co‐culture system of human CD4+ T cells with monocytes or myeloid dendritic cells, CXCL4 induced IL‐17 production upon triggering by superantigen. Moreover, when monocyte‐derived dendritic cells were differentiated in the presence of CXCL4, they orchestrated increased levels of IL‐17, IFN‐γ, and proliferation by CD4+ T cells. Furthermore, the CXCL4 levels in synovial fluid from psoriatic arthritis patients strongly correlated with IL‐17 and IL‐22 levels. A similar response to CXCL4 of enhanced IL‐17 production by CD4+ T cells was also observed in patients with psoriatic arthritis. Altogether, we demonstrate that CXCL4 boosts pro‐inflammatory cytokine production especially IL‐17 by human CD4+ T cells, either by acting directly or indirectly via myeloid antigen presenting cells, implicating a role for CXCL4 in PsA pathology. PMID:29193036

HIV-1 Infection of Primary CD4+ T Cells Regulates the Expression of Specific Human Endogenous Retrovirus HERV-K (HML-2) Elements.

PubMed

Young, George R; Terry, Sandra N; Manganaro, Lara; Cuesta-Dominguez, Alvaro; Deikus, Gintaras; Bernal-Rubio, Dabeiba; Campisi, Laura; Fernandez-Sesma, Ana; Sebra, Robert; Simon, Viviana; Mulder, Lubbertus C F

2018-01-01

Endogenous retroviruses (ERVs) occupy extensive regions of the human genome. Although many of these retroviral elements have lost their ability to replicate, those whose insertion took place more recently, such as the HML-2 group of HERV-K elements, still retain intact open reading frames and the capacity to produce certain viral RNA and/or proteins. Transcription of these ERVs is, however, tightly regulated by dedicated epigenetic control mechanisms. Nonetheless, it has been reported that some pathological states, such as viral infections and certain cancers, coincide with ERV expression, suggesting that transcriptional reawakening is possible. HML-2 elements are reportedly induced during HIV-1 infection, but the conserved nature of these elements has, until recently, rendered their expression profiling problematic. Here, we provide comprehensive HERV-K HML-2 expression profiles specific for productively HIV-1-infected primary human CD4 + T cells. We combined enrichment of HIV-1 infected cells using a reporter virus expressing a surface reporter for gentle and efficient purification with long-read single-molecule real-time sequencing. We show that three HML-2 proviruses-6q25.1, 8q24.3, and 19q13.42-are upregulated on average between 3- and 5-fold in HIV-1-infected CD4 + T cells. One provirus, HML-2 12q24.33, in contrast, was repressed in the presence of active HIV replication. In conclusion, this report identifies the HERV-K HML-2 loci whose expression profiles differ upon HIV-1 infection in primary human CD4 + T cells. These data will help pave the way for further studies on the influence of endogenous retroviruses on HIV-1 replication. IMPORTANCE Endogenous retroviruses inhabit big portions of our genome. Moreover, although they are mainly inert, some of the evolutionarily younger members maintain the ability to express both RNA and proteins. We have developed an approach using long-read single-molecule real-time (SMRT) sequencing that produces long reads that allow us to obtain detailed and accurate HERV-K HML-2 expression profiles. We applied this approach to study HERV-K expression in the presence or absence of productive HIV-1 infection of primary human CD4 + T cells. In addition to using SMRT sequencing, our strategy also includes the magnetic selection of the infected cells so that levels of background expression due to uninfected cells are kept at a minimum. The results presented here provide a blueprint for in-depth studies of the interactions of the authentic upregulated HERV-K HML-2 elements and HIV-1. Copyright © 2017 American Society for Microbiology.

Probing the Effector and Suppressive Functions of Human T Cell Subsets Using Antigen-Specific Engineered T Cell Receptors

PubMed Central

Imberg, Keren; Mercer, Frances; Zhong, Shi; Krogsgaard, Michelle; Unutmaz, Derya

2013-01-01

Activation of T cells through the engagement of the T cell receptors (TCRs) with specific peptide-MHC complexes on antigen presenting cells (APCs) is the major determinant for their proliferation, differentiation and display of effector functions. To assess the role of quantity and quality of peptide-MHC presentation in eliciting T cell activation and suppression functions, we genetically engineered human T cells with two TCRs that recognize HLA-A*0201-restricted peptides derived from either HIV or melanoma antigens. The engineered-TCRs are highly functional in both CD8+ and CD4+ T cells as assessed by the upregulation of activation markers, induction of cytokine secretion and cytotoxicity. We further demonstrated that engineered-TCRs can also be expressed on naïve human T cells, which are stimulated through APCs presenting specific peptides to induce T cell proliferation and acquire effector functions. Furthermore, regulatory T cells (Tregs) ectopically expressing the engineered-TCRs are activated in an antigen-specific fashion and suppress T cell proliferation. In this system, the inhibitory activity of peptide-stimulated Tregs require the presence of dendritic cells (DCs) in the culture, either as presenters or as bystander cells, pointing to a critical role for DCs in suppression by Tregs. In conclusion, the engineered-TCR system reported here advances our ability to understand the differentiation pathways of naïve T cells into antigen-specific effector cells and the role of antigen-specific signaling in Treg-mediated immune suppression. PMID:23437112

In Vitro T-Cell Generation From Adult, Embryonic, and Induced Pluripotent Stem Cells: Many Roads to One Destination.

PubMed

Smith, Michelle J; Webber, Beau R; Mohtashami, Mahmood; Stefanski, Heather E; Zúñiga-Pflücker, Juan Carlos; Blazar, Bruce R

2015-11-01

T lymphocytes are critical mediators of the adaptive immune system and have the capacity to serve as therapeutic agents in the areas of transplant and cancer immunotherapy. While T cells can be isolated and expanded from patients, T cells derived in vitro from both hematopoietic stem/progenitor cells (HSPCs) and human pluripotent stem cells (hPSCs) offer great potential advantages in generating a self-renewing source of T cells that can be readily genetically modified. T-cell differentiation in vivo is a complex process requiring tightly regulated signals; providing the correct signals in vitro to induce T-cell lineage commitment followed by their development into mature, functional, single positive T cells, is similarly complex. In this review, we discuss current methods for the in vitro derivation of T cells from murine and human HSPCs and hPSCs that use feeder-cell and feeder-cell-free systems. Furthermore, we explore their potential for adoption for use in T-cell-based therapies. © 2015 AlphaMed Press.

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 hypothesis that Mφ play crucial roles for the lifelong maintenance and expansion of HCMV-committed T cells in the human host.

Ex vivo expansion of human umbilical cord blood-derived T-lymphocytes with homologous cord blood plasma.

PubMed

Kim, Yong-Man; Jung, Min-Hyung; Song, Ha-Young; Yang, Hyun Ok; Lee, Sung-Tae; Kim, Jong-Hyeok; Kim, Young-Tak; Nam, Joo-Hyun; Mok, Jung-Eun

2005-02-01

This study was designed to establish a more effective and safe culture system for adoptive immunotherapy by investigating the use of homologous cord blood plasma (HCBP) instead of fetal bovine serum (FBS), which has various limitations including ethical problems for the ex vivo expansion of human umbilical T lymphocytes. Fresh human umbilical mononuclear cell fractions were isolated by Ficoll-Hypaque density centrifugation. Nonadherent mononuclear cell fractions were cultured with anti-CD3 antibody (5 microg/ml), IL-2 (175 U/ml), and either 10% FBS or 10% HCBP. On day 8, the cellular proliferation rate and cell surface markers were assessed. There was no significant difference in proliferation when human umbilical cord blood T lymphocytes were grown in medium supplemented with FBS or HCBP (p > 0.05). In medium containing FBS, the proportion of CD3(+)CD4(+) (markers for helper T cell), CD3(+)CD8(+) (cytotoxic T cell), CD3(+)CD25(+) (activated T cell), CD3(+)CD38(+) (immature T cell), and CD3(+)CD45RO(+) (memory T cell) cells was significantly increased (p < 0.05), whereas proportion of CD3(+)CD45RA(+) (naive T cell) and CD16(+)CD56(+) (NK cell) cells was significantly decreased (p < 0.05). In HCBP supplemented medium, the proportion of CD3(+)CD8(+), CD3(+)CD25(+), CD3(+)CD45RA(+), and CD3(+)CD45RO(+) cells was significantly increased (p < 0.05). The proportion of CD3(+)CD4(+), CD3(+)CD45RO(+) and CD3(+)CD38(+) cells was significantly higher, but proportion of CD3(+)CD45RA(+) and CD3(+)CD8(+) cells was significantly lower in FBS compared with HCBP supplemented medium (p < 0.05). Our results support the feasibility of ex vivo expansion of human umbilical cord blood T lymphocytes in medium supplemented with HCBP for future adoptive cellular immunotherapy.

Human renal adipose tissue induces the invasion and progression of renal cell carcinoma

PubMed Central

Campo-Verde-Arbocco, Fiorella; López-Laur, José D.; Romeo, Leonardo R.; Giorlando, Noelia; Bruna, Flavia A.; Contador, David E.; López-Fontana, Gastón; Santiano, Flavia E.; Sasso, Corina V.; Zyla, Leila E.; López-Fontana, Constanza M.; Calvo, Juan C.; Carón, Rubén W.; Creydt, Virginia Pistone

2017-01-01

We evaluated the effects of conditioned media (CMs) of human adipose tissue from renal cell carcinoma located near the tumor (hRATnT) or farther away from the tumor (hRATfT), on proliferation, adhesion and migration of tumor (786-O and ACHN) and non-tumor (HK-2) human renal epithelial cell lines. Human adipose tissues were obtained from patients with renal cell carcinoma (RCC) and CMs from hRATnT and hRATfT incubation. Proliferation, adhesion and migration were quantified in 786-O, ACHN and HK-2 cell lines incubated with hRATnT-, hRATfT- or control-CMs. We evaluated versican, adiponectin and leptin expression in CMs from hRATnT and hRATfT. We evaluated AdipoR1/2, ObR, pERK, pAkt y pPI3K expression on cell lines incubated with CMs. No differences in proliferation of cell lines was found after 24 h of treatment with CMs. All cell lines showed a significant decrease in cell adhesion and increase in cell migration after incubation with hRATnT-CMs vs. hRATfT- or control-CMs. hRATnT-CMs showed increased levels of versican and leptin, compared to hRATfT-CMs. AdipoR2 in 786-O and ACHN cells decreased significantly after incubation with hRATfT- and hRATnT-CMs vs. control-CMs. We observed a decrease in the expression of pAkt in HK-2, 786-O and ACHN incubated with hRATnT-CMs. This result could partially explain the observed changes in migration and cell adhesion. We conclude that hRATnT released factors, such as leptin and versican, could enhance the invasive potential of renal epithelial cell lines and could modulate the progression of the disease. PMID:29212223

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

Bet v 1-specific T-cell receptor/forkhead box protein 3 transgenic T cells suppress Bet v 1-specific T-cell effector function in an activation-dependent manner.

PubMed

Schmetterer, Klaus G; Haiderer, Daniela; Leb-Reichl, Victoria M; Neunkirchner, Alina; Jahn-Schmid, Beatrice; Küng, Hans J; Schuch, Karina; Steinberger, Peter; Bohle, Barbara; Pickl, Winfried F

2011-01-01

Regulatory T (Treg) cells establish and maintain tolerance to self-antigens and many foreign antigens, such as allergens, by suppressing effector T-cell proliferation and function. We have previously shown that human T-cell receptor (TCR) αβ-chains specific for allergen-derived epitopes confer allergen specificity on peripheral blood T cells of individuals with and without allergy. To study the feasibility of generating allergen-specific human Treg cells by retroviral transduction of a transcription unit encoding forkhead box protein 3 (FOXP3) and allergen-specific TCR αβ-chains. cDNAs encoding the α and β-chains of a Bet v 1(142-153)-specific TCR (TCR alpha variable region 6/TCR beta variable region 20) and human FOXP3 were linked via picornaviral 2A sequences and expressed as single translational unit from an internal ribosomal entry site-green fluorescence protein-containing retroviral vector. Retrovirally transduced peripheral blood T cells were tested for expression of transgenes, Treg phenotype, and regulatory capacity toward allergen-specific effector T cells. Transduced T cells displayed a Treg phenotype with clear-cut upregulation of CD25, CD39, and cytotoxic T-lymphocyte antigen 4. The transduced cells were hyporesponsive in cytokine production and secretion and, like naturally occurring Treg cells, did not proliferate after antigen-specific or antigen-mimetic stimulation. However, proliferation was inducible upon exposure to exogenous IL-2. In coculture experiments, TRAV6(+)TRBV20(+)FOXP3(+) transgenic T cells, unlike FOXP3(+) single transgenic T cells or naturally occurring Treg cells, highly significantly suppressed T cell cytokine production and proliferation of corresponding allergen-specific effector T cells in an allergen-specific, dose-dependent manner. We demonstrate a transgenic approach to engineer human allergen-specific Treg cells that exert their regulatory function in an activation-dependent manner. Customized Treg cells might become useful for tolerance induction therapies in individuals with allergic and other immune-mediated diseases. Copyright © 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Heterogeneity of Human CD4(+) T Cells Against Microbes.

PubMed

Sallusto, Federica

2016-05-20

CD4(+) T helper (Th) cells play a central role in the adaptive immune response by providing help to B cells and cytotoxic T cells and by releasing different types of cytokines in tissues to mediate protection against a wide range of pathogenic microorganisms. These functions are performed by different types of Th cells endowed with distinct migratory capacities and effector functions. Here we discuss how studies of the human T cell response to microbes have advanced our understanding of Th cell functional heterogeneity, in particular with the discovery of a distinct Th1 subset involved in the response to Mycobacteria and the characterization of two types of Th17 cells specific for extracellular bacteria or fungi. We also review new approaches to dissect at the clonal level the human CD4(+) T cell response induced by pathogens or vaccines that have revealed an unexpected degree of intraclonal diversification and propose a progressive and selective model of CD4(+) T cell differentiation.

Effects of PVA-coated nanoparticles on human T helper cell activity.

PubMed

Strehl, Cindy; Schellmann, Saskia; Maurizi, Lionel; Hofmann-Amtenbrink, Margarethe; Häupl, Thomas; Hofmann, Heinrich; Buttgereit, Frank; Gaber, Timo

2016-03-14

Superparamagnetic iron oxide nanoparticles (SPION) are used as high-sensitive enhancer for magnetic resonance imaging, where they represent a promising tool for early diagnosis of destructive diseases such as rheumatoid arthritis (RA). Since we could demonstrate that professional phagocytes are activated by amino-polyvinyl-alcohol-coated-SPION (a-PVA-SPION), the study here focuses on the influence of a-PVA-SPION on human T cells activity. Therefore, primary human CD4+ T cells from RA patients and healthy subjects were treated with varying doses of a-PVA-SPION for 20h or 72h. T cells were then analyzed for apoptosis, cellular energy, expression of the activation marker CD25 and cell proliferation. Although, we observed that T cells from RA patients are more susceptible to low-dose a-PVA-SPION-induced apoptosis than T cells from healthy subjects, in both groups a-PVA-SPION do not activate CD4+ T cells per se and do not influence mitogen-mediated T cells activation with regard to CD25 expression and cell proliferation. Nevertheless, our results demonstrate that CD4+ T cells from RA patients and healthy subjects differ in their response to mitogen stimulation and oxygen availability. We conclude from our data, that a-PVA-SPION do neither activate nor significantly influence mitogen-stimulated CD4+ T cells activation and have negligible influence on T cells apoptosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

p53 Dependent Centrosome Clustering Prevents Multipolar Mitosis in Tetraploid Cells

PubMed Central

Yi, Qiyi; Zhao, Xiaoyu; Huang, Yun; Ma, Tieliang; Zhang, Yingyin; Hou, Heli; Cooke, Howard J.; Yang, Da-Qing; Wu, Mian; Shi, Qinghua

2011-01-01

Background p53 abnormality and aneuploidy often coexist in human tumors, and tetraploidy is considered as an intermediate between normal diploidy and aneuploidy. The purpose of this study was to investigate whether and how p53 influences the transformation from tetraploidy to aneuploidy. Principal Findings Live cell imaging was performed to determine the fates and mitotic behaviors of several human and mouse tetraploid cells with different p53 status, and centrosome and spindle immunostaining was used to investigate centrosome behaviors. We found that p53 dominant-negative mutation, point mutation, or knockout led to a 2∼ 33-fold increase of multipolar mitosis in N/TERT1, 3T3 and mouse embryonic fibroblasts (MEFs), while mitotic entry and cell death were not significantly affected. In p53-/- tetraploid MEFs, the ability of centrosome clustering was compromised, while centrosome inactivation was not affected. Suppression of RhoA/ROCK activity by specific inhibitors in p53-/- tetraploid MEFs enhanced centrosome clustering, decreased multipolar mitosis from 38% to 20% and 16% for RhoA and ROCK, respectively, while expression of constitutively active RhoA in p53+/+ tetraploid 3T3 cells increased the frequency of multipolar mitosis from 15% to 35%. Conclusions p53 could not prevent tetraploid cells entering mitosis or induce tetraploid cell death. However, p53 abnormality impaired centrosome clustering and lead to multipolar mitosis in tetraploid cells by modulating the RhoA/ROCK signaling pathway. PMID:22076149

Sympathetic neural signaling via the β2-adrenergic receptor suppresses T-cell receptor-mediated human and mouse CD8(+) T-cell effector function.

PubMed

Estrada, Leonardo D; Ağaç, Didem; Farrar, J David

2016-08-01

Postganglionic sympathetic neurons innervate secondary lymphoid organs and secrete norepinephrine (NE) as the primary neurotransmitter. NE binds and signals through five distinct members of the adrenergic receptor family. In this study, we show elevated expression of the β2-adrenergic receptor (ADRB2) on primary human CD8(+) effector memory T cells. Treatment of both human and murine CD8(+) T cells with NE decreased IFN-γ and TNF-α secretion and suppressed their cytolytic capacity in response to T-cell receptor (TCR) activation. The effects of NE were specifically reversed by β2-specific antagonists. Adrb2(-/-) CD8(+) T cells were completely resistant to the effects of NE. Further, the ADRB2-specific pharmacological ligand, albuterol, significantly suppressed effector functions in both human and mouse CD8(+) T cells. While both TCR activation and stimulation with IL-12 + IL-18 were able to induce inflammatory cytokine secretion, NE failed to suppress IFN-γ secretion in response to IL-12 + IL18. Finally, the long-acting ADRB2-specific agonist, salmeterol, markedly reduced the cytokine secretion capacity of CD8(+) T cells in response to infection with vesicular stomatitis virus. This study reveals a novel intrinsic role for ADRB2 signaling in CD8(+) T-cell function and underscores the novel role this pathway plays in adaptive T-cell responses to infection. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pooled thrombin-activated platelet-rich plasma: a substitute for fetal bovine serum in the engineering of osteogenic/vasculogenic grafts.

PubMed

Tchang, Laurent A; Pippenger, Benjamin E; Todorov, Atanas; Wolf, Francine; Burger, Maximilian G; Jaquiery, Claude; Bieback, Karen; Martin, Ivan; Schaefer, Dirk J; Scherberich, Arnaud

2017-05-01

The use of fetal bovine serum (FBS) as a culture medium supplement in cell therapy and clinical tissue engineering is challenged by immunological concerns and the risk of disease transmission. Here we tested whether human, thrombin-activated, pooled, platelet-rich plasma (tPRP) can be substituted for FBS in the engineering of osteogenic and vasculogenic grafts, using cells from the stromal vascular fraction (SVF) of human adipose tissue. SVF cells were cultured under perfusion flow into porous hydroxyapatite scaffolds for 5 days, with the medium supplemented with either 10% tPRP or 10% FBS and implanted in an ectopic mouse model. Following in vitro culture, as compared to FBS, the use of tPRP did not modify the fraction of clonogenic cells or the different cell phenotypes, but increased by 1.9-fold the total number of cells. After 8 weeks in vivo, bone tissue was formed more reproducibly and in higher amounts (3.7-fold increase) in constructs cultured with tPRP. Staining for human-specific ALU sequences and for the human isoforms of CD31/CD34 revealed the human origin of the bone, the formation of blood vessels by human vascular progenitors and a higher density of human cells in implants cultured with tPRP. In summary, tPRP supports higher efficiency of bone formation by SVF cells than FBS, likely by enhancing cell expansion in vitro while maintaining vasculogenic properties. The use of tPRP may facilitate the clinical translation of osteogenic grafts with intrinsic capacity for vascularization, based on the use of adipose-derived cells. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Clinical and Mucosal Immune Correlates of HIV-1 Semen Levels in Antiretroviral-Naive Men

PubMed Central

Marsh, Angie K.; Huibner, Sanja; Shahabi, Kamnoosh; Liu, Cindy; Contente, Tania; Nagelkerke, Nico J. D.; Kovacs, Colin; Benko, Erika; Price, Lance; MacDonald, Kelly S.; Kaul, Rupert

2017-01-01

Abstract Background. This study was done to characterize parameters associated with semen human immunodeficiency virus (HIV)-1 ribonucleic acid (RNA) viral load (VL) variability in HIV-infected, therapy-naive men. Methods. Paired blood and semen samples were collected from 30 HIV-infected, therapy-naive men who have sex with men, and 13 participants were observed longitudinally for up to 1 year. Human immunodeficiency virus RNA, bacterial load by 16S RNA, herpesvirus (Epstein-Barr virus and cytomegalovirus [CMV]) shedding, and semen cytokines/chemokines were quantified, and semen T-cell subsets were assessed by multiparameter flow cytometry. Results. Semen HIV RNA was detected at 93% of visits, with >50% of men shedding high levels of virus (defined as >5000 copies/mL). In the baseline cross-sectional analysis, an increased semen HIV VL correlated with local CMV reactivation, the semen bacterial load, and semen inflammatory cytokines, particularly interleukin (IL)-8. T cells in semen were more activated than blood, and there was an increased frequency of Th17 cells and γδ-T-cells. Subsequent prospective analysis demonstrated striking interindividual variability in HIV and CMV shedding patterns, and only semen IL-8 levels and the blood VL were independently associated with semen HIV levels. Conclusions. Several clinical and immune parameters were associated with increased HIV semen levels in antiretroviral therapy-naive men, with induction of local proinflammatory cytokines potentially acting as a common pathway. PMID:28534034

Red blood cells inhibit activation-induced cell death and oxidative stress in human peripheral blood T lymphocytes.

PubMed

Fonseca, A M; Porto, G; Uchida, K; Arosa, F A

2001-05-15

Red blood cells (RBCs) are known to perform one prominent function: to carry and deliver oxygen to the tissues. Earlier studies, however, suggested a role for RBCs in potentiating T-cell proliferation in vitro. Here it is shown that the presence of RBCs in cultures of stimulated human peripheral blood lymphocytes strengthens T-cell proliferation and survival. Analysis of phosphatidylserine externalization and DNA fragmentation showed that RBCs inhibit T-cell apoptosis. This inhibition correlated with a reduction in CD71 but not CD95 expression. RBCs enhanced T-cell proliferation and survival upon activation with phytohemagglutinin and with OKT3 antibodies. Studies aimed at characterizing the cellular and molecular basis of the protection afforded to T cells by RBCs showed that (1) optimal protection required intact RBCs and red cell/T-cell contact but not monocytes; (2) RBCs markedly reduced the level of intracellular reactive oxygen species; and (3) RBCs inhibited the formation of protein-bound acrolein, a peroxidation adduct in biologic systems. Overall, these data indicate that human RBCs protect T cells from activation-induced cell death, at least in part by reducing the pro-oxidant state, and suggest a role for RBCs as conceivable modulators of T-cell homeostasis.

Human T lymphocytes express N-methyl-D-aspartate receptors functionally active in controlling T cell activation

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

Miglio, Gianluca; Varsaldi, Federica; Lombardi, Grazia

2005-12-30

The aim of this study was to investigate the expression and the functional role of N-methyl-D-aspartate (NMDA) receptors in human T cells. RT-PCR analysis showed that human resting peripheral blood lymphocytes (PBL) and Jurkat T cells express genes encoding for both NR1 and NR2B subunits: phytohemagglutinin (PHA)-activated PBL also expresses both these genes and the NR2A and NR2D genes. Cytofluorimetric analysis showed that NR1 expression increases as a consequence of PHA (10 {mu}g/ml) treatment. D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5), and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine [(+)-MK 801], competitive and non-competitive NMDA receptor antagonists, respectively, inhibited PHA-induced T cell proliferation, whereas they did not affect IL-2 (10more » U/ml)-induced proliferation of PHA blasts. These effects were due to the prevention of T cell activation (inhibition of cell aggregate formation and CD25 expression), but not to cell cycle arrest or death. These results demonstrate that human T lymphocytes express NMDA receptors, which are functionally active in controlling cell activation.« less

Regulation of human intestinal T-cell responses by type 1 interferon-STAT1 signaling is disrupted in inflammatory bowel disease.

PubMed

Giles, E M; Sanders, T J; McCarthy, N E; Lung, J; Pathak, M; MacDonald, T T; Lindsay, J O; Stagg, A J

2017-01-01

Type 1 interferon (IFN-1) promotes regulatory T-cell function to suppress inflammation in the mouse intestine, but little is known about IFN-1 in the human gut. We therefore assessed the influence of IFN-1 on CD4+ T-cells isolated from human colon tissue obtained from healthy controls or patients with inflammatory bowel disease (IBD). Immunofluorescent imaging revealed constitutive expression of IFNβ in human intestinal tissue, and colonic T-cells were responsive to exogenous IFN-1 as assessed by phosphorylation of signal transduction and activator of transcription 1 (pSTAT1) and induction of interferon stimulated genes (ISGs). Unlike their blood counterparts, intestinal T-cells from non-inflamed regions of IBD colon displayed enhanced responsiveness to IFN-1, increased frequency of pSTAT1+ cells, and greater induction of ISGs upon IFN-1 exposure in vitro. In healthy tissue, antibody neutralization of IFNβ selectively reduced T-cell production of the pro-regulatory cytokine interleukin-10 (IL-10) and increased IFNγ synthesis. In contrast, neutralization of IFNβ in IBD tissue cultures increased the frequency of T-cells producing inflammatory cytokines but did not alter IL-10 expression. These data support a role for endogenous IFN-1 as a context-dependent modulator of T-cell function that promotes regulatory activity in healthy human intestine, but indicate that the IFN-1/STAT1 pathway is dysregulated in inflammatory bowel disease.

Immune dysregulation in human subjects with heterozygous germline mutations in CTLA4

PubMed Central

Deenick, Elissa K.; Niemela, Julie E.; Avery, Danielle T.; Schickel, Jean-Nicolas; Tran, Dat Q.; Stoddard, Jennifer; Zhang, Yu; Frucht, David M.; Dumitriu, Bogdan; Scheinberg, Phillip; Folio, Les R.; Frein, Cathleen A.; Price, Susan; Koh, Christopher; Heller, Theo; Seroogy, Christine M.; Huttenlocher, Anna; Rao, V. Koneti; Su, Helen C.; Kleiner, David; Notarangelo, Luigi D.; Rampertaap, Yajesh; Olivier, Kenneth N.; McElwee, Joshua; Hughes, Jason; Pittaluga, Stefania; Oliveira, Joao B.; Meffre, Eric; Fleisher, Thomas A.; Holland, Steven M.; Lenardo, Michael J.; Tangye, Stuart G.; Uzel, Gulbu

2015-01-01

Cytotoxic T lymphocyte antigen–4 (CTLA-4) is an inhibitory receptor found on immune cells. The consequences of mutations in CTLA4 in humans are unknown. We identified germline heterozygous mutations in CTLA4 in subjects with severe immune dysregulation from four unrelated families. Whereas Ctla4 heterozygous mice have no obvious phenotype, human CTLA4 haploinsufficiency caused dysregulation of FoxP3+ regulatory T (Treg) cells, hyperactivation of effector T cells, and lymphocytic infiltration of target organs. Patients also exhibited progressive loss of circulating B cells, associated with an increase of predominantly autoreactive CD21lo B cells and accumulation of B cells in nonlymphoid organs. Inherited human CTLA4 haploinsufficiency demonstrates a critical quantitative role for CTLA-4 in governing T and B lymphocyte homeostasis. PMID:25213377

Physiologic and Genetic Evidence Links Hemopexin to Triglycerides in Mice and Humans

PubMed Central

Lawson, Heather A; Zayed, Mohamed; Wayhart, Jessica P; Fabbrini, Elisa; Love-Gregory, Latisha; Klein, Samuel; Semenkovich, Clay F

2017-01-01

Background/Objectives Elevated triglycerides predict insulin resistance and vascular disease in obesity, but how the inert triglyceride molecule is related to development of metabolic disease is unknown. To pursue novel potential mediators of triglyceride-associated metabolic disease, we used a forward genetics approach involving inbred mice and translated our findings to human subjects. Subjects/Methods Hemopexin was identified as a differentially expressed gene within a quantitative trait locus associated with serum triglycerides in an F16 advanced intercross between the LG/J and SM/J strains of mice. Hpx expression was evaluated in both reproductive fatpads and livers of mice representing three strains, LG/J (n = 25), SM/J (n = 27) and C57Bl/6J (n = 19), on high- and low-fat diets. The effect of altered Hpx expression on adipogenesis was studied in 3T3-L1 cells. Circulating HPX protein along with HPX expression were characterized in subcutaneous white adipose tissue samples obtained from a cohort of metabolically abnormal (n = 18) and of metabolically normal (n = 24) obese human subjects. We further examined the relationship between HPX and triglycerides in human atherosclerotic plaques (n = 18). Results Hemopexin expression in mouse adipose tissue, but not liver, was regulated by dietary fat regardless of genetic background. Hemopexin increased in concert with adipogenesis in 3T3-L1 cells, and disruption of its expression impaired adipocyte differentiation. RNAseq data from the adipose tissue of obese humans showed differential expression of hemopexin based on metabolic disease status (p < 0.05), and circulating hemopexin levels were correlated with serum triglycerides in these subjects (r = 0.33; p = 0.03). Hemopexin was also found in an unbiased proteomic screen of human atherosclerotic plaques, and shown to display differential abundance based on extent of disease and triglyceride content (p < 0.05). Conclusions Our findings suggest that hemopexin is associated with triglycerides and provide a framework for understanding mechanisms underlying lipid metabolism and metabolic disease. PMID:28119529

STAT3 is a critical cell-intrinsic regulator of human unconventional T cell numbers and function

PubMed Central

Wilson, Robert P.; Ives, Megan L.; Rao, Geetha; Lau, Anthony; Payne, Kathryn; Kobayashi, Masao; Arkwright, Peter D.; Peake, Jane; Wong, Melanie; Adelstein, Stephen; Smart, Joanne M.; French, Martyn A.; Fulcher, David A.; Picard, Capucine; Bustamante, Jacinta; Boisson-Dupuis, Stephanie; Gray, Paul; Stepensky, Polina; Warnatz, Klaus; Freeman, Alexandra F.; Rossjohn, Jamie; McCluskey, James; Holland, Steven M.; Casanova, Jean-Laurent; Uzel, Gulbu; Ma, Cindy S.

2015-01-01

Unconventional T cells such as γδ T cells, natural killer T cells (NKT cells) and mucosal-associated invariant T cells (MAIT cells) are a major component of the immune system; however, the cytokine signaling pathways that control their development and function in humans are unknown. Primary immunodeficiencies caused by single gene mutations provide a unique opportunity to investigate the role of specific molecules in regulating human lymphocyte development and function. We found that individuals with loss-of-function mutations in STAT3 had reduced numbers of peripheral blood MAIT and NKT but not γδ T cells. Analysis of STAT3 mosaic individuals revealed that this effect was cell intrinsic. Surprisingly, the residual STAT3-deficient MAIT cells expressed normal levels of the transcription factor RORγt. Despite this, they displayed a deficiency in secretion of IL-17A and IL-17F, but were able to secrete normal levels of cytokines such as IFNγ and TNF. The deficiency in MAIT and NKT cells in STAT3-deficient patients was mirrored by loss-of-function mutations in IL12RB1 and IL21R, respectively. Thus, these results reveal for the first time the essential role of STAT3 signaling downstream of IL-23R and IL-21R in controlling human MAIT and NKT cell numbers. PMID:25941256

Carbon Nanotubes and Human Cells?

ERIC Educational Resources Information Center

King, G. Angela

2005-01-01

Single-walled carbon nanotubes that were chemically altered to be water soluble are shown to enter fibroblasts, T cells, and HL60 cells. Nanoparticles adversely affect immortalized HaCaT human keratinocyte cultures, indicating that they may enter cells.

Identity and Diversity of Human Peripheral Th and T Regulatory Cells Defined by Single-Cell Mass Cytometry.

PubMed

Kunicki, Matthew A; Amaya Hernandez, Laura C; Davis, Kara L; Bacchetta, Rosa; Roncarolo, Maria-Grazia

2018-01-01

Human CD3 + CD4 + Th cells, FOXP3 + T regulatory (Treg) cells, and T regulatory type 1 (Tr1) cells are essential for ensuring peripheral immune response and tolerance, but the diversity of Th, Treg, and Tr1 cell subsets has not been fully characterized. Independent functional characterization of human Th1, Th2, Th17, T follicular helper (Tfh), Treg, and Tr1 cells has helped to define unique surface molecules, transcription factors, and signaling profiles for each subset. However, the adequacy of these markers to recapitulate the whole CD3 + CD4 + T cell compartment remains questionable. In this study, we examined CD3 + CD4 + T cell populations by single-cell mass cytometry. We characterize the CD3 + CD4 + Th, Treg, and Tr1 cell populations simultaneously across 23 memory T cell-associated surface and intracellular molecules. High-dimensional analysis identified several new subsets, in addition to the already defined CD3 + CD4 + Th, Treg, and Tr1 cell populations, for a total of 11 Th cell, 4 Treg, and 1 Tr1 cell subsets. Some of these subsets share markers previously thought to be selective for Treg, Th1, Th2, Th17, and Tfh cells, including CD194 (CCR4) + FOXP3 + Treg and CD183 (CXCR3) + T-bet + Th17 cell subsets. Unsupervised clustering displayed a phenotypic organization of CD3 + CD4 + T cells that confirmed their diversity but showed interrelation between the different subsets, including similarity between Th1-Th2-Tfh cell populations and Th17 cells, as well as similarity of Th2 cells with Treg cells. In conclusion, the use of single-cell mass cytometry provides a systems-level characterization of CD3 + CD4 + T cells in healthy human blood, which represents an important baseline reference to investigate abnormalities of different subsets in immune-mediated pathologies. Copyright © 2017 by The American Association of Immunologists, Inc.

Androgen-Induced Cell Migration: Role of Androgen Receptor/Filamin A Association

PubMed Central

Castoria, Gabriella; D'Amato, Loredana; Ciociola, Alessandra; Giovannelli, Pia; Giraldi, Tiziana; Sepe, Leandra; Paolella, Giovanni; Barone, Maria Vittoria; Migliaccio, Antimo; Auricchio, Ferdinando

2011-01-01

Background Androgen receptor (AR) controls male morphogenesis, gametogenesis and prostate growth as well as development of prostate cancer. These findings support a role for AR in cell migration and invasiveness. However, the molecular mechanism involved in AR-mediated cell migration still remains elusive. Methodology/Principal Findings Mouse embryo NIH3T3 fibroblasts and highly metastatic human fibrosarcoma HT1080 cells harbor low levels of transcriptionally incompetent AR. We now report that, through extra nuclear action, AR triggers migration of both cell types upon stimulation with physiological concentrations of the androgen R1881. We analyzed the initial events leading to androgen-induced cell migration and observed that challenging NIH3T3 cells with 10 nM R1881 rapidly induces interaction of AR with filamin A (FlnA) at cytoskeleton. AR/FlnA complex recruits integrin beta 1, thus activating its dependent cascade. Silencing of AR, FlnA and integrin beta 1 shows that this ternary complex controls focal adhesion kinase (FAK), paxillin and Rac, thereby driving cell migration. FAK-null fibroblasts migrate poorly and Rac inhibition by EHT impairs motility of androgen-treated NIH3T3 cells. Interestingly, FAK and Rac activation by androgens are independent of each other. Findings in human fibrosarcoma HT1080 cells strengthen the role of Rac in androgen signaling. The Rac inhibitor significantly impairs androgen-induced migration in these cells. A mutant AR, deleted of the sequence interacting with FlnA, fails to mediate FAK activation and paxillin tyrosine phosphorylation in androgen-stimulated cells, further reinforcing the role of AR/FlnA interaction in androgen-mediated motility. Conclusions/Significance The present report, for the first time, indicates that the extra nuclear AR/FlnA/integrin beta 1 complex is the key by which androgen activates signaling leading to cell migration. Assembly of this ternary complex may control organ development and prostate cancer metastasis. PMID:21359179

HTLV-I Tax-dependent and -independent events associated with immortalization of human primary T lymphocytes

PubMed Central

Bellon, Marcia; Baydoun, Hicham H.; Yao, Yuan

2010-01-01

Human T-cell leukemia virus type I (HTLV-I)–associated malignancies are seen in a small percentage of infected persons. Although in vitro immortalization by HTLV-I virus is very efficient, we report that Tax has poor oncogenic activity in human primary T cells and that immortalization by Tax is rare. Sustained telomerase activity represents one of the oncogenic steps required for Tax-mediated immortalization. Tax expression was required for the growth of primary T cells, but was not sufficient to propel T cells into cell cycle in the absence of exogenous interleukin-2 (IL-2). Tax was sufficient to activate the phosphoinositide-3 kinase (PI3K)/Akt pathway as shown by down regulation of Src homology phosphatase-1 and increased phosphorylation of Akt. We also found disruption of putative tumor suppressors IL-16 and translocated promoter region (TPR) in Tax-immortalized and HTLV-I–transformed cell lines. Our results confirmed previous observations that Tax activates the anaphase-promoting complex. However, Tax did not affect the mitotic spindle checkpoint, which was also functional in HTLV-I–transformed cells. These data provide a better understanding of Tax functions in human T cells, and highlight the limitations of Tax, suggesting that other viral proteins are key to T-cell transformation and development of adult T-cell leukemia. PMID:20093405

Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

PubMed Central

Malik, P; McQuiston, S A; Yu, X J; Pepper, K A; Krall, W J; Podsakoff, G M; Kurtzman, G J; Kohn, D B

1997-01-01

We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate for gene therapy of nondividing cells, a very high MOI or improvements in basic aspects of AAV-based vectors may be necessary to improve integration frequency in the rapidly dividing hematopoietic cell population. PMID:9032306

Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

PubMed

Malik, P; McQuiston, S A; Yu, X J; Pepper, K A; Krall, W J; Podsakoff, G M; Kurtzman, G J; Kohn, D B

1997-03-01

We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate for gene therapy of nondividing cells, a very high MOI or improvements in basic aspects of AAV-based vectors may be necessary to improve integration frequency in the rapidly dividing hematopoietic cell population.

Improving T-cell expansion and function for adoptive T-cell therapy using ex vivo treatment with PI3Kδ inhibitors and VIP antagonists

PubMed Central

Petersen, Christopher T.; Hassan, Mojibade; Morris, Anna B.; Jeffery, Jasmin; Lee, Kunhee; Jagirdar, Neera; Staton, Ashley D.; Raikar, Sunil S.; Spencer, Harold T.; Sulchek, Todd; Flowers, Christopher R.

2018-01-01

Adoptive therapy with ex vivo–expanded genetically modified antigen-specific T cells can induce remissions in patients with relapsed/refractory cancer. The clinical success of this therapy depends upon efficient transduction and expansion of T cells ex vivo and their homing, persistence and cytotoxicity following reinfusion. Lower rates of ex vivo expansion and clinical response using anti-CD19 chimeric antigen receptor (CAR) T cells have been seen in heavily pretreated lymphoma patients compared with B-cell acute lymphoblastic leukemia patients and motivate the development of novel strategies to enhance ex vivo T cell expansion and their persistence in vivo. We demonstrate that inhibition of phosphatidylinositol 3-kinase δ (PI3Kδ) and antagonism of vasoactive intestinal peptide (VIP) signaling partially inhibits the terminal differentiation of T cells during anti-CD3/CD28 bead-mediated expansion (mean, 54.4% CD27+CD28+ T cells vs 27.4% in control cultures; P < .05). This strategy results in a mean of 83.7% more T cells cultured from lymphoma patients in the presence of PI3Kδ and VIP antagonists, increased survival of human T cells from a lymphoma patient in a murine xenograft model, enhanced cytotoxic activity of antigen-specific human CAR T cells and murine T cells against lymphoma, and increased transduction and expansion of anti-CD5 human CAR T cells. PI3Kδ and VIP antagonist-expanded T cells from lymphoma patients show reduced terminal differentiation, enhanced polyfunctional cytokine expression, and preservation of costimulatory molecule expression. Taken together, synergistic blockade of these pathways is an attractive strategy to enhance the expansion and functional capacity of ex vivo–expanded cancer-specific T cells. PMID:29386194

Human umbilical cord mesenchymal stem cells increase interleukin-9 production of CD4+ T cells

PubMed Central

Yang, Zhou Xin; Chi, Ying; Ji, Yue Ru; Wang, You Wei; Zhang, Jing; Luo, Wei Feng; Li, Li Na; Hu, Cai Dong; Zhuo, Guang Sheng; Wang, Li Fang; Han, Zhi-Bo; Han, Zhong Chao

2017-01-01

Mesenchymal stem cells (MSC) are able to differentiate into cells of multiple lineage, and additionally act to modulate the immune response. Interleukin (IL)-9 is primarily produced by cluster of differentiation (CD)4+ T cells to regulate the immune response. The present study aimed to investigate the effect of human umbilical cord derived-MSC (UC-MSC) on IL-9 production of human CD4+ T cells. It was demonstrated that the addition of UC-MSC to the culture of CD4+ T cells significantly enhanced IL-9 production by CD4+ T cells. Transwell experiments suggested that UC-MSC promotion of IL-9 production by CD4+ T cells was dependent on cell-cell contact. Upregulated expression of CD106 was observed in UC-MSC co-cultured with CD4+ T cells, and the addition of a blocking antibody of CD106 significantly impaired the ability of UC-MSC to promote IL-9 production by CD4+ T cells. Therefore, the results of the present study demonstrated that UC-MSC promoted the generation of IL-9 producing cells, which may be mediated, in part by CD106. The findings may act to expand understanding and knowledge of the immune modulatory role of UC-MSC. PMID:29042945

Wip1 and p53 contribute to HTLV-1 Tax-induced tumorigenesis

PubMed Central

2012-01-01

Background Human T-cell Leukemia Virus type 1 (HTLV-1) infects 20 million individuals world-wide and causes Adult T-cell Leukemia/Lymphoma (ATLL), a highly aggressive T-cell cancer. ATLL is refractory to treatment with conventional chemotherapy and fewer than 10% of afflicted individuals survive more than 5 years after diagnosis. HTLV-1 encodes a viral oncoprotein, Tax, that functions in transforming virus-infected T-cells into leukemic cells. All ATLL cases are believed to have reduced p53 activity although only a minority of ATLLs have genetic mutations in their p53 gene. It has been suggested that p53 function is inactivated by the Tax protein. Results Using genetically altered mice, we report here that Tax expression does not achieve a functional equivalence of p53 inactivation as that seen with genetic mutation of p53 (i.e. a p53−/− genotype). Thus, we find statistically significant differences in tumorigenesis between Tax+p53+/+versus Tax+p53−/− mice. We also find a role contributed by the cellular Wip1 phosphatase protein in tumor formation in Tax transgenic mice. Notably, Tax+Wip1−/− mice show statistically significant reduced prevalence of tumorigenesis compared to Tax+Wip1+/+ counterparts. Conclusions Our findings provide new insights into contributions by p53 and Wip1 in the in vivo oncogenesis of Tax-induced tumors in mice. PMID:23256545

Human Epidermal Langerhans Cells Maintain Immune Homeostasis in Skin by Activating Skin Resident Regulatory T Cells

PubMed Central

Seneschal, Julien; Clark, Rachael A.; Gehad, Ahmed; Baecher-Allan, Clare M.; Kupper, Thomas S.

2013-01-01

Recent discoveries indicate that the skin of a normal individual contains 10-20 billion resident memory T cells ( which include various T helper, T cytotoxic, and T regulatory subsets, that are poised to respond to environmental antigens. Using only autologous human tissues, we report that both in vitro and in vivo, resting epidermal Langerhan cells (LC) selectively and specifically induced the activation and proliferation of skin resident regulatory T cells (Treg), a minor subset of skin resident memory T cells. In the presence of foreign pathogen, however, the same LC activated and induced proliferation of effector memory T (Tem) cells and limited Treg cells activation. These underappreciated properties of LC: namely maintenance of tolerance in normal skin, and activation of protective skin resident memory T cells upon infectious challenge, help clarify the role of LC in skin. PMID:22560445

Autoreactive T Cells and Chronic Fungal Infection Drive Esophageal Carcinogenesis

PubMed Central

Zhu, Feng; Willette-Brown, Jami; Song, Na-Young; Lomada, Dakshayani; Song, Yongmei; Xue, Liyan; Gray, Zane; Zhao, Zitong; Davis, Sean R.; Sun, Zhonghe; Zhang, Peilin; Wu, Xiaolin; Zhan, Qimin; Richie, Ellen R.; Hu, Yinling

2018-01-01

SUMMARY Humans with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a T cell–driven autoimmune disease caused by impaired central tolerance, are susceptible to developing chronic fungal infection and esophageal squamous cell carcinoma (ESCC). However, the relationship between autoreactive T cells and chronic fungal infection in ESCC development remains unclear. We find that kinase-dead Ikkα knockin mice develop phenotypes reminiscent of APECED, including impaired central tolerance, autoreactive T cells, chronic fungal infection, and ESCCs expressing specific human ESCC markers. Using this model, we investigated the potential link between ESCC and fungal infection. Autoreactive CD4 T cells permit fungal infection and incite tissue injury and inflammation. Antifungal treatment or depletion of autoreactive CD4 T cells rescues, whereas oral fungal administration promotes, ESCC development. Inhibition of inflammation or EGFR activity decreases fungal burden. Importantly, fungal infection is highly associated with ESCCs in non-autoimmune human patients. Therefore, autoreactive T cells and chronic fungal infection, fostered by inflammation and epithelial injury, promote ESCC development. PMID:28407484

Origin and differentiation of human memory CD8 T cells after vaccination.

PubMed

Akondy, Rama S; Fitch, Mark; Edupuganti, Srilatha; Yang, Shu; Kissick, Haydn T; Li, Kelvin W; Youngblood, Ben A; Abdelsamed, Hossam A; McGuire, Donald J; Cohen, Kristen W; Alexe, Gabriela; Nagar, Shashi; McCausland, Megan M; Gupta, Satish; Tata, Pramila; Haining, W Nicholas; McElrath, M Juliana; Zhang, David; Hu, Bin; Greenleaf, William J; Goronzy, Jorg J; Mulligan, Mark J; Hellerstein, Marc; Ahmed, Rafi

2017-12-21

The differentiation of human memory CD8 T cells is not well understood. Here we address this issue using the live yellow fever virus (YFV) vaccine, which induces long-term immunity in humans. We used in vivo deuterium labelling to mark CD8 T cells that proliferated in response to the virus and then assessed cellular turnover and longevity by quantifying deuterium dilution kinetics in YFV-specific CD8 T cells using mass spectrometry. This longitudinal analysis showed that the memory pool originates from CD8 T cells that divided extensively during the first two weeks after infection and is maintained by quiescent cells that divide less than once every year (doubling time of over 450 days). Although these long-lived YFV-specific memory CD8 T cells did not express effector molecules, their epigenetic landscape resembled that of effector CD8 T cells. This open chromatin profile at effector genes was maintained in memory CD8 T cells isolated even a decade after vaccination, indicating that these cells retain an epigenetic fingerprint of their effector history and remain poised to respond rapidly upon re-exposure to the pathogen.

CXCL4 is a novel inducer of human Th17 cells and correlates with IL-17 and IL-22 in psoriatic arthritis.

PubMed

Affandi, Alsya J; Silva-Cardoso, Sandra C; Garcia, Samuel; Leijten, Emmerik F A; van Kempen, Tessa S; Marut, Wioleta; van Roon, Joel A G; Radstake, Timothy R D J

2018-03-01

CXCL4 regulates multiple facets of the immune response and is highly upregulated in various Th17-associated rheumatic diseases. However, whether CXCL4 plays a direct role in the induction of IL-17 production by human CD4 + T cells is currently unclear. Here, we demonstrated that CXCL4 induced human CD4 + T cells to secrete IL-17 that co-expressed IFN-γ and IL-22, and differentiated naïve CD4 + T cells to become Th17-cytokine producing cells. In a co-culture system of human CD4 + T cells with monocytes or myeloid dendritic cells, CXCL4 induced IL-17 production upon triggering by superantigen. Moreover, when monocyte-derived dendritic cells were differentiated in the presence of CXCL4, they orchestrated increased levels of IL-17, IFN-γ, and proliferation by CD4 + T cells. Furthermore, the CXCL4 levels in synovial fluid from psoriatic arthritis patients strongly correlated with IL-17 and IL-22 levels. A similar response to CXCL4 of enhanced IL-17 production by CD4 + T cells was also observed in patients with psoriatic arthritis. Altogether, we demonstrate that CXCL4 boosts pro-inflammatory cytokine production especially IL-17 by human CD4 + T cells, either by acting directly or indirectly via myeloid antigen presenting cells, implicating a role for CXCL4 in PsA pathology. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

PubMed Central

Santana, Vinicius Canato; Almeida, Rafael Ribeiro; Ribeiro, Susan Pereira; Ferreira, Luís Carlos de Souza; Kalil, Jorge; Rosa, Daniela Santoro; Cunha-Neto, Edecio

2015-01-01

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity. PMID:26602876

A novel p53 mutational hotspot in skin tumors from UV-irradiated Xpc mutant mice alters transactivation functions.

PubMed

Inga, Alberto; Nahari, Dorit; Velasco-Miguel, Susana; Friedberg, Errol C; Resnick, Michael A

2002-08-22

A mutation in codon 122 of the mouse p53 gene resulting in a T to L amino acid substitution (T122-->L) is frequently associated with skin cancer in UV-irradiated mice that are both homozygous mutant for the nucleotide excision repair (NER) gene Xpc (Xpc(-/-)) and hemizygous mutant for the p53 gene. We investigated the functional consequences of the mouse T122-->L mutation when expressed either in mammalian cells or in the yeast Saccharomyces cerevisiae. Similar to a non-functional allele, high expression of the T122-->L allele in p53(-/-) mouse embryo fibroblasts and human Saos-2 cells failed to suppress growth. However, the T122-->L mutant p53 showed wild-type transactivation levels with Bax and MDM2 promoters when expressed in either cell type and retained transactivation of the p21 and the c-Fos promoters in one cell line. Using a recently developed rheostatable p53 induction system in yeast we assessed the T122-->L transactivation capacity at low levels of protein expression using 12 different p53 response elements (REs). Compared to wild-type p53 the T122-->L protein manifested an unusual transactivation pattern comprising reduced and enhanced activity with specific REs. The high incidence of the T122-->L mutant allele in the Xpc(-/-) background suggests that both genetic and epigenetic conditions may facilitate the emergence of particular functional p53 mutations. Furthermore, the approach that we have taken also provides for the dissection of functions that may be retained in many p53 tumor alleles.

Identification of a regulatory T cell specific cell surface molecule that mediates suppressive signals and induces Foxp3 expression.

PubMed

Wang, Rui; Wan, Qi; Kozhaya, Lina; Fujii, Hodaka; Unutmaz, Derya

2008-07-16

Regulatory T (T(reg)) cells control immune activation and maintain tolerance. How T(regs) mediate their suppressive function is unclear. Here we identified a cell surface molecule, called GARP, (or LRRC32), which within T cells is specifically expressed in T(regs) activated through the T cell receptor (TCR). Ectopic expression of GARP in human naïve T (T(N)) cells inhibited their proliferation and cytokine secretion upon TCR activation. Remarkably, GARP over-expression in T(N) cells induced expression of T(reg) master transcription factor Foxp3 and endowed them with a partial suppressive function. The extracellular but not the cytoplasmic region of GARP, was necessary for these functions. Silencing Foxp3 in human T(reg) cells reduced expression of GARP and attenuated their suppressive function. However, GARP function was not affected when Foxp3 was downregulated in GARP-overexpressing cells, while silencing GARP in Foxp3-overexpressing cells reduced their suppressive activity. These findings reveal a novel cell surface molecule-mediated regulatory mechanism, with implications for modulating aberrant immune responses.

Tumor-derived exosomes regulate expression of immune function-related genes in human T cell subsets.

PubMed

Muller, Laurent; Mitsuhashi, Masato; Simms, Patricia; Gooding, William E; Whiteside, Theresa L

2016-02-04

Tumor cell-derived exosomes (TEX) suppress functions of immune cells. Here, changes in the gene profiles of primary human T lymphocytes exposed in vitro to exosomes were evaluated. CD4(+) Tconv, CD8(+) T or CD4(+) CD39(+) Treg were isolated from normal donors' peripheral blood and co-incubated with TEX or exosomes isolated from supernatants of cultured dendritic cells (DEX). Expression levels of 24-27 immune response-related genes in these T cells were quantified by qRT-PCR. In activated T cells, TEX and DEX up-regulated mRNA expression levels of multiple genes. Multifactorial data analysis of ΔCt values identified T cell activation and the immune cell type, but not exosome source, as factors regulating gene expression by exosomes. Treg were more sensitive to TEX-mediated effects than other T cell subsets. In Treg, TEX-mediated down-regulation of genes regulating the adenosine pathway translated into high expression of CD39 and increased adenosine production. TEX also induced up-regulation of inhibitory genes in CD4(+) Tconv, which translated into a loss of CD69 on their surface and a functional decline. Exosomes are not internalized by T cells, but signals they carry and deliver to cell surface receptors modulate gene expression and functions of human T lymphocytes.

Multiple sclerosis and human T-cell lymphotropic retroviruses

NASA Astrophysics Data System (ADS)

Koprowski, Hilary; Defreitas, Elaine C.; Harper, Mary E.; Sandberg-Wollheim, Magnhild; Sheremata, William A.; Robert-Guroff, Marjorie; Saxinger, Carl W.; Feinberg, Mark B.; Wong-Staal, Flossie; Gallo, Robert C.

1985-11-01

A combination of different types of data suggests that some multiple sclerosis patients respond immunologically to, and have cerebrospinal T cells containing, a retrovirus that is related to, but distinct from, the three types of human T-cell lymphotropic viruses. The role of this virus in multiple sclerosis is uncertain.

Single Cell Dissection of Human Pancreatic Islet Dysfunction in Diabetes

DTIC Science & Technology

2017-06-01

of memory T cells , innate cells and the differentiation potential of naive T cells during ME/CFS; and 3) To determine the T cell and innate cell ...apoptosis and the innate immune response in human pancreatic β- cells . Diabetes 64: 3808–3817. Marselli L, Thorne J, Dahiya S, Sgroi DC, Sharma A, Bonner-Weir...interactive nature of CellView aids in cell doublet identification. In the PBMC data, ‘Subcluster-analysis’ reveals a mixture of lymphoid and myeloid

Chimeric antigen receptor containing ICOS signaling domain mediates specific and efficient antitumor effect of T cells against EGFRvIII expressing glioma

PubMed Central

2013-01-01

Background Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells appears to be a promising immunotherapeutic strategy. CAR combines the specificity of antibody and cytotoxicity of cytotoxic T lymphocytes, enhancing T cells’ ability to specifically target antigens and to effectively kill cancer cells. Recent efforts have been made to integrate the costimulatory signals in the CAR to improve the antitumor efficacy. Epidermal growth factor receptor variant III (EGFRvIII) is an attractive therapeutic target as it frequently expresses in glioma and many other types of cancers. Our current study aimed to investigate the specific and efficient antitumor effect of T cells modified with CAR containing inducible costimulator (ICOS) signaling domain. Methods A second generation of EGFRvIII/CAR was generated and it contained the EGFRvIII single chain variable fragment, ICOS signaling domain and CD3ζ chain. Lentiviral EGFRvIII/CAR was prepared and human CD3+ T cells were infected by lentivirus encoding EGFRvIII/CAR. The expression of EGFRvIII/CAR on CD3+ T cells was confirmed by flow cytometry and Western blot. The functions of EGFRvIII/CAR+ T cells were evaluated using in vitro and in vivo methods including cytotoxicity assay, cytokine release assay and xenograft tumor mouse model. Results Chimeric EGFRvIIIscFv-ICOS-CD3ζ (EGFRvIII/CAR) was constructed and lentiviral EGFRvIII/CAR were made to titer of 106 TU/ml. The transduction efficiency of lentiviral EGFRvIII/CAR on T cells reached around 70% and expression of EGFRvIII/CAR protein was verified by immunoblotting as a band of about 57 kDa. Four hour 51Cr release assays demonstrated specific and efficient cytotoxicity of EGFRvIII/CAR+ T cells against EGFRvIII expressing U87 cells. A robust increase in the IFN-γ secretion was detected in the co-culture supernatant of the EGFRvIII/CAR+ T cells and the EGFRvIII expressing U87 cells. Intravenous and intratumor injection of EGFRvIII/CAR+ T cells inhibited the in vivo growth of the EGFRvIII expressing glioma cells. Conclusions Our study demonstrates that the EGFRvIII/CAR-modified T cells can destroy glioma cells efficiently in an EGFRvIII specific manner and release IFN-γ in an antigen dependent manner. The specific recognition and effective killing activity of the EGFRvIII-directed T cells with ICOS signaling domain lays a foundation for us to employ such approach in future cancer treatment. PMID:23656794

GARP: a key receptor controlling FOXP3 in human regulatory T cells.

PubMed

Probst-Kepper, M; Geffers, R; Kröger, A; Viegas, N; Erck, C; Hecht, H-J; Lünsdorf, H; Roubin, R; Moharregh-Khiabani, D; Wagner, K; Ocklenburg, F; Jeron, A; Garritsen, H; Arstila, T P; Kekäläinen, E; Balling, R; Hauser, H; Buer, J; Weiss, S

2009-09-01

Recent evidence suggests that regulatory pathways might control sustained high levels of FOXP3 in regulatory CD4(+)CD25(hi) T (T(reg)) cells. Based on transcriptional profiling of ex vivo activated T(reg) and helper CD4(+)CD25(-) T (T(h)) cells we have identified GARP (glycoprotein-A repetitions predominant), LGALS3 (lectin, galactoside-binding, soluble, 3) and LGMN (legumain) as novel genes implicated in human T(reg) cell function, which are induced upon T-cell receptor stimulation. Retroviral overexpression of GARP in antigen-specific T(h) cells leads to an efficient and stable re-programming of an effector T cell towards a regulatory T cell, which involves up-regulation of FOXP3, LGALS3, LGMN and other T(reg)-associated markers. In contrast, overexpression of LGALS3 and LGMN enhance FOXP3 and GARP expression, but only partially induced a regulatory phenotype. Lentiviral down-regulation of GARP in T(reg) cells significantly impaired the suppressor function and was associated with down-regulation of FOXP3. Moreover, down-regulation of FOXP3 resulted in similar phenotypic changes and down-regulation of GARP. This provides compelling evidence for a GARP-FOXP3 positive feedback loop and provides a rational molecular basis for the known difference between natural and transforming growth factor-beta induced T(reg) cells as we show here that the latter do not up-regulate GARP. In summary, we have identified GARP as a key receptor controlling FOXP3 in T(reg) cells following T-cell activation in a positive feedback loop assisted by LGALS3 and LGMN, which represents a promising new system for the therapeutic manipulation of T cells in human disease.

CD8+ T Cells Contribute to the Development of Coronary Arteritis in the Lactobacillus casei Cell Wall Extract-Induced Murine Model of Kawasaki Disease.

PubMed

Noval Rivas, Magali; Lee, Youngho; Wakita, Daiko; Chiba, Norika; Dagvadorj, Jargalsaikhan; Shimada, Kenichi; Chen, Shuang; Fishbein, Michael C; Lehman, Thomas J A; Crother, Timothy R; Arditi, Moshe

2017-02-01

Kawasaki disease (KD) is the leading cause of acquired heart disease among children in developed countries. Coronary lesions in KD in humans are characterized by an increased presence of infiltrating CD3+ T cells; however, the specific contributions of the different T cell subpopulations in coronary arteritis development remain unknown. Therefore, we sought to investigate the function of CD4+ and CD8+ T cells, Treg cells, and natural killer (NK) T cells in the pathogenesis of KD. We addressed the function of T cell subsets in KD development by using a well-established murine model of Lactobacillus casei cell wall extract (LCWE)-induced KD vasculitis. We determined which T cell subsets were required for development of KD vasculitis by using several knockout murine strains and depleting monoclonal antibodies. LCWE-injected mice developed coronary lesions characterized by the presence of inflammatory cell infiltrates. Frequently, this chronic inflammation resulted in complete occlusion of the coronary arteries due to luminal myofibroblast proliferation (LMP) as well as the development of coronary arteritis and aortitis. We found that CD8+ T cells, but not CD4+ T cells, NK T cells, or Treg cells, were required for development of KD vasculitis. The LCWE-induced murine model of KD vasculitis mimics many histologic features of the disease in humans, such as the presence of CD8+ T cells and LMP in coronary artery lesions as well as epicardial coronary arteritis. Moreover, CD8+ T cells functionally contribute to the development of KD vasculitis in this murine model. Therapeutic strategies targeting infiltrating CD8+ T cells might be useful in the management of KD in humans. © 2016, American College of Rheumatology.

Evidence for a stepwise program of extrathymic T cell development within the human tonsil

PubMed Central

McClory, Susan; Hughes, Tiffany; Freud, Aharon G.; Briercheck, Edward L.; Martin, Chelsea; Trimboli, Anthony J.; Yu, Jianhua; Zhang, Xiaoli; Leone, Gustavo; Nuovo, Gerard; Caligiuri, Michael A.

2012-01-01

The development of a broad repertoire of T cells, which is essential for effective immune function, occurs in the thymus. Although some data suggest that T cell development can occur extrathymically, many researchers remain skeptical that extrathymic T cell development has an important role in generating the T cell repertoire in healthy individuals. However, it may be important in the setting of poor thymic function or congenital deficit and in the context of autoimmunity, cancer, or regenerative medicine. Here, we report evidence that a stepwise program of T cell development occurs within the human tonsil. We identified 5 tonsillar T cell developmental intermediates: (a) CD34+CD38dimLin– cells, which resemble multipotent progenitors in the bone marrow and thymus; (b) more mature CD34+CD38brightLin– cells; (c) CD34+CD1a+CD11c– cells, which resemble committed T cell lineage precursors in the thymus; (d) CD34–CD1a+CD3–CD11c– cells, which resemble CD4+CD8+ double-positive T cells in the thymus; and (e) CD34–CD1a+CD3+CD11c– cells. The phenotype of each subset closely resembled that of its thymic counterpart. The last 4 populations expressed RAG1 and PTCRA, genes required for TCR rearrangement, and all 5 subsets were capable of ex vivo T cell differentiation. TdT+ cells found within the tonsillar fibrous scaffold expressed CD34 and/or CD1a, indicating that this distinct anatomic region contributes to pre–T cell development, as does the subcapsular region of the thymus. Thus, we provide evidence of a role for the human tonsil in a comprehensive program of extrathymic T cell development. PMID:22378041

Human T-cell leukemia virus type 1 Tax oncoprotein represses the expression of the BCL11B tumor suppressor in T-cells

PubMed Central

Takachi, Takayuki; Takahashi, Masahiko; Takahashi-Yoshita, Manami; Higuchi, Masaya; Obata, Miki; Mishima, Yukio; Okuda, Shujiro; Tanaka, Yuetsu; Matsuoka, Masao; Saitoh, Akihiko; Green, Patrick L; Fujii, Masahiro

2015-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia (ATL), which is an aggressive form of T-cell malignancy. HTLV-1 oncoproteins, Tax and HBZ, play crucial roles in the immortalization of T-cells and/or leukemogenesis by dysregulating the cellular functions in the host. Recent studies show that HTLV-1-infected T-cells have reduced expression of the BCL11B tumor suppressor protein. In the present study, we explored whether Tax and/or HBZ play a role in downregulating BCL11B in HTLV-1-infected T-cells. Lentiviral transduction of Tax in a human T-cell line repressed the expression of BCL11B at both the protein and mRNA levels, whereas the transduction of HBZ had little effect on the expression. Tax mutants with a decreased activity for the NF-κB, CREB or PDZ protein pathways still showed a reduced expression of the BCL11B protein, thereby implicating a different function of Tax in BCL11B downregulation. In addition, the HTLV-2 Tax2 protein reduced the BCL11B protein expression in T-cells. Seven HTLV-1-infected T-cell lines, including three ATL-derived cell lines, showed reduced BCL11B mRNA and protein expression relative to an uninfected T-cell line, and the greatest reductions were in the cells expressing Tax. Collectively, these results indicate that Tax is responsible for suppressing BCL11B protein expression in HTLV-1-infected T-cells; Tax-mediated repression of BCL11B is another mechanism that Tax uses to promote oncogenesis of HTLV-1-infected T-cells. PMID:25613934

Tributyltin (TBT) and Dibutyltin (DBT) Alter Secretion of Tumor Necrosis Factor Alpha (TNFα) from Human Natural Killer (NK) Cells and a Mixture of T cells and NK Cells

PubMed Central

Hurt, Kelsi; Hurd-Brown, Tasia; Whalen, Margaret

2012-01-01

Butyltins (BTs) have been in widespread use. Tributyltin (TBT) has been used as a biocide in a variety of applications and is found in human blood samples. Dibutyltin (DBT) has been used as a stabilizer in polyvinyl chloride plastics and as a de-worming agent in poultry. DBT, like TBT, is found in human blood. Human natural killer (NK) cells are the earliest defense against tumors and viral infections and secrete the cytokine tumor necrosis factor (TNF) alpha (α). TNFα is an important regulator of adaptive and innate immune responses. TNFα promotes inflammation and an association between malignant transformation and inflammation has been established. Previously, we have shown that TBT and DBT were able to interfere with the ability of NK cells to lyse tumor target cells. Here we show that BTs alter cytokine secretion by NK cells as well as a mixture of T and NK lymphocytes (T/NK cells). We examined 24 h, 48 h, and 6 day exposures to TBT (200- 2.5 nM) and DBT (5- 0.05 µM) on TNFα secretion by highly enriched human NK cells and T/NK cells. The results indicate that TBT (200 - 2.5 nM) decreased TNFα secretion from NK cells. In the T/NK cells 200 nM TBT decreased secretion while 100-5 nM TBT increased secretion of TNFα. NK cells or T/NK cells exposed to higher concentrations of DBT showed decreased TNFα secretion while lower concentrations showed increased secretion. The effects of BTs on TNFα secretion are seen at concentrations present in human blood. PMID:23047847

Examination of the signal transduction pathways leading to upregulation of tissue type plasminogen activator by Porphyromonas endodontalis in human pulp cells.

PubMed

Huang, F-M; Chen, Y-J; Chou, M-Y; Chang, Y-C

2005-12-01

To investigate the tissue type plasminogen activator (t-PA) activity in human pulp cells stimulated with Porphyromonas endodontalis (P. endodontalis) in the absence or presence of p38 inhibitor SB203580, mitogen-activated protein kinase kinase (MEK) inhibitor U0126 and phosphatidylinositaol 3-kinase (PI3K) inhibitor LY294002. The supernatants of P. endodontalis were used to evaluate t-PA activity in human pulp cells using casein zymography and enzyme-linked immunosorbent assay (ELISA). Furthermore, to search for possible signal transduction pathways, SB203580, U0126 and LY294002 were added to test how they modulated the t-PA activity. The main casein secreted by human pulp cells migrated at 70 kDa and represented t-PA. Secretion of t-PA was found to be stimulated with P. endodontalis during 2-day cultured period (P < 0.05). From the results of casein zymography and ELISA, SB203580 and U0126 significantly reduced the P. endodontalis stimulated t-PA production respectively (P < 0.05). However, LY294002 lacked the ability to change the P. endodontalis stimulated t-PA production (P > 0.05). Porphyromonas endodontalis enhances t-PA production in human pulp cells, and the signal transduction pathways p38 and MEK are involved in the inhibition of t-PA.

Interleukin 4-producing CD4+ T cells in the skin of cats with allergic dermatitis.

PubMed

Roosje, P J; Dean, G A; Willemse, T; Rutten, V P M G; Thepen, T

2002-03-01

Lesional skin of cats with allergic dermatitis has a cellular infiltrate and a CD4/CD8 ratio comparable to that in humans with atopic dermatitis. CD4+ helper T cells and in particular cells belonging to the Th2 subset play an important role in disease pathogenesis in humans. We investigated the cytokine pattern of CD4+ T cells in situ, with special emphasis on the putative presence of cells producing interleukin 4 (IL4), in cats with allergic dermatitis. Immunohistochemical procedures were used to determine that CD4+ T cells in lesional and nonlesional skin of cats with allergic dermatitis can produce IL4, as occurs in humans. Lesional and nonlesional skin of cats with allergic dermatitis had significantly more IL4+ T cells (P = 0.001) than did skin of healthy control cats. Double staining indicated that all IL4+ cells were positive for pan-T or CD4 markers. Double labeling for mast cell chymase and IL4 stained primarily different cells. Western blotting demonstrated cross-reactivity between the antibody against human IL4 and a feline recombinant IL4. These results indicate that IL4 is primarily produced by CD4+ T cells and is also present in clinically uninvolved skin, indicating a role in the pathogenesis of allergic dermatitis in cats.

A novel CBL-Bflox/flox mouse model allows tissue-selective fully conditional CBL/CBL-B double-knockout: CD4-Cre mediated CBL/CBL-B deletion occurs in both T-cells and hematopoietic stem cells

PubMed Central

Goetz, Benjamin; An, Wei; Mohapatra, Bhopal; Zutshi, Neha; Iseka, Fany; Storck, Matthew D.; Meza, Jane; Sheinin, Yuri; Band, Vimla; Band, Hamid

2016-01-01

CBL-family ubiquitin ligases are critical negative regulators of tyrosine kinase signaling, with a clear redundancy between CBL and CBL-B evident in the immune cell and hematopoietic stem cell studies. Since CBL and CBL-B are negative regulators of immune cell activation, elimination of their function to boost immune cell activities could be beneficial in tumor immunotherapy. However, mutations of CBL are associated with human leukemias, pointing to tumor suppressor roles of CBL proteins; hence, it is critical to assess the tumor-intrinsic roles of CBL and CBL-B in cancers. This has not been possible since the only available whole-body CBL-B knockout mice exhibit constitutive tumor rejection. We engineered a new CBL-Bflox/flox mouse, combined this with an existing CBLflox/flox mouse to generate CBLflox/flox; CBL-Bflox/flox mice, and tested the tissue-specific concurrent deletion of CBL and CBL-B using the widely-used CD4-Cre transgenic allele to produce a T-cell-specific double knockout. Altered T-cell development, constitutive peripheral T-cell activation, and a lethal multi-organ immune infiltration phenotype largely resembling the previous Lck-Cre driven floxed-CBL deletion on a CBL-B knockout background establish the usefulness of the new model for tissue-specific CBL/CBL-B deletion. Unexpectedly, CD4-Cre-induced deletion in a small fraction of hematopoietic stem cells led to expansion of certain non-T-cell lineages, suggesting caution in the use of CD4-Cre for T-cell-restricted gene deletion. The establishment of a new model of concurrent tissue-selective CBL/CBL-B deletion should allow a clear assessment of the tumor-intrinsic roles of CBL/CBL-B in non-myeloid malignancies and help test the potential for CBL/CBL-B inactivation in immunotherapy of tumors. PMID:27276677

Beta-lactam antibiotics modulate T-cell functions and gene expression via covalent binding to cellular albumin.

PubMed

Mor, Felix; Cohen, Irun R

2013-02-19

Recent work has suggested that beta-lactam antibiotics might directly affect eukaryotic cellular functions. Here, we studied the effects of commonly used beta-lactam antibiotics on rodent and human T cells in vitro and in vivo on T-cell-mediated experimental autoimmune diseases. We now report that experimental autoimmune encephalomyelitis and adjuvant arthritis were significantly more severe in rats treated with cefuroxime and other beta-lactams. T cells appeared to mediate the effect: an anti-myelin basic protein T-cell line treated with cefuroxime or penicillin was more encephalitogenic in adoptive transfer experiments. The beta-lactam ampicillin, in contrast to cefuroxime and penicillin, did not enhance encephalomyelitis, but did inhibit the autoimmune diabetes developing spontaneously in nonobese diabetic mice. Gene expression analysis of human peripheral blood T cells showed that numerous genes associated with T helper 2 (Th2) and T regulatory (Treg) differentiation were down-regulated in T cells stimulated in the presence of cefuroxime; these genes were up-regulated in the presence of ampicillin. The T-cell protein that covalently bound beta-lactam antibiotics was found to be albumin. Human and rodent T cells expressed albumin mRNA and protein, and penicillin-modified albumin was taken up by rat T cells, leading to enhanced encephalitogenicity. Thus, beta-lactam antibiotics in wide clinical use have marked effects on T-cell behavior; beta-lactam antibiotics can function as immunomodulators, apparently through covalent binding to albumin.

T cell receptor αβ diversity inversely correlates with pathogen-specific antibody levels in human cytomegalovirus infection.

PubMed

Wang, George C; Dash, Pradyot; McCullers, Jonathan A; Doherty, Peter C; Thomas, Paul G

2012-04-04

A diverse T cell receptor (TCR) repertoire capable of recognizing a broad range of antigenic peptides is thought to be central to effective pathogen-specific immunity by counteracting escape mutations, selecting high-avidity T cells, and providing T cell specificities with comprehensive functional characteristics. However, evidence that TCR diversity is important for the successful control of human infections is limited. A single-cell strategy for the clonotypic analysis of human CD8⁺ TCRαβ repertoires was used to probe the diversity and magnitude of individual human cytomegalovirus (CMV)-specific CD8⁺ T cells recovered directly ex vivo. We found that CD8⁺ TCRαβ repertoire diversity, but not the size of the CD8⁺ T cell response, was inversely related to circulating CMV-specific antibody levels, a measure that has been correlated epidemiologically with differential mortality risks and found here to be higher in persons with detectable (versus undetectable) CMV viral loads. Overall, our findings indicate that CD8⁺ T cell diversity may be more important than T cell abundance in limiting the negative consequences of CMV persistence, demonstrate high prevalence of both TCRα and TCRβ public motif usage, and suggest that a highly diverse TCRαβ repertoire may be an important benchmark and target in the success of immunotherapeutic strategies.

Butyrate Conditions Human Dendritic Cells to Prime Type 1 Regulatory T Cells via both Histone Deacetylase Inhibition and G Protein-Coupled Receptor 109A Signaling

PubMed Central

Kaisar, Maria M. M.; Pelgrom, Leonard R.; van der Ham, Alwin J.; Yazdanbakhsh, Maria; Everts, Bart

2017-01-01

Recently, it has become clear that short-chain fatty acids (SCFAs), and in particular butyrate, have anti-inflammatory properties. Murine studies have shown that butyrate can promote regulatory T cells via the induction of tolerogenic dendritic cells (DCs). However, the effects of SCFAs on human DCs and how they affect their capacity to prime and polarize T-cell responses have not been addressed. Here, we report that butyrate suppresses LPS-induced maturation and metabolic reprogramming of human monocyte-derived DCs (moDCs) and conditions them to polarize naive CD4+ T cells toward IL-10-producing type 1 regulatory T cells (Tr1). This effect was dependent on induction of the retinoic acid-producing enzyme retinaldehyde dehydrogenase 1 in DCs. The induction of retinaldehyde dehydrogenase activity and Tr1 cell differentiation by butyrate was dependent on simultaneous inhibition of histone deacetylases and signaling through G protein-coupled receptor 109A. Taken together, we reveal that butyrate is a potent inducer of tolerogenic human DCs, thereby shedding new light on the cellular and molecular mechanisms through which SCFAs can exert their immunomodulatory effects in humans. PMID:29163504

Differential activation of human T cells to allogeneic endothelial cells, epithelial cells and fibroblasts in vitro

PubMed Central

2012-01-01

Background In the direct pathway, T cells recognize intact donor major histocompatability complexes and allogeneic peptide on the surface of donor antigen presenting cells (APCs). Indirect allorecognition results from the recognition of processed alloantigen by self MHC complexes on self APCs. In this study, we wished to evaluate the relative contribution of different intragraft cells to the alloactivation of nave and memory T cells though the direct and the indirect pathway of allorecognition. Methods The processing of membrane fragments from IFN-treated single donor endothelial cells (EC), fibroblasts or renal epithelial cells (RPTEC) was evaluated by DiOC labeling of each cell type and flow cytometry following interaction with PBMC. Direct pathway activation of nave CD45RA+ or memory CD45RO+ CD4+ T cells was evaluated following coculture with IFN-treated and MHC class II-expressing EC, fibroblasts or RPTEC. Indirect pathway activation was assessed using CD45RA+ or CD45RO+ CD4+ T cells cocultured with autologous irradiated APCs in the absence or presence of sonicates derived from IFN-treated allogeneic EC, fibroblasts or RPTEC. Activation of T cells was assessed by [3H]thymidine incorporation and by ELISpot assays. Results We find that CD14+ APCs readily acquire membrane fragments from fibroblasts and RPTEC, but fail to acquire membrane fragments from intact EC. However, APCs process membranes from EC undergoing apoptosis.There was a notable direct pathway alloproliferative response of CD45RO+ CD4+ T cells to IFN-treated EC, but not to fibroblasts or RPTEC. Also, there was a minimal direct pathway response of CD45RA+ CD4+ T cells to all cell types. In contrast, we found that both CD45RA+ and CD45RO+ CD4+ T cells proliferated following coculture with autologous APCs in the presence of sonicates derived from IFN-treated EC, fibroblasts or RPTEC. By ELISpot, we found that these T cells stimulated via the indirect pathway also produced the cytokines IFN, IL-2, IL-4 and IL-5. Conclusions Recipient APCs may readily process membrane fragments from allogeneic intragraft cells, but not from EC unless they are undergoing apoptosis. This processing is sufficient for indirect pathway alloactivation of both CD45RA+ and CD45RO+ CD4+ T cells. Only graft vascular EC mediate direct pathway reactivation of CD4+ T cells. PMID:23369287

T Cell Inactivation by Poxviral B22 Family Proteins Increases Viral Virulence

PubMed Central

Alzhanova, Dina; Hammarlund, Erika; Reed, Jason; Meermeier, Erin; Rawlings, Stephanie; Ray, Caroline A.; Edwards, David M.; Bimber, Ben; Legasse, Alfred; Planer, Shannon; Sprague, Jerald; Axthelm, Michael K.; Pickup, David J.; Lewinsohn, David M.; Gold, Marielle C.; Wong, Scott W.; Sacha, Jonah B.; Slifka, Mark K.; Früh, Klaus

2014-01-01

Infections with monkeypox, cowpox and weaponized variola virus remain a threat to the increasingly unvaccinated human population, but little is known about their mechanisms of virulence and immune evasion. We now demonstrate that B22 proteins, encoded by the largest genes of these viruses, render human T cells unresponsive to stimulation of the T cell receptor by MHC-dependent antigen presentation or by MHC-independent stimulation. In contrast, stimuli that bypass TCR-signaling are not inhibited. In a non-human primate model of monkeypox, virus lacking the B22R homologue (MPXVΔ197) caused only mild disease with lower viremia and cutaneous pox lesions compared to wild type MPXV which caused high viremia, morbidity and mortality. Since MPXVΔ197-infected animals displayed accelerated T cell responses and less T cell dysregulation than MPXV US2003, we conclude that B22 family proteins cause viral virulence by suppressing T cell control of viral dissemination. PMID:24832205

T cell inactivation by poxviral B22 family proteins increases viral virulence.

PubMed

Alzhanova, Dina; Hammarlund, Erika; Reed, Jason; Meermeier, Erin; Rawlings, Stephanie; Ray, Caroline A; Edwards, David M; Bimber, Ben; Legasse, Alfred; Planer, Shannon; Sprague, Jerald; Axthelm, Michael K; Pickup, David J; Lewinsohn, David M; Gold, Marielle C; Wong, Scott W; Sacha, Jonah B; Slifka, Mark K; Früh, Klaus

2014-05-01

Infections with monkeypox, cowpox and weaponized variola virus remain a threat to the increasingly unvaccinated human population, but little is known about their mechanisms of virulence and immune evasion. We now demonstrate that B22 proteins, encoded by the largest genes of these viruses, render human T cells unresponsive to stimulation of the T cell receptor by MHC-dependent antigen presentation or by MHC-independent stimulation. In contrast, stimuli that bypass TCR-signaling are not inhibited. In a non-human primate model of monkeypox, virus lacking the B22R homologue (MPXVΔ197) caused only mild disease with lower viremia and cutaneous pox lesions compared to wild type MPXV which caused high viremia, morbidity and mortality. Since MPXVΔ197-infected animals displayed accelerated T cell responses and less T cell dysregulation than MPXV US2003, we conclude that B22 family proteins cause viral virulence by suppressing T cell control of viral dissemination.

Meta-Analysis of the Changes of Peripheral Blood T Cell Subsets in Patients with Brucellosis

PubMed Central

Zheng, Rongjiong; Xie, Songsong; Niyazi, Shaniya; Lu, Xiaobo; Zhou, Yan

2018-01-01

Brucellosis is one of the most prevalent zoonotic diseases in the world, but its pathogenesis is not very clear. At present, it is thought that it may be related to the immunity of T cells. The conclusions of related studies are inconsistent, and its clinical significance is not explicit. We searched published articles in electronic databases up to December 2017 identified as relating to the clinical features of human brucellosis in China. Only eight studies had sufficient quality for data extraction. Meta-analysis showed a significantly decreased proportion of CD4+ T cells in human brucellosis patients compared to healthy subject individuals. The frequency of CD8+ T cells was significantly higher in human brucellosis patients than that in the healthy control group. The pooled analysis presented a significant decrease of the CD4+/CD8+ ratio in human brucellosis patients compared to healthy subjects. There is immunologic dysfunction of T lymphocyte in patients with human brucellosis, the CD4+ and CD8+ T cells might be the important factors affecting the progress of brucellosis. PMID:29888294

Role for granulocyte colony-stimulating factor in the generation of human T regulatory type 1 cells.

PubMed

Rutella, Sergio; Pierelli, Luca; Bonanno, Giuseppina; Sica, Simona; Ameglio, Franco; Capoluongo, Ettore; Mariotti, Andrea; Scambia, Giovanni; d'Onofrio, Giuseppe; Leone, Giuseppe

2002-10-01

Granulocyte colony-stimulating factor (G-CSF) may affect T-cell homeostasis by multiple mechanisms, inducing polarization of cytokine secretion, inhibition of T-cell proliferation, and enhancement of T-cell apoptosis. We analyzed the production of interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-beta1) by T cells from healthy volunteer donors treated with recombinant human G-CSF. Highly purified CD4(+) T cells obtained before and after G-CSF administration (pre-G and post-G, respectively) were activated using the allogeneic mixed leukocyte reaction. Post-G CD4(+) T cells produced high levels of IL-10 but undetectable levels of IL-2 and IL-4, whereas the level of TGF-beta1 release was comparable to that of pre-G CD4(+) T cells. Notably, post-G CD4(+) T cells proliferated poorly in response to alloantigens and to recall antigens and suppressed the proliferation of autologous CD4(+) T cells in a cell contact-independent and an antigen-nonspecific manner. TGF-beta1 and IL-10 were not dispensable for post-G CD4(+) T cells to mediate suppression, as shown by neutralization studies. Compared with pre-G CD4(+) T cells, alloantigen-activated post-G CD4(+) T cells preferentially expressed markers associated with memory T cells, in conjunction with reduced levels of CD28 and CD62L. Collectively, these data demonstrate that CD4(+) T cells exposed to G-CSF in vivo acquire the properties of T regulatory (Tr) cells once triggered in vitro through the T-cell receptor, including a peculiar cytokine production profile (IL-10(++)TGF-beta1(+)IL-2(low/-)IL-4(low/-)), an intrinsic low proliferative capacity, and a contact-independent suppression of antigen-driven proliferation. Tr cells generated ex vivo after exposure to G-CSF might be clinically relevant for transplantation medicine and for the treatment of human immune-mediated diseases.

Interluekin-12 enhances the function and anti-tumor activity in murine and human CD8+ T cells

PubMed Central

Rubinstein, Mark P.; Su, Ee Wern; Suriano, Samantha; Cloud, Colleen A.; Andrijauskaite, Kristina; Kesarwani, Pravin; Schwartz, Kristina M.; Williams, Katelyn; Johnson, C. Bryce; Li, Mingli; Scurti, Gina M.; Salem, Mohamed L.; Paulos, Chrystal M.; Garrett-Mayer, Elizabeth; Mehrotra, Shikhar; Cole, David J.

2016-01-01

Mouse CD8+ T cells conditioned with Interleukin (IL)-12 ex vivo mediate the potent regression of established melanoma when transferred into lymphodepleted mice. However, the quantitative and qualitative changes induced by IL-12 in the responding mouse CD8+ T cells have not been well defined. Moreover, the mechanisms by which IL-12-conditioning impacts human CD8+ T cells, and how such cells might be expanded prior to infusion into patients is not known. We found that ex vivo IL-12-conditioning of mouse CD8+ T cells led to a 10- to 100-fold increase in persistence and anti-tumor efficacy upon adoptive transfer into lymphodepleted mice. The enhancing effect of IL-12 was associated with maintenance of functional avidity. Importantly, in the context of ongoing ACT clinical trials, human CD8+ T cells genetically modified with a tyrosinase-specific T-cell receptor exhibited significantly enhanced functional activity when conditioned with IL-12 as indicated by heightened granzyme B expression and elevated peptide-specific CD107a degranulation. This effect was sustainable despite the 20 days of in vitro cellular expansion required to expand cells over 1,000-fold allowing adequate cell numbers for administration to cancer patients. Overall, these findings support the efficacy and feasibility of ex vivo IL-12-conditioning of TCR-modified human CD8+ T cells for adoptive transfer and cancer therapy. PMID:25676709

Insights into human CD8(+) T-cell memory using the yellow fever and smallpox vaccines.

PubMed

Ahmed, Rafi; Akondy, Rama S

2011-03-01

Live virus vaccines provide a unique opportunity to study human CD8(+) T-cell memory in the context of a controlled, primary acute viral infection. Yellow fever virus-17D and Dryvax are two such live-virus vaccines that are highly efficacious, used worldwide and provide long-term immunity against yellow fever and smallpox respectively. In this review, we describe the properties of virus-specific memory CD8(+) T cells generated in smallpox and yellow fever vaccinees. We address fundamental questions regarding magnitude, functional quality and longevity of the CD8(+) T-cell response, which are otherwise challenging to address in humans. These findings provide insights into the attributes of the human immune system as well as provide a benchmark for the optimal quality of a CD8(+) T-cell response that can be used to evaluate novel candidate vaccines.

Aflatoxin B1-induced DNA adduct formation and p53 mutations in CYP450-expressing human liver cell lines.

PubMed

Macé, K; Aguilar, F; Wang, J S; Vautravers, P; Gómez-Lechón, M; Gonzalez, F J; Groopman, J; Harris, C C; Pfeifer, A M

1997-07-01

Epidemiological evidence has been supporting a relationship between dietary aflatoxin B1 (AFB1) exposure, development of human primary hepatocellular carcinoma (HCC) and mutations in the p53 tumor suppressor gene. However, the correlation between the observed p53 mutations, the AFB1 DNA adducts and their activation pathways has not been elucidated. Development of relevant cellular in vitro models, taking into account species and tissue specificity, could significantly contribute to the knowledge of cytotoxicity and genotoxicity mechanisms of chemical procarcinogens, such as AFB1, in humans. For this purpose a non-tumorigenic SV40-immortalized human liver epithelial cell line (THLE cells) which retained most of the phase II enzymes, but had markedly reduced phase I activities was used for stable expression of the human CYP1A2, CYP2A6, CYP2B6 and CYP3A4 cDNA. The four genetically engineered cell lines (T5-1A2, T5-2A6, T5-2B6 and T5-3A4) produced high levels of the specific CYP450 proteins and showed comparable or higher catalytic activities related to the CYP450 expression when compared to human hepatocytes. The T5-1A2, T5-2A6, T5-2B6 and T5-3A4 cell lines exhibited a very high sensitivity to the cytotoxic effects of AFB1 and were approximately 125-, 2-, 2- and 15-fold, respectively, more sensitive than the control T5-neo cells, transfected with an expressing vector which does not contain CYP450 cDNA. In the CYP450-expressing cells, nanomolar doses of AFB1-induced DNA adduct formation including AFB1-N7-guanine, -pyrimidyl and -diol adducts. In addition, the T5-1A2 cells showed AFM1-DNA adducts. At similar levels of total DNA adducts, both the T5-1A2 and T5-3A4 cells showed, at codon 249 of the p53 gene, AGG to AGT transversions at a relative frequency of 15x10(-6). In contrast, only the T5-3A4 cells showed CCC to ACC transversion at codon 250 at a high frequency, whereas the second most frequent mutations found in the T5-1A2 cells were C to T transitions at the first and second position of the codon 250. No significant AFB1-induced p53 mutations could be detected in the T5-2A6 cells. Therefore, the differential expression of specific CYP450 genes in human hepatocytes can modulate the cytotoxicity, DNA adduct levels and frequency of p53 mutations produced by AFB1.

Transformation to continuous growth of primary human T lymphocytes by human T-cell leukemia virus type I X-region genes transduced by a herpesvirus saimiri vector

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

Grassmann, R.; Dengler, C.; Mueller-Fleckenstein, I.

1989-05-01

The role of the X region of the genome of the human T-cell leukemia virus type I (HTLV-I) in the immortalization of lymphocytes has been difficult to distinguish from its role in viral replication as this region encodes at least two genes, tax and rex, required for replication and the expression of viral proteins. To determine whether the X region does encode immortalizing functions, a fragment of the HTLV-I provirus capable of expressing known X-region proteins was inserted into the genome of a transformation-defective, replication-competent Herpesvirus saimiri. Infection of fresh mitogen-activated human cord blood and thymocytes yielded immortal T-cell linesmore » that had the same phenotype (CD4{sup +}, Cd5{sup +}, HLA class II{sup +}, interleukin 2 receptor {alpha}-chain +) as lymphocytes transformed by cocultivation with HTLV-I. These experiments demonstrate that the X region encodes the functions of HTLV-I that immortalize a distinct subpopulation of human T cells. The experiments also demonstrate the utility of the H. saimiri vector for the transduction of heterologous genes into human T cells.« less

PD-1 ligand expression by human colonic myofibroblasts/fibroblasts regulates CD4+ T-cell activity.

PubMed

Pinchuk, Irina V; Saada, Jamal I; Beswick, Ellen J; Boya, Gushyalatha; Qiu, Sumin M; Mifflin, Randy C; Raju, Gottumukkala S; Reyes, Victor E; Powell, Don W

2008-10-01

A prominent role for inhibitory molecules PD-L1 and PD-L2 in peripheral tolerance has been proposed. However, the phenotype and function of PD-L-expressing cells in human gut remains unclear. Recent studies suggest that colonic myofibroblasts (CMFs) and fibroblasts are important in the switch from acute inflammation to adaptive immunity. In the normal human colon, CMFs represent a distinct population of major histocompatibility complex class II(+) cells involved in the regulation of mucosal CD4(+) T-cell responses. PD-L1 and PD-L2 expression on human CMFs was determined using Western blot, fluorescence-activated cell sorter analysis and confocal microscopy. Lymphoproliferation assays and cytokine enzyme-linked immunosorbent assays were used to evaluate the role of B7 costimulators expressed by CMFs with regard to the regulation of preactivated T-helper cell responses. We demonstrate here the expression of PD-L1/2 molecules by normal human CMF and fibroblasts in situ and in culture. Both molecules support suppressive functions of CMFs in the regulation of activated CD4(+) T-helper cell proliferative responses; blocking this interaction reverses the suppressive effect of CMFs on T-cell proliferation and leads to increased production of the major T-cell growth factor, interleukin (IL)-2. PD-L1/2-mediated CMF suppressive functions are mainly due to the inhibition of IL-2 production, because supplementation of the coculture media with exogenous IL-2 led to partial recovery of activated T-cell proliferation. Our data suggest that stromal myofibroblasts and fibroblasts may limit T-helper cell proliferative activity in the gut and, thus, might play a prominent role in mucosal intestinal tolerance.

Assessment of porcine endogenous retrovirus transmission across an alginate barrier used for the encapsulation of porcine islets.

PubMed

Crossan, Claire; Mourad, Nizar I; Smith, Karen; Gianello, Pierre; Scobie, Linda

2018-05-21

Subcutaneous implantation of a macroencapsulated patch containing human allogenic islets has been successfully used to alleviate type 1 diabetes mellitus (T1DM) in a human recipient without the need for immunosuppression. The use of encapsulated porcine islets to treat T1DM has also been reported. Although no evidence of pathogen transfer using this technology has been reported to date, we deemed it appropriate to determine if the encapsulation technology would prevent the release of virus, in particular, the porcine endogenous retrovirus (PERV). HEK293 (human epithelial kidney) and swine testis (ST) cells were co-cultured with macroencapsulated pig islets embedded in an alginate patch, macroencapsulated PK15 (swine kidney epithelial) cells embedded in an alginate patch and free PK15 cells. Cells and supernatant were harvested at weekly time points from the cultures for up to 60 days and screened for evidence of PERV release using qRT-PCR to detect PERV RNA and SG-PERT to detect reverse transcriptase (RT). No PERV virus, or evidence of PERV replication, was detected in the culture medium of HEK293 or pig cells cultured with encapsulated porcine islets. Increased PERV activity relative to the background was not detected in ST cells cultured with encapsulated PK15 cells. However, PERV was detected in 1 of the 3 experimental replicates of HEK293 cells cultured with encapsulated PK15 cells. Both HEK293 and ST cells cultured with free PK15 cells showed an increase in RT detection. With the exception of 1 replicate, there does not appear to be evidence of transmission of replication competent PERV from the encapsulated islet cells or the positive control PK15 cells across the alginate barrier. The detection of PERV would suggest the alginate barrier of this replicate may have become compromised, emphasizing the importance of quality control when producing encapsulated islet patches. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Effect of high electromagnetic fields on cellular growth

NASA Astrophysics Data System (ADS)

Albalawi, Abdullah; Mustafa, Mohammed; Masood, Samina

It is already known that high-intensity electromagnetic field affect the human lung growth and forces the T-cells to decrease by 20-30 percent. The electromagnetic field had a severe impact on human T-cells in contrast to lung cells. Due to the high-intensity electromagnetic field, the growth of T-cells becomes low and release of Ca+2 increases up to 3.5 times more than the lung cells. The high-intensity electromagnetic radiations do not directly produce cancer cells but had a severe impact on the growth of T-cells. It can also be said that electromagnetic field acts a role in the cancer initiation. It creates disordered in the structure of membranes and gesture transduction. The higher exposure to electromagnetic field increases PKC-alpha and this larger release from membranes cannot be controlled. It was concluded that greater exposure to the electromagnetic field is dangerous and had a severe impact on T-cells growth and lung cells growth and due to this greater possibility of leukemia occurrence. We show a similar effect of electromagnetic fields single celled bacteria to compare the bacterial cellular growth with the human cells using the bacteria strains which are commonly found in human body.

Engineering human T cells for resistance to methotrexate and mycophenolate mofetil as an in vivo cell selection strategy.

PubMed

Jonnalagadda, Mahesh; Brown, Christine E; Chang, Wen-Chung; Ostberg, Julie R; Forman, Stephen J; Jensen, Michael C

2013-01-01

Gene transfer and drug selection systems that enforce ongoing transgene expression in vitro and in vivo which are compatible with human pharmaceutical drugs are currently underdeveloped. Here, we report on the utility of incorporating human enzyme muteins that confer resistance to the lymphotoxic/immunosuppressive drugs methotrexate (MTX) and mycophenolate mofetil (MMF) in a multicistronic lentiviral vector for in vivo T lymphocyte selection. We found that co-expression of human dihydrofolate reductase (DHFR(FS); L22F, F31S) and inosine monophosphate dehydrogenase II (IMPDH2(IY); T333I, S351Y) conferred T cell resistance to the cytocidal and anti-proliferative effects of these drugs at concentrations that can be achieved clinically (up to 0.1 µM MTX and 1.0 µM MPA). Furthermore, using a immunodeficient mouse model that supports the engraftment of central memory derived human T cells, in vivo selection studies demonstrate that huEGFRt(+)DHFR(FS+)IMPDH2(IY+) T cells could be enriched following adoptive transfer either by systemic administration of MTX alone (4.4 -fold), MMF alone (2.9-fold), or combined MTX and MMF (4.9-fold). These findings demonstrate the utility of both DHFR(FS)/MTX and IMPDH2(IY)/MMF for in vivo selection of lentivirally transduced human T cells. Vectors incorporating these muteins in combination with other therapeutic transgenes may facilitate the selective engraftment of therapeutically active cells in recipients.

HLA-E–restricted regulatory CD8+ T cells are involved in development and control of human autoimmune type 1 diabetes

PubMed Central

Jiang, Hong; Canfield, Steve M.; Gallagher, Mary P.; Jiang, Hong H.; Jiang, Yihua; Zheng, Zongyu; Chess, Leonard

2010-01-01

A key feature of the immune system is its ability to discriminate self from nonself. Breakdown in any of the mechanisms that maintain unresponsiveness to self (a state known as self-tolerance) contributes to the development of autoimmune conditions. Recent studies in mice show that CD8+ T cells specific for the unconventional MHC class I molecule Qa-1 bound to peptides derived from the signal sequence of Hsp60 (Hsp60sp) contribute to self/nonself discrimination. However, it is unclear whether they exist in humans and play a role in human autoimmune diseases. Here we have shown that CD8+ T cells specific for Hsp60sp bound to HLA-E (the human homolog of Qa-1) exist and play an important role in maintaining peripheral self-tolerance by discriminating self from nonself in humans. Furthermore, in the majority of type 1 diabetes (T1D) patients tested, there was a specific defect in CD8+ T cell recognition of HLA-E/Hsp60sp, which was associated with failure of self/nonself discrimination. However, the defect in the CD8+ T cells from most of the T1D patients tested could be corrected in vitro by exposure to autologous immature DCs loaded with the Hsp60sp peptide. These data suggest that HLA-E–restricted CD8+ T cells may play an important role in keeping self-reactive T cells in check. Thus, correction of this defect could be a potentially effective and safe approach in the therapy of T1D. PMID:20877010

Construction and evaluation of a novel humanized HER2-specific chimeric receptor

PubMed Central

2014-01-01

Introduction The human epidermal growth factor receptor 2 (HER2) represents one of the most studied tumor-associated antigens (TAAs) for cancer immunotherapy. The monoclonal antibody (mAb) trastuzumab has improved the outcomes of patients with HER2+ breast cancer. However, a large number of HER2+ tumors are not responsive to, or become resistant to, trastuzumab-based therapy, and thus more effective therapies targeting HER2 are needed. Methods HER2-specific T cells were generated by the transfer of genes that encode chimeric antigen receptor (CAR). Using a multistep overlap extension PCR method, we constructed a novel, humanized HER2 CAR-containing, chA21 single-chain variable fragment (scFv) region of antigen-specific mAb and T-cell intracellular signaling chains made up of CD28 and CD3ζ. An interferon γ and interleukin 2 enzyme-linked immunosorbent assay and a chromium-51 release assay were used to evaluate the antitumor immune response of CAR T cells in coculture with tumor cells. Furthermore, SKBR3 tumor–bearing nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were treated with HER2 CAR T cells to evaluate antitumor activity. Human CD3+ T cell accumulation in tumor xenograft was detected by immunohistochemistry. Results chA21-28z CAR was successfully constructed, and both CD4+ and CD8+ T cells were transduced. The expanded HER2 CAR T cells expressed a central memory phenotype and specifically reacted against HER2+ tumor cell lines. Furthermore, the SKBR3 tumor xenograft model revealed that HER2 CAR T cells significantly inhibited tumor growth in vivo. Immunohistochemical analysis showed robust accumulation of human CD3+ T cells in regressing SKBR3 lesions. Conclusions The results of this study show that novel chA21 scFv-based, HER2-specific CAR T cells not only recognized and killed HER2+ breast and ovarian cancer cells ex vivo but also induced regression of experimental breast cancer in vivo. Our data support further exploration of the HER2 CAR T-cell therapy for HER2-expressing cancers. PMID:24919843

Viral microRNA effects on persistent infection of human lymphoid cells by polyomavirus SV40

PubMed Central

McNees, Adrienne L.; Harrigal, Lindsay J.; Kelly, Aoife; Minard, Charles G.; Wong, Connie

2018-01-01

Background Polyomaviruses, including simian virus 40 (SV40), display evidence of lymphotropic properties. This study analyzed the nature of SV40–human lymphocyte interactions in established cell lines and in primary lymphocytes. The effects of viral microRNA and the structure of the viral regulatory region on SV40 persistence were examined. Results SV40 DNA was maintained in infected B cell and myeloid cell lines during cell growth for at least 28 days. Limiting dilution analysis showed that low amounts of SV40 DNA (~2 copies per cell) were retained over time. Infected B cells remained viable and able to proliferate. Genome copies of the SV40 microRNA-null mutant persisted at higher levels than the DNA of wild-type viruses. Complex viral regulatory regions produced modestly higher DNA levels than simple regulatory regions. Viral large T-antigen protein was detected at low frequency and at low levels in infected B cells. Following infection of primary lymphocytes, SV40 DNA was detected in CD19+ B cells and CD14+ monocytes, but not in CD3+ T cells. Rescue attempts using either lysates of SV40-infected B lymphocytes, coculture of live cells, or infectious center assays all showed that replication-competent SV40 could be recovered on rare occasions. SV40 infections altered the expression of several B cell surface markers, with more pronounced changes following infections with the microRNA-null mutant. Conclusion These findings indicate that SV40 can establish persistent infections in human B lymphocytes. The cells retain low copy numbers of viral DNA; the infections are nonproductive and noncytolytic but can occasionally produce infectious virus. SV40 microRNA negatively regulates the degree of viral effects on B cells. Significance Lymphocytes may serve as viral reservoirs and may function to disseminate polyomaviruses to different tissues in a host. To our knowledge, this report is the first extensive analysis of viral microRNA effects on SV40 infection of human lymphocytes. PMID:29432481

Reduced Plasmodium Parasite Burden Associates with CD38+ CD4+ T Cells Displaying Cytolytic Potential and Impaired IFN-γ Production

PubMed Central

Burel, Julie G.; Apte, Simon H.; Groves, Penny L.; Klein, Kerenaftali; McCarthy, James S.; Doolan, Denise L.

2016-01-01

Using a unique resource of samples from a controlled human malaria infection (CHMI) study, we identified a novel population of CD4+ T cells whose frequency in the peripheral blood was inversely correlated with parasite burden following P. falciparum infection. These CD4+ T cells expressed the multifunctional ectoenzyme CD38 and had unique features that distinguished them from other CD4+ T cells. Specifically, their phenotype was associated with proliferation, activation and cytotoxic potential as well as significantly impaired production of IFN-γ and other cytokines and reduced basal levels of activated STAT1. A CD38+ CD4+ T cell population with similar features was identified in healthy uninfected individuals, at lower frequency. CD38+ CD4+ T cells could be generated in vitro from CD38- CD4+ T cells after antigenic or mitogenic stimulation. This is the first report of a population of CD38+ CD4+ T cells with a cytotoxic phenotype and markedly impaired IFN-γ capacity in humans. The expansion of this CD38+ CD4+ T population following infection and its significant association with reduced blood-stage parasite burden is consistent with an important functional role for these cells in protective immunity to malaria in humans. Their ubiquitous presence in humans suggests that they may have a broad role in host-pathogen defense. Trial Registration ClinicalTrials.gov clinical trial numbers ACTRN12612000814875, ACTRN12613000565741 and ACTRN12613001040752 PMID:27662621

Activated Pancreatic Stellate Cells Sequester CD8+ T-Cells to Reduce Their Infiltration of the Juxtatumoral Compartment of Pancreatic Ductal Adenocarcinoma

PubMed Central

Ene-Obong, Abasi; Clear, Andrew J.; Watt, Jennifer; Wang, Jun; Fatah, Rewas; Riches, John C.; Marshall, John F.; Chin-Aleong, Joanne; Chelala, Claude; Gribben, John G.; Ramsay, Alan G.; Kocher, Hemant M.

2013-01-01

Background & Aims Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prominent desmoplastic microenvironment that contains many different immune cells. Activated pancreatic stellate cells (PSCs) contribute to the desmoplasia. We investigated whether distinct stromal compartments are differentially infiltrated by different types of immune cells. Method We used tissue microarray analysis to compare immune cell infiltration of different pancreatico-biliary diseased tissues (PDAC, ampullary carcinoma, cholangiocarcinoma, mucinous cystic neoplasm, chronic inflammation, and chronic pancreatitis), and juxtatumoral stromal (<100 μm from tumor) and panstromal compartments. We investigated the association between immune infiltrate and patient survival times. We analyzed T-cell migration and tumor infiltration in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mice, and the effects of all-trans retinoic acid (ATRA) on these processes. Results Juxtatumoral compartments in PDAC samples from 2 independent groups of patients contained increased numbers of myeloperoxidase+ and CD68+ cells, compared with panstromal compartments. However, juxtatumoral compartments of PDACs contained fewer CD8+, FoxP3+, CD56+, or CD20+ cells than panstromal compartments, a distinction absent in ampullary carcinomas and cholangiocarcinomas. Patients with PDACs that had high densities of CD8+ T-cells in the juxtatumoral compartment had longer survival times than patients with lower densities. In KPC mice, administration of ATRA, which renders PSCs quiescent, increased numbers of CD8+ T-cells in juxtatumoral compartments. We found that activated PSCs express cytokines, chemokines, and adhesion molecules that regulate T-cell migration. In vitro migration assays showed that CD8+ T-cells from PDAC patients had increased chemotaxis towards activated PSCs, which secrete CXCL12, compared with quiescent PSC or tumor cells. These effects could be reversed by knockdown of CXCL12 or treatment of PSCs with ATRA. Conclusion Based on studies of human PDAC samples and KPC mice, activated PSCs appear to reduce migration of CD8+ T-cells to juxtatumoral stromal compartments, preventing their access to cancer cells. Deregulated signaling by activated PSCs could prevent an effective anti-tumor immune response. PMID:23891972

Downregulation of proapoptotic Bim augments IL-2-independent T-cell transformation by human T-cell leukemia virus type-1 Tax

PubMed Central

Higuchi, Masaya; Takahashi, Masahiko; Tanaka, Yuetsu; Fujii, Masahiro

2014-01-01

Human T-cell leukemia virus type 1 (HTLV-1), an etiological agent of adult T-cell leukemia, immortalizes and transforms primary human T cells in vitro in both an interleukin (IL)-2-dependent and IL-2-independent manner. Expression of the HTLV-1 oncoprotein Tax transforms the growth of the mouse T-cell line CTLL-2 from being IL-2-dependent to IL-2-independent. Withdrawal of IL-2 from normal activated T cells induces apoptosis, which is mediated through the inducible expression of several proapoptotic proteins, including Bim. In this study, we found that Tax protects IL-2-depleted T cells against Bim-induced apoptosis. Withdrawal of IL-2 from CTLL-2 cells induced a prominent increase in the level of Bim protein in CTLL-2 cells, but not in Tax-transformed CTLL-2 cells. This inhibition of Bim in Tax-transformed CTLL-2 cells was mediated by two mechanisms: downregulation of Bim mRNA and posttranscriptional reduction of Bim protein. Transient expression of Tax in CTLL-2 cells also inhibited IL-2 depletion–induced expression of Bim, however, this decrease in Bim protein expression was not due to downregulation of Bim mRNA, thus indicating that Bim mRNA downregulation in Tax-transformed CTLL-2 occurs only after long-term expression of Tax. Transient expression of Tax in CTLL-2 cells also induced Erk activation, however, this was not involved in the reduction of Bim protein. Knockdown of Bim expression in CTLL-2 cells augmented Tax-induced IL-2-independent transformation. HTLV-1 infection of human T cells also reduced their levels of Bim protein, and restoring Bim expression in HTLV-1-infected cells reduced their proliferation by inducing apoptosis. Taken together, these results indicate that Tax-induced downregulation of Bim in HTLV-1-infected T cells promotes their IL-2-independent growth, thereby supporting the persistence of HTLV-1 infection in vivo. PMID:25175936

Trypanosoma brucei Invasion and T-Cell Infiltration of the Brain Parenchyma in Experimental Sleeping Sickness: Timing and Correlation with Functional Changes

PubMed Central

Laperchia, Claudia; Palomba, Maria; Seke Etet, Paul F.; Rodgers, Jean; Bradley, Barbara; Montague, Paul; Grassi-Zucconi, Gigliola; Bentivoglio, Marina

2016-01-01

Background The timing of Trypanosoma brucei entry into the brain parenchyma to initiate the second, meningoencephalitic stage of human African trypanosomiasis or sleeping sickness is currently debated and even parasite invasion of the neuropil has been recently questioned. Furthermore, the relationship between neurological features and disease stage are unclear, despite the important diagnostic and therapeutic implications. Methodology Using a rat model of chronic Trypanosoma brucei brucei infection we determined the timing of parasite and T-cell neuropil infiltration and its correlation with functional changes. Parasite DNA was detected using trypanosome-specific PCR. Body weight and sleep structure alterations represented by sleep-onset rapid eye movement (SOREM) periods, reported in human and experimental African trypanosomiasis, were monitored. The presence of parasites, as well as CD4+ and CD8+ T-cells in the neuropil was assessed over time in the brain of the same animals by immunocytochemistry and quantitative analyses. Principal findings Trypanosome DNA was present in the brain at day 6 post-infection and increased more than 15-fold by day 21. Parasites and T-cells were observed in the parenchyma from day 9 onwards. Parasites traversing blood vessel walls were observed in the hypothalamus and other brain regions. Body weight gain was reduced from day 7 onwards. SOREM episodes started in most cases early after infection, with an increase in number and duration after parasite neuroinvasion. Conclusion These findings demonstrate invasion of the neuropil over time, after an initial interval, by parasites and lymphocytes crossing the blood-brain barrier, and show that neurological features can precede this event. The data thus challenge the current clinical and cerebrospinal fluid criteria of disease staging. PMID:28002454

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

Effects of Natalizumab Treatment on Foxp3+ T Regulatory Cells

PubMed Central

Buck, Dorothea; Doerck, Sebastian; Einsele, Hermann; Toyka, Klaus V.; Wiendl, Heinz

2008-01-01

Background Natalizumab, a monoclonal humanized antibody targeting the alpha-4 chain of very late activation antigen 4 (VLA-4) exerts impressive therapeutic effects in patients with relapsing-remitting multiple sclerosis. Our objective was to study impacts of Natalizumab therapy on Foxp3+ T regulatory cells (Tregs) in multiple sclerosis (MS) patients. Methodology A combined approach of in vitro and ex vivo experiments using T cells isolated from the peripheral blood of healthy donors and Natalizumab treated MS patients was chosen. We determined binding of Natalizumab and its effects on the frequency, transmigratory behaviour and suppressive function of Tregs. Principal Findings Binding of Natalizumab and expression of CD49d (alpha-4 chain of VLA-4) differed between non-regulatory and regulatory cells. Albeit Foxp3+ Tregs had lower levels of CD49d, Natalizumab blocked the transmigration of Foxp3+ Tregs similar to non-regulatory T cells. The frequency of peripheral blood Tregs was unaffected by Natalizumab treatment. Natalizumab does not alter the suppressive capacity of CD4+CD25highCD127lowFoxp3+ Tregs under in vitro conditions. Furthermore, the impaired function of Tregs in MS patients is not restored by Natalizumab treatment. Conclusions We provide a first detailed analysis of Natalizumab effects on the regulatory T cell population. Our prospective study shows that Foxp3+ Tregs express lower levels of VLA-4 and bind less Natalizumab. We further the understanding of the mechanisms of action of Natalizumab by demonstrating that unlike other immunomodulatory drugs the beneficial therapeutic effects of the monoclonal antibody are largely independent of alterations in Treg frequency or function. PMID:18836525

Contribution of herpesvirus specific CD8 T cells to anti-viral T cell response in humans.

PubMed

Sandalova, Elena; Laccabue, Diletta; Boni, Carolina; Tan, Anthony T; Fink, Katja; Ooi, Eng Eong; Chua, Robert; Shafaeddin Schreve, Bahar; Ferrari, Carlo; Bertoletti, Antonio

2010-08-19

Herpesviruses infect most humans. Their infections can be associated with pathological conditions and significant changes in T cell repertoire but evidences of symbiotic effects of herpesvirus latency have never been demonstrated. We tested the hypothesis that HCMV and EBV-specific CD8 T cells contribute to the heterologous anti-viral immune response. Volume of activated/proliferating virus-specific and total CD8 T cells was evaluated in 50 patients with acute viral infections: 20 with HBV, 12 with Dengue, 12 with Influenza, 3 with Adenovirus infection and 3 with fevers of unknown etiology. Virus-specific (EBV, HCMV, Influenza) pentamer+ and total CD8 T cells were analyzed for activation (CD38/HLA-DR), proliferation (Ki-67/Bcl-2(low)) and cytokine production. We observed that all acute viral infections trigger an expansion of activated/proliferating CD8 T cells, which differs in size depending on the infection but is invariably inflated by CD8 T cells specific for persistent herpesviruses (HCMV/EBV). CD8 T cells specific for other non-related non persistent viral infection (i.e. Influenza) were not activated. IL-15, which is produced during acute viral infections, is the likely contributing mechanism driving the selective activation of herpesvirus specific CD8 T cells. In addition we were able to show that herpesvirus specific CD8 T cells displayed an increased ability to produce the anti-viral cytokine interferon-gamma during the acute phase of heterologous viral infection. Taken together, these data demonstrated that activated herpesvirus specific CD8 T cells inflate the activated/proliferating CD8 T cells population present during acute viral infections in human and can contribute to the heterologous anti-viral T cell response.

Galectin-1 Prevents Infection and Damage Induced by Trypanosoma cruzi on Cardiac Cells

PubMed Central

Benatar, Alejandro F.; García, Gabriela A.; Bua, Jacqeline; Cerliani, Juan P.; Postan, Miriam; Tasso, Laura M.; Scaglione, Jorge; Stupirski, Juan C.; Toscano, Marta A.

2015-01-01

Background Chronic Chagas cardiomyopathy caused by Trypanosoma cruzi is the result of a pathologic process starting during the acute phase of parasite infection. Among different factors, the specific recognition of glycan structures by glycan-binding proteins from the parasite or from the mammalian host cells may play a critical role in the evolution of the infection. Methodology and Principal Findings Here we investigated the contribution of galectin–1 (Gal–1), an endogenous glycan-binding protein abundantly expressed in human and mouse heart, to the pathophysiology of T. cruzi infection, particularly in the context of cardiac pathology. We found that exposure of HL–1 cardiac cells to Gal–1 reduced the percentage of infection by two different T. cruzi strains, Tulahuén (TcVI) and Brazil (TcI). In addition, Gal–1 prevented exposure of phosphatidylserine and early events in the apoptotic program by parasite infection on HL–1 cells. These effects were not mediated by direct interaction with the parasite surface, suggesting that Gal–1 may act through binding to host cells. Moreover, we also observed that T. cruzi infection altered the glycophenotype of cardiac cells, reducing binding of exogenous Gal–1 to the cell surface. Consistent with these data, Gal–1 deficient (Lgals1 -/-) mice showed increased parasitemia, reduced signs of inflammation in heart and skeletal muscle tissues, and lower survival rates as compared to wild-type (WT) mice in response to intraperitoneal infection with T. cruzi Tulahuén strain. Conclusion/Significance Our results indicate that Gal–1 modulates T. cruzi infection of cardiac cells, highlighting the relevance of galectins and their ligands as regulators of host-parasite interactions. PMID:26451839

Innate T cell responses in human gut.

PubMed

Meresse, Bertrand; Cerf-Bensussan, Nadine

2009-06-01

One arm of the gut-associated immune system is represented by a vast collection of T lymphocytes which participate in the subtle interplay between innate and adaptive immune mechanisms and maintain homeostasis at the main body external surface. Mounting data are providing exciting new insight into the innate-like mechanisms which enable intestinal T cells to rapidly sense local conditions and which broaden the spectrum of their functions and regulation at this strategic location. Herein we discuss how innate-like T cell recognition by unconventional T cell subsets and expression of innate NK receptors might modulate immune T cell responses in the human normal or diseased intestine.

Pathogenic bacteria induce colonic PepT1 expression: an implication in host defense response

PubMed Central

Nguyen, Hang Thi Thu; Dalmasso, Guillaume; Powell, Kimberly R.; Yan, Yutao; Bhatt, Shantanu; Kalman, Daniel; Sitaraman, Shanthi; Merlin, Didier

2009-01-01

Background & Aims Expression of the di/tripeptide transporter PepT1 has been observed in the colon under inflammatory conditions, however, the inducing factors and underlying mechanisms remain unknown. Here, we address the effects of pathogenic bacteria on colonic PepT1 expression together with its functional consequences. Methods Human colonic HT29-Cl.19A cells were infected with the attaching and effacing (A/E) enteropathogenic E. coli (EPEC). Wild-type and PepT1 transgenic mice or cultured colonic tissues derived from these mice were infected with Citrobacter rodentium, a murine A/E pathogen related to EPEC. Results EPEC induced PepT1 expression and activity in HT29-Cl.19A cells by intimately attaching to host cells through lipid rafts. Induction of PepT1 expression by EPEC required the transcription factor Cdx2. PepT1 expression reduced binding of EPEC to lipid rafts, as well as activation of NF-κB and MAP kinase and production of IL-8. Accordingly, ex vivo and in vivo experiments revealed that C. rodentium induced colonic PepT1 expression and that, compared to their wild-type counterparts, PepT1 transgenic mice infected with C. rodentium exhibited decreased bacterial colonization, production of pro-inflammatory cytokines, and neutrophil infiltration into the colon. Conclusions Our findings demonstrate a molecular mechanism underlying the regulation of colonic PepT1 expression under pathological conditions and reveal a novel role for PepT1 in host defense via its capacity to modulate bacterial-epithelial interactions and intestinal inflammation. PMID:19549526

Influence of antiretroviral therapy on programmed death-1 (CD279) expression on T cells in lymph nodes of human immunodeficiency virus-infected individuals.

PubMed

Ehrhard, Simone; Wernli, Marion; Dürmüller, Ursula; Battegay, Manuel; Gudat, Fred; Erb, Peter

2009-10-01

Human immunodeficiency virus infection leads to T-cell exhaustion and involution of lymphoid tissue. Recently, the programmed death-1 pathway was found to be crucial for virus-specific T-cell exhaustion during human immunodeficiency virus infection. Programmed death-1 expression was elevated on human immunodeficiency virus-specific peripheral blood CD8+ and CD4+ T cells and correlated with disease severity. During human immunodeficiency infection, lymphoid tissue acts as a major viral reservoir and is an important site for viral replication, but it is also essential for regulatory processes important for immune recovery. We compared programmed death-1 expression in 2 consecutive inguinal lymph nodes of 14 patients, excised before antiretroviral therapy (antiretroviral therapy as of 1997-1999) and 16 to 20 months under antiretroviral therapy. In analogy to lymph nodes of human immunodeficiency virus-negative individuals, in all treated patients, the germinal center area decreased, whereas the number of germinal centers did not significantly change. Programmed death-1 expression was mostly found in germinal centers. The absolute extent of programmed death 1 expression per section was not significantly altered after antiretroviral therapy resulting in a significant-relative increase of programmed death 1 per shrunken germinal center. In colocalization studies, CD45R0+ cells that include helper/inducer T cells strongly expressed programmed death-1 before and during therapy, whereas CD8+ T cells, fewer in numbers, showed a weak expression for programmed death-1. Thus, although antiretroviral therapy seems to reduce the number of programmed death-1-positive CD8+ T lymphocytes within germinal centers, it does not down-regulate programmed death-1 expression on the helper/inducer T-cell subset that may remain exhausted and therefore unable to trigger immune recovery.

Perspectives on Regulatory T Cell Therapies.

PubMed

Probst-Kepper, Michael; Kröger, Andrea; Garritsen, Henk S P; Buer, Jan

2009-01-01

Adoptive transfer in animal models clearly indicate an essential role of CD4+ CD25+ FOXP3+ regulatory T (T(reg)) cells in prevention and treatment of autoimmune and graft-versus-host disease. Thus, T(reg) cell therapies and development of drugs that specifically enhance T(reg) cell function and development represent promising tools to establish dominant tolerance. So far, lack of specific markers to differentiate human T(reg) cells from activated CD4+ CD25+ effector T cells, which also express FOXP3 at different levels, hampered such an approach. Recent identification of the orphan receptor glycoprotein-A repetitions predominant (GARP or LRRC32) as T(reg) cell-specific key molecule that dominantly controls FOXP3 via a positive feedback loop opens up new perspectives for molecular and cellular therapies. This brief review focuses on the role of GARP as a safeguard of a complex regulatory network of human T(reg) cells and its implications for regulatory T cell therapies in autoimmunity and graft-versus-host disease.

Human leukemia antigen-A*0201-restricted epitopes of human endogenous retrovirus W family envelope (HERV-W env) induce strong cytotoxic T lymphocyte responses.

PubMed

Tu, Xiaoning; Li, Shan; Zhao, Lijuan; Xiao, Ran; Wang, Xiuling; Zhu, Fan

2017-08-01

Human endogenous retrovirus W family (HERV-W) envelope (env) has been reported to be related to several human diseases, including autoimmune disorders, and it could activate innate immunity. However, there are no reports investigating whether human leukemia antigen (HLA)-A*0201 + restriction is involved in the immune response caused by HERV-W env in neuropsychiatric diseases. In the present study, HERV-W env-derived epitopes presented by HLA-A*0201 are described with the potential for use in adoptive immunotherapy. Five peptides displaying HLA-A*0201-binding motifs were predicted using SYFEPITHI and BIMAS, and synthesized. A CCK-8 assay showed peptides W, Q and T promoted lymphocyte proliferation. Stimulation of peripheral blood mononuclear cells from HLA-A*0201 + donors with each of these peptides induced peptide-specific CD8 + T cells. High numbers of IFN-γ-secreting T cells were also detectable after several weekly stimulations with W, Q and T. Besides lysis of HERV-W env-loaded target cells, specific apoptosis was also observed. These data demonstrate that human T cells can be sensitized toward HERV-W env peptides (W, Q and T) and, moreover, pose a high killing potential toward HERV-W env-expressing U251 cells. In conclusion, peptides W Q and T, which are HERV-W env antigenic epitopes, have both antigenicity and immunogenicity, and can cause strong T cell immune responses. Our data strengthen the view that HERV-W env should be considered as an autoantigen that can induce autoimmunity in neuropsychiatric diseases, such as multiple sclerosis and schizophrenia. These data might provide an experimental foundation for a HERV-W env peptide vaccine and new insight into the treatment of neuropsychiatric diseases.

Human T Lymphocytes Are Permissive for Dengue Virus Replication.

PubMed

Silveira, Guilherme F; Wowk, Pryscilla F; Cataneo, Allan H D; Dos Santos, Paula F; Delgobo, Murilo; Stimamiglio, Marco A; Lo Sarzi, Maria; Thomazelli, Ana Paula F S; Conchon-Costa, Ivete; Pavanelli, Wander R; Antonelli, Lis R V; Báfica, André; Mansur, Daniel S; Dos Santos, Claudia N Duarte; Bordignon, Juliano

2018-05-15

Dengue virus (DV) infection can cause either a self-limiting flu-like disease or a threatening hemorrhage that may evolve to shock and death. A variety of cell types, such as dendritic cells, monocytes, and B cells, can be infected by DV. However, despite the role of T lymphocytes in the control of DV replication, there remains a paucity of information on possible DV-T cell interactions during the disease course. In the present study, we have demonstrated that primary human naive CD4 + and CD8 + T cells are permissive for DV infection. Importantly, both T cell subtypes support viral replication and secrete viable virus particles. DV infection triggers the activation of both CD4 + and CD8 + T lymphocytes, but preactivation of T cells reduces the susceptibility of T cells to DV infection. Interestingly, the cytotoxicity-inducing protein granzyme A is highly secreted by human CD4 + but not CD8 + T cells after exposure to DV in vitro Additionally, using annexin V and polycaspase assays, we have demonstrated that T lymphocytes, in contrast to monocytes, are resistant to DV-induced apoptosis. Strikingly, both CD4 + and CD8 + T cells were found to be infected with DV in acutely infected dengue patients. Together, these results show that T cells are permissive for DV infection in vitro and in vivo , suggesting that this cell population may be a viral reservoir during the acute phase of the disease. IMPORTANCE Infection by dengue virus (DV) causes a flu-like disease that can evolve to severe hemorrhaging and death. T lymphocytes are important cells that regulate antibody secretion by B cells and trigger the death of infected cells. However, little is known about the direct interaction between DV and T lymphocytes. Here, we show that T lymphocytes from healthy donors are susceptible to infection by DV, leading to cell activation. Additionally, T cells seem to be resistant to DV-induced apoptosis, suggesting a potential role as a viral reservoir in humans. Finally, we show that both CD4 + and CD8 + T lymphocytes from acutely infected DV patients are infected by DV. Our results raise new questions about DV pathogenesis and vaccine development. Copyright © 2018 American Society for Microbiology.

Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells.

PubMed

Wang, Rui; Kozhaya, Lina; Mercer, Frances; Khaitan, Alka; Fujii, Hodaka; Unutmaz, Derya

2009-08-11

The molecules that define human regulatory T cells (Tregs) phenotypically and functionally remain to be fully characterized. We recently showed that activated human Tregs express mRNA for a transmembrane protein called glycoprotein A repetitions predominant (GARP, or LRRC32). Here, using a GARP-specific mAb, we demonstrate that expression of GARP on activated Tregs correlates with their suppressive capacity. However, GARP was not induced on T cells activated in the presence of TGFbeta, which expressed high levels of FOXP3 and lacked suppressive function. Ectopic expression of FOXP3 in conventional T cells was also insufficient for induction of GARP expression in most donors. Functionally, silencing GARP in Tregs only moderately attenuated their suppressive activity. CD25+ T cells sorted for high GARP expression displayed more potent suppressive activity compared with CD25+GARP- cells. Remarkably, CD25+GARP- T cells expanded in culture contained 3-5 fold higher IL-17-secreting cells compared with either CD25+GARP+ or CD25-GARP- cells, suggesting that high GARP expression can potentially discriminate Tregs from those that have switched to Th17 lineage. We also determined whether GARP expression correlates with FOXP3-expressing T cells in human immunodeficiency virus (HIV) -infected subjects. A subset of HIV+ individuals with high percentages of FOXP3+ T cells did not show proportionate increase in GARP+ T cells. This finding suggests that higher FOXP3 levels observed in these HIV+ individuals is possibly due to immune activation rather than to an increase in Tregs. Our findings highlight the significance of GARP both in dissecting duality of Treg/Th17 cell differentiation and as a marker to identify bona fide Tregs during diseases with chronic immune activation.

Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells

PubMed Central

Wang, Rui; Kozhaya, Lina; Mercer, Frances; Khaitan, Alka; Fujii, Hodaka; Unutmaz, Derya

2009-01-01

The molecules that define human regulatory T cells (Tregs) phenotypically and functionally remain to be fully characterized. We recently showed that activated human Tregs express mRNA for a transmembrane protein called glycoprotein A repetitions predominant (GARP, or LRRC32). Here, using a GARP-specific mAb, we demonstrate that expression of GARP on activated Tregs correlates with their suppressive capacity. However, GARP was not induced on T cells activated in the presence of TGFβ, which expressed high levels of FOXP3 and lacked suppressive function. Ectopic expression of FOXP3 in conventional T cells was also insufficient for induction of GARP expression in most donors. Functionally, silencing GARP in Tregs only moderately attenuated their suppressive activity. CD25+ T cells sorted for high GARP expression displayed more potent suppressive activity compared with CD25+GARP− cells. Remarkably, CD25+GARP− T cells expanded in culture contained 3–5 fold higher IL-17-secreting cells compared with either CD25+GARP+ or CD25−GARP− cells, suggesting that high GARP expression can potentially discriminate Tregs from those that have switched to Th17 lineage. We also determined whether GARP expression correlates with FOXP3-expressing T cells in human immunodeficiency virus (HIV) −infected subjects. A subset of HIV+ individuals with high percentages of FOXP3+ T cells did not show proportionate increase in GARP+ T cells. This finding suggests that higher FOXP3 levels observed in these HIV+ individuals is possibly due to immune activation rather than to an increase in Tregs. Our findings highlight the significance of GARP both in dissecting duality of Treg/Th17 cell differentiation and as a marker to identify bona fide Tregs during diseases with chronic immune activation. PMID:19666573

Upregulation of Glucose Uptake and Hexokinase Activity of Primary Human CD4+ T Cells in Response to Infection with HIV-1

PubMed Central

Kavanagh Williamson, Maia; Coombes, Naomi; Juszczak, Florian; Athanasopoulos, Marios; Khan, Mariam B.; Eykyn, Thomas R.; Srenathan, Ushani; Dias Zeidler, Julianna; Huthoff, Hendrik

2018-01-01

Infection of primary CD4+ T cells with HIV-1 coincides with an increase in glycolysis. We investigated the expression of glucose transporters (GLUT) and glycolytic enzymes in human CD4+ T cells in response to infection with HIV-1. We demonstrate the co-expression of GLUT1, GLUT3, GLUT4, and GLUT6 in human CD4+ T cells after activation, and their concerted overexpression in HIV-1 infected cells. The investigation of glycolytic enzymes demonstrated activation-dependent expression of hexokinases HK1 and HK2 in human CD4+ T cells, and a highly significant increase in cellular hexokinase enzyme activity in response to infection with HIV-1. HIV-1 infected CD4+ T cells showed a marked increase in expression of HK1, as well as the functionally related voltage-dependent anion channel (VDAC) protein, but not HK2. The elevation of GLUT, HK1, and VDAC expression in HIV-1 infected cells mirrored replication kinetics and was dependent on virus replication, as evidenced by the use of reverse transcription inhibitors. Finally, we demonstrated that the upregulation of HK1 in HIV-1 infected CD4+ T cells is independent of the viral accessory proteins Vpu, Vif, Nef, and Vpr. Though these data are consistent with HIV-1 dependency on CD4+ T cell glucose metabolism, a cellular response mechanism to infection cannot be ruled out. PMID:29518929

T helper 1 immunity requires complement-driven NLRP3 inflammasome activity in CD4⁺ T cells.

PubMed

Arbore, Giuseppina; West, Erin E; Spolski, Rosanne; Robertson, Avril A B; Klos, Andreas; Rheinheimer, Claudia; Dutow, Pavel; Woodruff, Trent M; Yu, Zu Xi; O'Neill, Luke A; Coll, Rebecca C; Sher, Alan; Leonard, Warren J; Köhl, Jörg; Monk, Pete; Cooper, Matthew A; Arno, Matthew; Afzali, Behdad; Lachmann, Helen J; Cope, Andrew P; Mayer-Barber, Katrin D; Kemper, Claudia

2016-06-17

The NLRP3 inflammasome controls interleukin-1β maturation in antigen-presenting cells, but a direct role for NLRP3 in human adaptive immune cells has not been described. We found that the NLRP3 inflammasome assembles in human CD4(+) T cells and initiates caspase-1-dependent interleukin-1β secretion, thereby promoting interferon-γ production and T helper 1 (T(H)1) differentiation in an autocrine fashion. NLRP3 assembly requires intracellular C5 activation and stimulation of C5a receptor 1 (C5aR1), which is negatively regulated by surface-expressed C5aR2. Aberrant NLRP3 activity in T cells affects inflammatory responses in human autoinflammatory disease and in mouse models of inflammation and infection. Our results demonstrate that NLRP3 inflammasome activity is not confined to "innate immune cells" but is an integral component of normal adaptive T(H)1 responses. Copyright © 2016, American Association for the Advancement of Science.

L1 stimulation of human glioma cell motility correlates with FAK activation

PubMed Central

Yang, Muhua; Li, Yupei; Chilukuri, Kalyani; Brady, Owen A.; Boulos, Magdy I.; Kappes, John C.

2011-01-01

The neural adhesion/recognition protein L1 (L1CAM; CD171) has been shown or implicated to function in stimulation of cell motility in several cancer types, including high-grade gliomas. Our previous work demonstrated the expression and function of L1 protein in stimulation of cell motility in rat glioma cells. However, the mechanism of this stimulation is still unclear. This study further investigated the function of L1 and L1 proteolysis in human glioblastoma multiforme (GBM) cell migration and invasion, as well as the mechanism of this stimulation. L1 mRNA was found to be present in human T98G GBM cell line but not in U-118 MG grade III human glioma cell line. L1 protein expression, proteolysis, and release were found in T98G cells and human surgical GBM cells by Western blotting. Exosome-like vesicles released by T98G cells were purified and contained full-length L1. In a scratch assay, T98G cells that migrated into the denuded scratch area exhibited upregulation of ADAM10 protease expression coincident with loss of surface L1. GBM surgical specimen cells exhibited a similar loss of cell surface L1 when xenografted into the chick embryo brain. When lentivirally introduced shRNA was used to attenuate L1 expression, such T98G/shL1 cells exhibited significantly decreased cell motility by time lapse microscopy in our quantitative Super Scratch assay. These cells also showed a decrease in FAK activity and exhibited increased focal complexes. L1 binding integrins which activate FAK were found in T98G and U-118 MG cells. Addition of L1 ectodomain-containing media (1) rescued the decreased cell motility of T98G/shL1 cells and (2) increased cell motility of U-118 MG cells but (3) did not further increase T98G cell motility. Injection of L1-attenuated T98G/shL1 cells into embryonic chick brains resulted in the absence of detectable invasion compared to control cells which invaded brain tissue. These studies support a mechanism where glioma cells at the edge of a cell mass upregulate ADAM10 to proteolyze surface L1 and the resultant ectodomain increases human glioma cell migration and invasion by binding to integrin receptors, activating FAK, and increasing turnover of focal complexes. PMID:21373966

L1 stimulation of human glioma cell motility correlates with FAK activation.

PubMed

Yang, Muhua; Li, Yupei; Chilukuri, Kalyani; Brady, Owen A; Boulos, Magdy I; Kappes, John C; Galileo, Deni S

2011-10-01

The neural adhesion/recognition protein L1 (L1CAM; CD171) has been shown or implicated to function in stimulation of cell motility in several cancer types, including high-grade gliomas. Our previous work demonstrated the expression and function of L1 protein in stimulation of cell motility in rat glioma cells. However, the mechanism of this stimulation is still unclear. This study further investigated the function of L1 and L1 proteolysis in human glioblastoma multiforme (GBM) cell migration and invasion, as well as the mechanism of this stimulation. L1 mRNA was found to be present in human T98G GBM cell line but not in U-118 MG grade III human glioma cell line. L1 protein expression, proteolysis, and release were found in T98G cells and human surgical GBM cells by Western blotting. Exosome-like vesicles released by T98G cells were purified and contained full-length L1. In a scratch assay, T98G cells that migrated into the denuded scratch area exhibited upregulation of ADAM10 protease expression coincident with loss of surface L1. GBM surgical specimen cells exhibited a similar loss of cell surface L1 when xenografted into the chick embryo brain. When lentivirally introduced shRNA was used to attenuate L1 expression, such T98G/shL1 cells exhibited significantly decreased cell motility by time lapse microscopy in our quantitative Super Scratch assay. These cells also showed a decrease in FAK activity and exhibited increased focal complexes. L1 binding integrins which activate FAK were found in T98G and U-118 MG cells. Addition of L1 ectodomain-containing media (1) rescued the decreased cell motility of T98G/shL1 cells and (2) increased cell motility of U-118 MG cells but (3) did not further increase T98G cell motility. Injection of L1-attenuated T98G/shL1 cells into embryonic chick brains resulted in the absence of detectable invasion compared to control cells which invaded brain tissue. These studies support a mechanism where glioma cells at the edge of a cell mass upregulate ADAM10 to proteolyze surface L1 and the resultant ectodomain increases human glioma cell migration and invasion by binding to integrin receptors, activating FAK, and increasing turnover of focal complexes.

In Vitro Measles Virus Infection of Human Lymphocyte Subsets Demonstrates High Susceptibility and Permissiveness of both Naive and Memory B Cells.

PubMed

Laksono, Brigitta M; Grosserichter-Wagener, Christina; de Vries, Rory D; Langeveld, Simone A G; Brem, Maarten D; van Dongen, Jacques J M; Katsikis, Peter D; Koopmans, Marion P G; van Zelm, Menno C; de Swart, Rik L

2018-04-15

Measles is characterized by a transient immune suppression, leading to an increased risk of opportunistic infections. Measles virus (MV) infection of immune cells is mediated by the cellular receptor CD150, expressed by subsets of lymphocytes, dendritic cells, macrophages, and thymocytes. Previous studies showed that human and nonhuman primate memory T cells express higher levels of CD150 than naive cells and are more susceptible to MV infection. However, limited information is available about the CD150 expression and relative susceptibility to MV infection of B-cell subsets. In this study, we assessed the susceptibility and permissiveness of naive and memory T- and B-cell subsets from human peripheral blood or tonsils to in vitro MV infection. Our study demonstrates that naive and memory B cells express CD150, but at lower frequencies than memory T cells. Nevertheless, both naive and memory B cells proved to be highly permissive to MV infection. Furthermore, we assessed the susceptibility and permissiveness of various functionally distinct T and B cells, such as helper T (T H ) cell subsets and IgG- and IgA-positive memory B cells, in peripheral blood and tonsils. We demonstrated that T H 1T H 17 cells and plasma and germinal center B cells were the subsets most susceptible and permissive to MV infection. Our study suggests that both naive and memory B cells, along with several other antigen-experienced lymphocytes, are important target cells of MV infection. Depletion of these cells potentially contributes to the pathogenesis of measles immune suppression. IMPORTANCE Measles is associated with immune suppression and is often complicated by bacterial pneumonia, otitis media, or gastroenteritis. Measles virus infects antigen-presenting cells and T and B cells, and depletion of these cells may contribute to lymphopenia and immune suppression. Measles has been associated with follicular exhaustion in lymphoid tissues in humans and nonhuman primates, emphasizing the importance of MV infection of B cells in vivo However, information on the relative susceptibility of B-cell subsets is scarce. Here, we compared the susceptibility and permissiveness to in vitro MV infection of human naive and memory T- and B-cell subsets isolated from peripheral blood or tonsils. Our results demonstrate that both naive and memory B cells are more permissive to MV infection than T cells. The highest infection levels were detected in plasma cells and germinal center B cells, suggesting that infection and depletion of these populations contribute to reduced host resistance. Copyright © 2018 Laksono et al.

Effects of 13 T Static Magnetic Fields (SMF) in the Cell Cycle Distribution and Cell Viability in Immortalized Hamster Cells and Human Primary Fibroblasts Cells

NASA Astrophysics Data System (ADS)

Zhao, Guoping; Chen, Shaopeng; Zhao, Ye; Zhu, Lingyan; Huang, Pei; Bao, Lingzhi; Wang, Jun; Wang, Lei; Wu, Lijun; Wu, Yuejin; Xu, An

2010-02-01

Magnetic resonance image (MRI) systems with a much higher magnetic flux density were developed and applied for potential use in medical diagnostic. Recently, much attention has been paid to the biological effects of static, strong magnetic fields (SMF). With the 13 T SMF facility in the Institute of Plasma Physics, Chinese Academy of Sciences, the present study focused on the cellular effects of the SMF with 13 T on the cell viability and the cell cycle distribution in immortalized hamster cells, such as human-hamster hybrid (AL) cells, Chinese hamster ovary (CHO) cells, DNA double-strand break repair deficient mutant (XRS-5) cells, and human primary skin fibroblasts (AG1522) cells. It was found that the exposure of 13 T SMF had less effect on the colony formation in either nonsynchronized or synchronized AL cells. Moreover, as compared to non-exposed groups, there were slight differences in the cell cycle distribution no matter in either synchronized or nonsynchronized immortalized hamster cells after exposure to 13 T SMF. However, it should be noted that the percentage of exposed AG1522 cells at G0/G1 phase was decreased by 10% as compared to the controls. Our data indicated that although 13 T SMF had minimal effects in immortalized hamster cells, the cell cycle distribution was slightly modified by SMF in human primary fibroblasts.

Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

PubMed Central

Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

2015-01-01

Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

Downregulation of cell surface molecules during noncytopathic infection of T cells with human immunodeficiency virus.

PubMed Central

Stevenson, M; Zhang, X H; Volsky, D J

1987-01-01

Noncytopathic infection of human T-lymphoid cell line CR-10 with human immunodeficiency virus (HIV) (CEM-N1T isolate) resulted in a gradual loss of cell surface receptors for OKT4/OKT4A (HIV receptor), OKT8, OKT3, and OKT11 but not for OKT9 (transferrin receptor) within 10 days after infection. Surface receptor decline was accompanied by a rapid increase in HIV antigens and mRNA expression. Multireceptor downregulation was also observed in three T-lymphoid cell lines (MT-4, CEM, and HBD-1) cytopathically infected with the HIV/N1T virus and in HUT-78 cells infected with the HIV/SF-2 isolate. HIV-infected and uninfected CR-10 cells contained similar levels of mRNAs coding for T3, T8, T9, T11, HLA-A2, and HLA-B7 proteins. By densitometry, fully infected CR-10 cells showed approximately 75% reduction in T4 and tubulin (beta chain) mRNA levels when compared with uninfected CR-10 cells. No such reduction was detected in HIV-infected MT-4 and HBD-1 cells. A T-cell receptor gene (beta chain) rearrangement study revealed that no distinct CR-10 subpopulation was selected out upon infection with HIV. Our results suggest that the reduction in cell surface receptors observed between 1 and 2 weeks postinfection cannot be directly attributed to similar reductions in mRNA levels coding for these receptor proteins. We conclude that HIV infection induces posttranscriptional downregulation of several T-cell surface receptors. Images PMID:3500327

Hemicholinium-3 sensitive choline transport in human T lymphocytes: Evidence for use as a proxy for brain choline transporter (CHT) capacity.

PubMed

Koshy Cherian, Ajeesh; Parikh, Vinay; Wu, Qi; Mao-Draayer, Yang; Wang, Qin; Blakely, Randy D; Sarter, Martin

2017-09-01

The synaptic uptake of choline via the high-affinity, hemicholinium-3-dependent choline transporter (CHT) strongly influences the capacity of cholinergic neurons to sustain acetylcholine (ACh) synthesis and release. To advance research on the impact of CHT capacity in humans, we established the presence of the neuronal CHT protein in human T lymphocytes. Next, we demonstrated CHT-mediated choline transport in human T cells. To address the validity of T cell-based choline uptake as a proxy for brain CHT capacity, we isolated T cells from the spleen, and synaptosomes from cortex and striatum, of wild type and CHT-overexpressing mice (CHT-OXP). Choline uptake capacity in T cells from CHT-OXP mice was two-fold higher than in wild type mice, mirroring the impact of CHT over-expression on synaptosomal CHT-mediated choline uptake. Monitoring T lymphocyte CHT protein and activity may be useful for estimating human CNS cholinergic capacity and for testing hypotheses concerning the contribution of CHT and, more generally, ACh signaling in cognition, neuroinflammation and disease. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Combined Omics Approach to Generate the Surface Atlas of Human Naive CD4+ T Cells during Early T-Cell Receptor Activation*

PubMed Central

Graessel, Anke; Hauck, Stefanie M.; von Toerne, Christine; Kloppmann, Edda; Goldberg, Tatyana; Koppensteiner, Herwig; Schindler, Michael; Knapp, Bettina; Krause, Linda; Dietz, Katharina; Schmidt-Weber, Carsten B.; Suttner, Kathrin

2015-01-01

Naive CD4+ T cells are the common precursors of multiple effector and memory T-cell subsets and possess a high plasticity in terms of differentiation potential. This stem-cell-like character is important for cell therapies aiming at regeneration of specific immunity. Cell surface proteins are crucial for recognition and response to signals mediated by other cells or environmental changes. Knowledge of cell surface proteins of human naive CD4+ T cells and their changes during the early phase of T-cell activation is urgently needed for a guided differentiation of naive T cells and may support the selection of pluripotent cells for cell therapy. Periodate oxidation and aniline-catalyzed oxime ligation technology was applied with subsequent quantitative liquid chromatography-tandem MS to generate a data set describing the surface proteome of primary human naive CD4+ T cells and to monitor dynamic changes during the early phase of activation. This led to the identification of 173 N-glycosylated surface proteins. To independently confirm the proteomic data set and to analyze the cell surface by an alternative technique a systematic phenotypic expression analysis of surface antigens via flow cytometry was performed. This screening expanded the previous data set, resulting in 229 surface proteins, which were expressed on naive unstimulated and activated CD4+ T cells. Furthermore, we generated a surface expression atlas based on transcriptome data, experimental annotation, and predicted subcellular localization, and correlated the proteomics result with this transcriptional data set. This extensive surface atlas provides an overall naive CD4+ T cell surface resource and will enable future studies aiming at a deeper understanding of mechanisms of T-cell biology allowing the identification of novel immune targets usable for the development of therapeutic treatments. PMID:25991687

A novel anti-EMMPRIN function-blocking antibody reduces T cell proliferation and neurotoxicity: relevance to multiple sclerosis.

PubMed

Agrawal, Smriti M; Silva, Claudia; Wang, Janet; Tong, Jade Pui-Wai; Yong, V Wee

2012-04-05

Extracellular matrix metalloproteinase inducer (EMMPRIN; CD147, basigin) is an inducer of the expression of several matrix metalloproteinases (MMPs). We reported previously that blocking EMMPRIN activity reduced neuroinflammation and severity of disease in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). To improve upon EMMPRIN blockade, and to help unravel the biological functions of EMMPRIN in inflammatory disorders, we have developed several anti-EMMPRIN monoclonal antibodies. Of these monoclonal antibodies, a particular one, clone 10, was efficient in binding mouse and human cells using several methods of detection. The specificity of clone 10 was demonstrated by its lack of staining of EMMPRIN-null embryos compared to heterozygous and wild-type mouse samples. Functionally, human T cells activated with anti-CD3 and anti-CD28 elevated their expression of EMMPRIN and the treatment of these T cells with clone 10 resulted in decreased proliferation and matrix metalloproteinase- 9 (MMP-9) production. Activated human T cells were toxic to human neurons in culture and clone 10 pretreatment reduced T cell cytotoxicity correspondent with decrease of granzyme B levels within T cells. In vivo, EAE mice treated with clone 10 had a markedly reduced disease score compared to mice treated with IgM isotype control. We have produced a novel anti-EMMPRIN monoclonal antibody that blocks several aspects of T cell activity, thus highlighting the multiple roles of EMMPRIN in T cell biology. Moreover, clone 10 reduces EAE scores in mice compared to controls, and has activity on human cells, potentially allowing for the testing of anti-EMMPRIN treatment not only in EAE, but conceivably also in MS.

TNF-α blockade induces IL-10 expression in human CD4+ T cells

NASA Astrophysics Data System (ADS)

Evans, Hayley G.; Roostalu, Urmas; Walter, Gina J.; Gullick, Nicola J.; Frederiksen, Klaus S.; Roberts, Ceri A.; Sumner, Jonathan; Baeten, Dominique L.; Gerwien, Jens G.; Cope, Andrew P.; Geissmann, Frederic; Kirkham, Bruce W.; Taams, Leonie S.

2014-02-01

IL-17+ CD4+ T (Th17) cells contribute to the pathogenesis of several human inflammatory diseases. Here we demonstrate that TNF inhibitor (TNFi) drugs induce the anti-inflammatory cytokine IL-10 in CD4+ T cells including IL-17+ CD4+ T cells. TNFi-mediated induction of IL-10 in IL-17+ CD4+ T cells is Treg-/Foxp3-independent, requires IL-10 and is overcome by IL-1β. TNFi-exposed IL-17+ CD4+ T cells are molecularly and functionally distinct, with a unique gene signature characterized by expression of IL10 and IKZF3 (encoding Aiolos). We show that Aiolos binds conserved regions in the IL10 locus in IL-17+ CD4+ T cells. Furthermore, IKZF3 and IL10 expression levels correlate in primary CD4+ T cells and Aiolos overexpression is sufficient to drive IL10 in these cells. Our data demonstrate that TNF-α blockade induces IL-10 in CD4+ T cells including Th17 cells and suggest a role for the transcription factor Aiolos in the regulation of IL-10 in CD4+ T cells.

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.

Characterization of resident lymphocytes in human pancreatic islets

PubMed Central

Radenkovic, M.; Uvebrant, K.; Skog, O.; Sarmiento, L.; Avartsson, J.; Storm, P.; Vickman, P.; Bertilsson, P.‐A.; Fex, M.; Korgsgren, O.

2016-01-01

Summary The current view of type 1 diabetes (T1D) is that it is an immune‐mediated disease where lymphocytes infiltrate the pancreatic islets, promote killing of beta cells and cause overt diabetes. Although tissue resident immune cells have been demonstrated in several organs, the composition of lymphocytes in human healthy pancreatic islets have been scarcely studied. Here we aimed to investigate the phenotype of immune cells associated with human islets of non‐diabetic organ donors. A flow cytometry analysis of isolated islets from perfused pancreases (n = 38) was employed to identify alpha, beta, T, natural killer (NK) and B cells. Moreover, the expression of insulin and glucagon transcripts was evaluated by RNA sequencing. Up to 80% of the lymphocytes were CD3+ T cells with a remarkable bias towards CD8+ cells. Central memory and effector memory phenotypes dominated within the CD8+ and CD4+ T cells and most CD8+ T cells were positive for CD69 and up to 50–70% for CD103, both markers of resident memory cells. The frequency of B and NK cells was low in most islet preparations (12 and 3% of CD45+ cells, respectively), and the frequency of alpha and beta cells varied between donors and correlated clearly with insulin and glucagon mRNA expression. In conclusion, we demonstrated the predominance of canonical tissue resident memory CD8+ T cells associated with human islets. We believe that these results are important to understand more clearly the immunobiology of human islets and the disease‐related phenotypes observed in diabetes. PMID:27783386

Antitumor activity of EGFR-specific CAR T cells against non-small-cell lung cancer cells in vitro and in mice.

PubMed

Li, He; Huang, Yao; Jiang, Du-Qing; Cui, Lian-Zhen; He, Zhou; Wang, Chao; Zhang, Zhi-Wei; Zhu, Hai-Li; Ding, Yong-Mei; Li, Lin-Fang; Li, Qiang; Jin, Hua-Jun; Qian, Qi-Jun

2018-02-07

Effective control of non-small-cell lung cancer (NSCLC) remains clinically challenging, especially during advanced stages of the disease. This study developed an adoptive T-cell treatment through expression of a chimeric antigen receptor (CAR) to target human epidermal growth factor receptor (EGFR) in NSCLC. We optimized the non-viral piggyBac transposon system to engineer human T cells for the expression of EGFR-CAR, consisting of EGFR scFv, transmembrane domain, and intracellular 4-1BB-CD3ζ signaling domains. The modified CAR T cells exhibited expansion capability and anticancer efficacy in a time- and antigen-dependent manner in vitro as well as regression of EGFR-positive human lung cancer xenografts in vivo. EGFR-CAR T therapy is a promising strategy to improve the efficacy and potency of the adoptive immunotherapy in NSCLC. Moreover, EGFR-CAR T therapy could become a clinical application for NSCLC patients in the future.

CD8 T cells and Mycobacterium tuberculosis infection

PubMed Central

Lin, Philana Ling; Flynn, JoAnne L.

2015-01-01

Tuberculosis is primarily a respiratory disease that is caused by Mycobacterium tuberculosis. M. tuberculosis can persist and replicate in macrophages in vivo, usually in organized cellular structures called granulomas. There is substantial evidence for the importance of CD4 T cells in control of tuberculosis, but the evidence for a requirement for CD8 T cells in this infection has not been proven in humans. However, animal model data support a non-redundant role for CD8 T cells in control of M. tuberculosis infection, and in humans, infection with this pathogen leads to generation of specific CD8 T cell responses. These responses include classical (MHC Class I restricted) and non-classical CD8 T cells. Here, we discuss the potential roles of CD8 T cells in defense against tuberculosis, and our current understanding of the wide range of CD8 T cell types seen in M. tuberculosis infection. PMID:25917388

Amniotic membrane extract differentially regulates human peripheral blood T cell subsets, monocyte subpopulations and myeloid dendritic cells.

PubMed

Laranjeira, Paula; Duque, Marta; Vojtek, Martin; Inácio, Maria J; Silva, Isabel; Mamede, Ana C; Laranjo, Mafalda; Pedreiro, Susana; Carvalho, Maria J; Moura, Paulo; Abrantes, Ana M; Maia, Cláudio J; Domingues, Pedro; Domingues, Rosário; Martinho, António; Botelho, Maria F; Trindade, Hélder; Paiva, Artur

2018-03-26

The discovery of the immunoregulatory potential of human amniotic membrane (hAM) propelled several studies focusing on its application for the treatment of immunological disorders. However, there is little information regarding the effects of hAM on distinct activation and differentiation stages of immune cells. Here, we aim to investigate the effect of human amniotic membrane extract (hAME) on the pattern of cytokine production by T cells, monocytes and myeloid dendritic cells (mDCs). For this purpose, peripheral blood mononuclear cells (PBMCs) from eight healthy individuals were stimulated in vitro in the presence or absence of hAME. Mitogen-induced proliferation of PBMCs and cytokine production among the distinct T cell functional compartments, monocyte subpopulations and mDCs were evaluated. hAME displayed an anti-proliferative effect and decreased the frequency of T cells producing tumor necrosis factor (TNF)α, interferon (IFN)γ and interleukin (IL)-2, for all T cell functional compartments. The frequency of IL-17 and IL-9-producing T cells was also reduced. The inhibition of mRNA expression of granzyme B, perforin and NKG2D by CD8 + T cells and γδ T cells and the augment of FoxP3 and IL-10 in CD4 + T cells and IL-10 in regulatory T cells were also observed. Furthermore, hAME inhibited IFNγ-induced protein (IP)-10 expression by classical and non-classical monocytes, without hampering the production of TNFα and IL-6 by monocytes and mDCs. These results suggest that hAME exerts an anti-inflammatory effect on T cells, still at a different extent for distinct T cell functional compartments.

Age-dependent metabolic and immunosuppressive effects of Tacrolimus

PubMed Central

Krenzien, Felix; Quante, Markus; Heinbokel, Timm; Seyda, Midas; Minami, Koichiro; Uehara, Hirohito; Biefer, Hector Rodriguez Cetina; Schuitenmaker, Jeroen M.; Gabardi, Steven; Splith, Katrin; Schmelzle, Moritz; Petrides, Athena K.; Azuma, Haruhito; Pratschke, Johann; Li, Xian C.; ElKhal, Abdallah; Tullius, Stefan G.

2016-01-01

Immunosuppression in elderly recipients has been underappreciated in clinical trials. Here, we assessed age-specific effects of the calcineurin inhibitor Tacrolimus (TAC) in a murine transplant model and assessed its clinical relevance on human T-cells. Old recipient mice exhibited prolonged skin graft survival when compared to young animals following TAC administration. More importantly, half of the TAC dose was sufficient in old mice to achieve comparable systemic trough levels. TAC administration was able to reduce pro-inflammatory IFN-γ cytokine production and promote IL-10 production in old CD4+ T-cells. In addition, TAC administration decreased IL-2 secretion in old CD4+ T-cells more effectively while inhibiting the proliferation of CD4+ T-cells in old mice. Both, TAC treated murine and human CD4+ T-cells demonstrated an age-specific suppression of intracellular calcineurin levels and Ca2+-influx, two critical pathways in T-cell activation. Of note, depletion of CD8+ T-cells did not alter allograft survival outcome in old TAC treated mice, suggesting that TAC age-specific effects were mainly CD4+ T-cell mediated. Collectively, our study demonstrates age-specific immunosuppressive capacities of TAC that are CD4+ T-cell mediated. The suppression of calcineurin levels and Ca2+-influx in both, old murine and human T-cells emphasizes on the clinical relevance of age-specific effects when utilizing TAC. PMID:27754593

Action of the photosensitizer QLT0074 upon human T lymphocytes in vitro

NASA Astrophysics Data System (ADS)

Hunt, David W. C.; Jiang, Huijun; Salmon, Ruth A.; Granville, David J.; North, John R.; Richter, Anna M.

2001-04-01

A new photosensitizer, presently designated QLT0074, may be useful for the treatment of skin conditions, particularly those mediated by T lymphocytes, with photodynamic therapy (PDT). QLT0074 was tested against human peripheral blood T cells and Jurkat T lymphoma cells. Low concentrations of QLT0074 and blue light were sufficient to induce apoptosis in peripheral blood T cells or Jurkat T lymphoma cells as indicated by expression of the apoptosis-associated mitochondrial 7A6 marker, annexin-V labeling or activation of capsase-3 and cleavage of the capsase substrate poly(ADP-ribose)polymerase (PARP). Flow cytometry studies performed following PDT showed that peripheral blood T cells with high expression of the interleukin-2 receptor (CD25) took up greater amounts of QLT0074 and were eliminated to a greater degree than T cells with low CD25 levels. This effect of PDT was also shown by the reduction in the percentage of T cells that expressed other activation-associated markers such as very late activation antigen-4 (CD49d), human leukocyte antigen DR (HLA-DR), intercellular adhesion molecule-1 (CD54) and Fas (CD95). In the case of T cells that remained viable following PDT, CD25 expression was lower while CD54, CD95 and HLA-DR levels were unchanged. Thus, PDT with QLT0074 has selective, dose-dependent effects on T cells in vitro.

Trichomonas vaginalis infection induces vaginal CD4+ T-cell infiltration in a mouse model: a vaccine strategy to reduce vaginal infection and HIV transmission.

PubMed

Smith, Jeffrey D; Garber, Gary E

2015-07-15

Complications related to the diagnosis and treatment of Trichomonas vaginalis infection, as well as the association between T. vaginalis infection and increased transmission of and susceptibility to human immunodeficiency virus, highlight the need for alternative interventions. We tested a human-safe, aluminum hydroxide-adjuvanted whole-cell T. vaginalis vaccine for efficacy in a BALB/c mouse model of vaginal infection. A whole-cell T. vaginalis vaccine was administered subcutaneously to BALB/c mice, using a prime-boost vaccination schedule. CD4(+) T-cell infiltration in the murine vaginal tissue and local and systemic levels of immunoglobulins were measured at time points up to 4 weeks following infection. Vaccination reduced the incidence and increased the clearance of T. vaginalis infection and induced both systemic and local humoral immune responses. CD4(+) T cells were detected in vaginal tissues following intravaginal infection with T. vaginalis but were not seen in uninfected mice. The presence of CD4(+) T cells following T. vaginalis infection can potentially increase susceptibility to and transmission of human immunodeficiency virus. The vaccine induces local and systemic immune responses and confers significantly greater protection against vaginal infection than seen in unvaccinated mice (P < .005). These data support the potential for a human vaccine against T. vaginalis infection that could also influence the incidence of human immunodeficiency virus infection. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Characterization of simian T-cell leukemia virus type 1 in naturally infected Japanese macaques as a model of HTLV-1 infection

PubMed Central

2013-01-01

Background Human T-cell leukemia virus type 1 (HTLV-1) causes chronic infection leading to development of adult T-cell leukemia (ATL) and inflammatory diseases. Non-human primates infected with simian T-cell leukemia virus type 1 (STLV-1) are considered to constitute a suitable animal model for HTLV-1 research. However, the function of the regulatory and accessory genes of STLV-1 has not been analyzed in detail. In this study, STLV-1 in naturally infected Japanese macaques was analyzed. Results We identified spliced transcripts of STLV-1 corresponding to HTLV-1 tax and HTLV-1 bZIP factor (HBZ). STLV-1 Tax activated the NFAT, AP-1 and NF-κB signaling pathways, whereas STLV-1 bZIP factor (SBZ) suppressed them. Conversely, SBZ enhanced TGF-β signaling and induced Foxp3 expression. Furthermore, STLV-1 Tax activated the canonical Wnt pathway while SBZ suppressed it. STLV-1 Tax enhanced the viral promoter activity while SBZ suppressed its activation. Then we addressed the clonal proliferation of STLV-1+ cells by massively sequencing the provirus integration sites. Some clones proliferated distinctively in monkeys with higher STLV-1 proviral loads. Notably, one of the monkeys surveyed in this study developed T-cell lymphoma in the brain; STLV-1 provirus was integrated in the lymphoma cell genome. When anti-CCR4 antibody, mogamulizumab, was administered into STLV-1-infected monkeys, the proviral load decreased dramatically within 2 weeks. We observed that some abundant clones recovered after discontinuation of mogamulizumab administration. Conclusions STLV-1 Tax and SBZ have functions similar to those of their counterparts in HTLV-1. This study demonstrates that Japanese macaques naturally infected with STLV-1 resemble HTLV-1 carriers and are a suitable model for the investigation of persistent HTLV-1 infection and asymptomatic HTLV-1 carrier state. Using these animals, we verified that mogamulizumab, which is currently used as a drug for relapsed ATL, is also effective in reducing the proviral load in asymptomatic individuals. PMID:24156738

Expression of checkpoint molecules on myeloid-derived suppressor cells.

PubMed

Ballbach, Marlene; Dannert, Angelika; Singh, Anurag; Siegmund, Darina M; Handgretinger, Rupert; Piali, Luca; Rieber, Nikolaus; Hartl, Dominik

2017-12-01

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous cell population expanded in cancer, infection and autoimmunity capable of suppressing T-cell functions. Checkpoint inhibitors have emerged as a key therapeutic strategy in immune-oncology. While checkpoint molecules were initially associated with T cell functions, recent evidence suggests a broader expression and function in innate myeloid cells. Previous studies provided first evidence for a potential role for checkpoints on MDSCs, yet the human relevance remained poorly understood. Therefore, we investigated the expression and functional relevance of checkpoint molecules in human MDSC-T-cell interactions. Our studies demonstrate that programmed death-ligand 1 (PD-L1) is expressed on granulocytic MDSCs upon co-culture with T cells. Transwell experiments showed that cell-to-cell contact was required for MDSC-T-cell interactions and antibody blocking studies showed that targeting PD-L1 partially impaired MDSC-mediated T-cell suppression. Collectively, these studies suggest a role for PD-L1 in human MDSC function and thereby expand the functionality of this checkpoint beyond T cells, which could pave the way for further understanding and therapeutic targeting of PD-1/PD-L1 in innate immune-mediated diseases. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

CD28-Negative CD4+ and CD8+ T Cells in Antiretroviral Therapy–Naive HIV-Infected Adults Enrolled in Adult Clinical Trials Group Studies

PubMed Central

Tassiopoulos, Katherine; Landay, Alan; Collier, Ann C.; Connick, Elizabeth; Deeks, Steven G.; Hunt, Peter; Lewis, Dorothy E.; Wilson, Cara; Bosch, Ronald

2012-01-01

Background Individuals infected with human immunodeficiency virus (HIV) have higher risk than HIV-negative individuals for diseases associated with aging. T-cell senescence, characterized by expansion of cells lacking the costimulatory molecule CD28, has been hypothesized to mediate these risks. Methods We measured the percentage of CD28−CD4+ and CD8+ T cells from HIV-infected treatment-naive adults from 5 Adult Clinical Trials Group (ACTG) antiretroviral therapy (ART) studies and the ALLRT (ACTG Longitudinal Linked Randomized Trials) cohort, and from 48 HIV-negative adults. Pretreatment and 96-week posttreatment %CD28− cells were assessed using linear regression for associations with age, sex, race/ethnicity, CD4 count, HIV RNA, ART regimen, and hepatitis C virus (HCV) infection. Results In total, 1291 chronically HIV-infected adults were studied. Pretreatment, lower CD4 count was associated with higher %CD28−CD4+ and %CD28−CD8+ cells. For CD8+ cells, younger age and HCV infection were associated with a lower %CD28−. ART reduced %CD28− levels at week 96 among virally suppressed individuals. Older age was strongly predictive of higher %CD28−CD8+. Compared to HIV-uninfected individuals, HIV-infected individuals maintained significantly higher %CD28−. Conclusions Effective ART reduced the proportion of CD28− T cells. However, levels remained abnormally high and closer to levels in older HIV-uninfected individuals. This finding may inform future research of increased rates of age-associated disease in HIV-infected adults. PMID:22448010

Factors secreted by human T lymphotropic virus type I (HTLV-I)-infected cells can enhance or inhibit replication of HIV-1 in HTLV-I-uninfected cells: implications for in vivo coinfection with HTLV-I and HIV-1.

PubMed

Moriuchi, H; Moriuchi, M; Fauci, A S

1998-05-18

It remains controversial whether human T lymphotropic virus type I (HTLV-I) coinfection leads to more rapid progression of human immunodeficiency virus (HIV) disease in dually infected individuals. To investigate whether HTLV-I infection of certain cells can modulate HIV-1 infection of surrounding cells, primary CD4(+) T cells were treated with cell-free supernatants from HTLV-I-infected MT-2 cell cultures. The primary CD4+ T cells became resistant to macrophage (M)-tropic HIV-1 but highly susceptible to T cell (T)-tropic HIV-1. The CC chemokines RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta in the MT-2 cell supernatants were identified as the major suppressive factors for M-tropic HIV-1 as well as the enhancers of T-tropic HIV-1 infection, whereas soluble Tax protein increased susceptibility to both M- and T-tropic HIV-1. The effect of Tax or CC chemokines on T-tropic HIV-1 was mediated, at least in part, by increasing HIV Env-mediated fusogenicity. Our data suggest that the net effect of HTLV-I coinfection in HIV-infected individuals favors the transition from M- to T-tropic HIV phenotype, which is generally indicative of progressive HIV disease.

A different representation of natural T cells and natural killer cells between tumor-infiltrating and periphery lymphocytes in human hepatocellular carcinoma.

PubMed

Li, Xiao-Feng; Dai, Dong; Song, Xiu-Yu; Liu, Jian-Jing; Zhu, Lei; Zhu, Xiang; Ma, Wenchao; Xu, Wengui

2017-05-01

Natural T cells [cluster of differentiation (CD) 3 + CD56 + ] and natural killer (NK) cells (CD3 - CD56 + ) are particularly abundant in the human liver and serve an important role in immune responses in the liver. The aim of the present study was to extensively determine the phenotypic and functional characteristics of natural T and NK cells in human hepatocellular carcinoma (HCC). Tumorous and non-tumorous tissue infiltrating lymphocytes (TILs and NILs, respectively) and peripheral blood mononuclear cells (PBMCs) from patients with hepatocellular carcinoma (HCC) were obtained to determine the frequency and phenotype of natural T/NK cells by a multicolor fluorescence activated cell sorting analysis. The abundance of natural T cells and NK cells was decreased in TILs vs. NILs (natural T cells, 6.315±1.002 vs. 17.16±1.804; NK cells, 6.324±1.559 vs. 14.52±2.336, respectively). However such results were not observed in PBMCs from HCC patients vs. that of healthy donors. Notably, a substantial fraction of the natural T cells (21.96±5.283) in TILs acquired forkhead box P3 (FOXP3) expression, and the FOXP3 + natural T cells lost the expression of interferon-γ and perforin. Conversely, being similar to the conventional FOXP3 + regulatory T cells, the FOXP3 + natural T cells assumed a specific phenotype that was characteristic of CD25 + , CD45RO + and cytotoxic T-lymphocyte-associated protein 4 + . Consistent with the phenotypic conversion, the present functional results indicate that FOXP3 expression in natural T cells contributes to the acquisition of a potent immunosuppressive capability. In conclusion, the present study describes a different representation of natural T cells and NK cells in local tumor tissues and in the periphery blood of patients with HCC, and identified a new type of FOXP3-expressing natural T cell spontaneously arising in the TILs of HCC.

Suppressive effects of chlorphenesin on lymphocyte function in mice and humans.

PubMed

Stites, D P; Brecher, G; Schmidt, L; Berger, F M

1979-12-01

The immunosuppressive action of chlorphenesin was investigated in a wide variety of in vitro assays for cellular immunity in humans and mice. Chlorphenesin, at doses of 20-50 micrograms/ml, inhibited mitogenic responses of both mouse and human B and T cells. These doses did not kill cells exposed to the drug for 72 hr. Mixed lymphocyte reactions in inbred strains of mice and in unrelated humans were also inhibited at concentrations of about 50 micrograms/ml. However, the generation of cytotoxic T cells in cell-mediated lympholysis assays was not inhibited to the same degree as proliferation in mixed lymphocyte reaction and the cytotoxic potential of presensitized mouse T cells for allogeneic targets was totally unaffected. These studies suggest that chlorphenesin may have a broad spectrum of suppressive effects both on T and B cells and that the predominant inhibition of proliferative responses in these cells may reduce the expansion of clones of immunocompetent cells in vivo.

Both cell fusion and transdifferentiation account for the transformation of human peripheral blood CD34-positive cells into cardiomyocytes in vivo.

PubMed

Zhang, Sui; Wang, Dachun; Estrov, Zeev; Raj, Sean; Willerson, James T; Yeh, Edward T H

2004-12-21

Adult human peripheral blood CD34-positive (CD34+) cells appear to transform into cardiomyocytes in the injured hearts of severe combined immunodeficient mice. It remains unclear, however, whether the apparent transformation is the result of transdifferentiation of the donor stem cells or of fusion of the donor cell with the cardiomyocyte of the recipients. We performed flow cytometry analyses of cells isolated from the hearts of mice that received human CD34+ cells. Human HLA-ABC antigen and cardiac troponin T or Nkx2.5 were used as markers for cardiomyocytes derived from human CD34+ cells, and HLA-ABC and VE-cadherin were used to identify the transformed endothelial cells. The double-positive cells were collected and interphase fluorescence in situ hybridization was used to detect the expression of human and mouse X chromosomes in these cells. We found that 73.3% of nuclei derived from HLA+ and troponin T+ or Nkx2.5+ cardiomyocytes contain both human and mouse X chromosomes and 23.7% contain only human X chromosome. In contrast, the nuclei of HLA-, troponin T+ cells contain only mouse X chromosomes. Furthermore, 97.3% of endothelial cells derived from CD34+ cells contained human X chromosome only. Thus, both cell fusion and transdifferentiation may account for the transformation of peripheral blood CD34+ cells into cardiomyocytes in vivo.

Divergent effects of norepinephrine, dopamine and substance P on the activation, differentiation and effector functions of human cytotoxic T lymphocytes

PubMed Central

2009-01-01

Background Neurotransmitters are important regulators of the immune system, with very distinct and varying effects on different leukocyte subsets. So far little is known about the impact of signals mediated by neurotransmitters on the function of CD8+ T lymphocytes. Therefore, we investigated the influence of norepinephrine, dopamine and substance P on the key tasks of CD8+ T lymphocytes: activation, migration, extravasation and cytotoxicity. Results The activation of naïve CD8+ T lymphocytes by CD3/CD28 cross-linking was inhibited by norepinephrine and dopamine, which was caused by a downregulation of interleukin (IL)-2 expression via Erk1/2 and NF-κB inhibition. Furthermore, all of the investigated neurotransmitters increased the spontaneous migratory activity of naïve CD8+ T lymphocytes with dopamine being the strongest inducer. In contrast, activated CD8+ T lymphocytes showed a reduced migratory activity in the presence of norepinephrine and substance P. With regard to extravasation we found norepinephrine to induce adhesion of activated CD8+ T cells: norepinephrine increased the interleukin-8 release from endothelium, which in turn had effect on the activated CXCR1+ CD8+ T cells. At last, release of cytotoxic granules from activated cells in response to CD3 cross-linking was not influenced by any of the investigated neurotransmitters, as we have analyzed by measuring the β-hexosamidase release. Conclusion Neurotransmitters are specific modulators of CD8+ T lymphocytes not by inducing any new functions, but by fine-tuning their key tasks. The effect can be either stimulatory or suppressive depending on the activation status of the cells. PMID:19968887

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.

Dendritic cell based vaccines: progress in immunotherapy studies for prostate cancer.

PubMed

Ragde, Haakon; Cavanagh, William A; Tjoa, Benjamin A

2004-12-01

No effective treatment is currently available for metastatic prostate cancer. Dendritic cell (DC) based cancer vaccine research has emerged from the laboratories to human clinical trials. We describe progress in the development of DC based prostate cancer vaccine. The literature was reviewed for major contributions to a growing number of studies that demonstrate the potential of DC based immunotherapeutics for prostate cancer. Background topics relating to DC based immunotherapy theory and practice are also addressed. DCs have been recognized as the most efficient antigen presenting cells that have the capacity to initiate naive T cell response in vitro and in vivo. During their differentiation and maturation pathways, dendritic cells can efficiently capture, process and present antigens for T cell activation. These characteristics make DC an attractive choice as the cellular adjuvant for cancer vaccines. Advances in DC generation, loading, and maturation methodologies have made it possible to generate clinical grade vaccines for various human trials. More than 100 DC vaccine trials, including 7 studies of patients with advanced prostate cancer have been reported to date. These vaccines were generally well tolerated with no significant adverse toxicity reported. Clinical responders have been identified in these studies. The new prospects opened by DC based vaccines for prostate cancer are fascinating. When compared to conventional treatments, DC vaccinations have few side effects. Improvements in patient selection, vaccine delivery strategies, immune monitoring and vaccine manufacturing will be crucial in moving DC based prostate cancer vaccines closer to the clinics.

Effect of constituents from samaras of Austroplenckia populnea (Celastraceae) on human cancer cells

PubMed Central

Caneschi, Carolina Milagres; Muniyappa, Mohan K.; Duarte, Lucienir P.; Silva, Grácia D. F.; dos Santos, Orlando David Henrique; Spillane, Charles; Filho, Sidney Augusto Vieira

2015-01-01

Background: Aiming the continuity of the studies of Austroplenckia populnea, Brazilian species of the Celastraceae family, in the present study, it was investigated the effect of crude extracts obtained with ethanol, ethyl acetate and chloroform and two purified constituents, proanthocyanidin A and 4’-O-methylepigallocatechin, both isolated from its samaras, on cancer cell proliferation assays. Materials and Methods: The human cancer cells lines MCF-7 (ductal breast carcinoma), A549 (lung cancer), HS578T (ductal breast carcinoma) and non-cancer HEK293 (embryonic kidney cells) were treated with different concentrations of extracts and constituents and the effect was observed through the acid phosphatase method. The chemical structures of the purified compounds were identified by the respective IR and 1H and 13C nuclear magnetic resonance spectral data. Results: While crude extracts from samaras of the folk medicine A. populnea can trigger cell proliferative effects in human cell lines, the purified compounds (proanthocyanidin A and 4’-O-methyl-epigallocatechin) isolated from the same extracts can have an opposite (anti-proliferative) effect. Conclusion: Based on the results, it was possible to suggest that extracts from samaras of A. populnea should be further investigated for possible cancer-promoting activities; and the active extracts can also represent a source of compounds that have anti-cancer properties. PMID:26401377

Human tRNA genes function as chromatin insulators

PubMed Central

Raab, Jesse R; Chiu, Jonathan; Zhu, Jingchun; Katzman, Sol; Kurukuti, Sreenivasulu; Wade, Paul A; Haussler, David; Kamakaka, Rohinton T

2012-01-01

Insulators help separate active chromatin domains from silenced ones. In yeast, gene promoters act as insulators to block the spread of Sir and HP1 mediated silencing while in metazoans most insulators are multipartite autonomous entities. tDNAs are repetitive sequences dispersed throughout the human genome and we now show that some of these tDNAs can function as insulators in human cells. Using computational methods, we identified putative human tDNA insulators. Using silencer blocking, transgene protection and repressor blocking assays we show that some of these tDNA-containing fragments can function as barrier insulators in human cells. We find that these elements also have the ability to block enhancers from activating RNA pol II transcribed promoters. Characterization of a putative tDNA insulator in human cells reveals that the site possesses chromatin signatures similar to those observed at other better-characterized eukaryotic insulators. Enhanced 4C analysis demonstrates that the tDNA insulator makes long-range chromatin contacts with other tDNAs and ETC sites but not with intervening or flanking RNA pol II transcribed genes. PMID:22085927

Characterization of mutagen-activated cellular oncogenes that confer anchorage independence to human fibroblasts and tumorigenicity to NIH 3T3 cells: Sequence analysis of an enzymatically amplified mutant HRAS allele

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

Stevens, C.W.; Manoharan, T.H.; Fahl, W.E.

1988-06-01

Treatment of diploid human fibroblasts with an alkylating mutagen has been shown to induce stable, anchorage-independent cell populations at frequencies consistent with an activating mutation. After treatment of human foreskin fibroblasts with the mutagen benzo({alpha})pyrene ({plus minus})anti-7,8-dihydrodiol 9,10-epoxide and selection in soft agar, 17 anchorage-independent clones were isolated and expanded, and their cellular DNA was used to cotransfect NIH 3T3 cells along with pSV2neo. DNA from 11 of the 17 clones induced multiple NIH 3T3 cell tumors in recipient nude mice. Southern blot analyses showed the presence of human Alu repetitive sequences in all of the NIH 3T3 tumor cellmore » DNAs. Intact, human HRAS sequences were observed in 2 of the 11 tumor groups, whereas no hybridization was detected when human KRAS or NRAS probes were used. Slow-migrating ras p21 proteins, consistent with codon 12 mutations, were observed in the same two NIH 3T3 tumor cell groups that contained the human HRAS bands. Genomic DNA from one of these two human anchorage-independent cell populations (clone 21A) was used to enzymatically amplify a portion of exon 1 of the HRAS gene. The results demonstrate that exposure of normal human cells to a common environmental mutagen yields HRAS GC {yields} TA codon 12 transversions that have been commonly observed in human tumors.« less

Temporal dynamics of the primary human T cell response to yellow fever virus 17D as it matures from an effector- to a memory-type response.

PubMed

Blom, Kim; Braun, Monika; Ivarsson, Martin A; Gonzalez, Veronica D; Falconer, Karolin; Moll, Markus; Ljunggren, Hans-Gustaf; Michaëlsson, Jakob; Sandberg, Johan K

2013-03-01

The live attenuated yellow fever virus (YFV) 17D vaccine provides a good model to study immune responses to an acute viral infection in humans. We studied the temporal dynamics, composition, and character of the primary human T cell response to YFV. The acute YFV-specific effector CD8 T cell response was broad and complex; it was composed of dominant responses that persisted into the memory population, as well as of transient subdominant responses that were not detected at the memory stage. Furthermore, HLA-A2- and HLA-B7-restricted YFV epitope-specific effector cells predominantly displayed a CD45RA(-)CCR7(-)PD-1(+)CD27(high) phenotype, which transitioned into a CD45RA(+)CCR7(-)PD-1(-)CD27(low) memory population phenotype. The functional profile of the YFV-specific CD8 T cell response changed in composition as it matured from an effector- to a memory-type response, and it tended to become less polyfunctional during the course of this transition. Interestingly, activation of CD4 T cells, as well as FOXP3(+) T regulatory cells, in response to YFV vaccination preceded the kinetics of the CD8 T cell response. The present results contribute to our understanding of how immunodominance patterns develop, as well as the phenotypic and functional characteristics of the primary human T cell response to a viral infection as it evolves and matures into memory.

Potent Immune Modulation by MEDI6383, an Engineered Human OX40 Ligand IgG4P Fc Fusion Protein.

PubMed

Oberst, Michael D; Augé, Catherine; Morris, Chad; Kentner, Stacy; Mulgrew, Kathy; McGlinchey, Kelly; Hair, James; Hanabuchi, Shino; Du, Qun; Damschroder, Melissa; Feng, Hui; Eck, Steven; Buss, Nicholas; de Haan, Lolke; Pierce, Andrew J; Park, Haesun; Sylwester, Andrew; Axthelm, Michael K; Picker, Louis; Morris, Nicholas P; Weinberg, Andrew; Hammond, Scott A

2018-05-01

Ligation of OX40 (CD134, TNFRSF4) on activated T cells by its natural ligand (OX40L, CD252, TNFSF4) enhances cellular survival, proliferation, and effector functions such as cytokine release and cellular cytotoxicity. We engineered a recombinant human OX40L IgG4P Fc fusion protein termed MEDI6383 that assembles into a hexameric structure and exerts potent agonist activity following engagement of OX40. MEDI6383 displayed solution-phase agonist activity that was enhanced when the fusion protein was clustered by Fc gamma receptors (FcγRs) on the surface of adjacent cells. The resulting costimulation of OX40 on T cells induced NFκB promoter activity in OX40-expressing T cells and induced Th1-type cytokine production, proliferation, and resistance to regulatory T cell (Treg)-mediated suppression. MEDI6383 enhanced the cytolytic activity of tumor-reactive T cells and reduced tumor growth in the context of an alloreactive human T cell:tumor cell admix model in immunocompromised mice. Consistent with the role of OX40 costimulation in the expansion of memory T cells, MEDI6383 administered to healthy nonhuman primates elicited peripheral blood CD4 and CD8 central and effector memory T-cell proliferation as well as B-cell proliferation. Together, these results suggest that OX40 agonism has the potential to enhance antitumor immunity in human malignancies. Mol Cancer Ther; 17(5); 1024-38. ©2018 AACR . ©2018 American Association for Cancer Research.

Mutations in STAT3 and IL12RB1 impair the development of human IL-17–producing T cells

PubMed Central

de Beaucoudrey, Ludovic; Puel, Anne; Filipe-Santos, Orchidée; Cobat, Aurélie; Ghandil, Pegah; Chrabieh, Maya; Feinberg, Jacqueline; von Bernuth, Horst; Samarina, Arina; Jannière, Lucile; Fieschi, Claire; Stéphan, Jean-Louis; Boileau, Catherine; Lyonnet, Stanislas; Jondeau, Guillaume; Cormier-Daire, Valérie; Le Merrer, Martine; Hoarau, Cyrille; Lebranchu, Yvon; Lortholary, Olivier; Chandesris, Marie-Olivia; Tron, François; Gambineri, Eleonora; Bianchi, Lucia; Rodriguez-Gallego, Carlos; Zitnik, Simona E.; Vasconcelos, Julia; Guedes, Margarida; Vitor, Artur Bonito; Marodi, Laszlo; Chapel, Helen; Reid, Brenda; Roifman, Chaim; Nadal, David; Reichenbach, Janine; Caragol, Isabel; Garty, Ben-Zion; Dogu, Figen; Camcioglu, Yildiz; Gülle, Sanyie; Sanal, Ozden; Fischer, Alain; Abel, Laurent; Stockinger, Birgitta; Picard, Capucine; Casanova, Jean-Laurent

2008-01-01

The cytokines controlling the development of human interleukin (IL) 17–producing T helper cells in vitro have been difficult to identify. We addressed the question of the development of human IL-17–producing T helper cells in vivo by quantifying the production and secretion of IL-17 by fresh T cells ex vivo, and by T cell blasts expanded in vitro from patients with particular genetic traits affecting transforming growth factor (TGF) β, IL-1, IL-6, or IL-23 responses. Activating mutations in TGFB1, TGFBR1, and TGFBR2 (Camurati-Engelmann disease and Marfan-like syndromes) and loss-of-function mutations in IRAK4 and MYD88 (Mendelian predisposition to pyogenic bacterial infections) had no detectable impact. In contrast, dominant-negative mutations in STAT3 (autosomal-dominant hyperimmunoglobulin E syndrome) and, to a lesser extent, null mutations in IL12B and IL12RB1 (Mendelian susceptibility to mycobacterial diseases) impaired the development of IL-17–producing T cells. These data suggest that IL-12Rβ1– and STAT-3–dependent signals play a key role in the differentiation and/or expansion of human IL-17–producing T cell populations in vivo. PMID:18591412

Broadly targeted human cytomegalovirus-specific CD4+ and CD8+ T cells dominate the memory compartments of exposed subjects

PubMed Central

Sylwester, Andrew W.; Mitchell, Bridget L.; Edgar, John B.; Taormina, Cara; Pelte, Christian; Ruchti, Franziska; Sleath, Paul R.; Grabstein, Kenneth H.; Hosken, Nancy A.; Kern, Florian; Nelson, Jay A.; Picker, Louis J.

2005-01-01

Human cytomegalovirus (HCMV) infections of immunocompetent hosts are characterized by a dynamic, life-long interaction in which host immune responses, particularly of T cells, restrain viral replication and prevent disease but do not eliminate the virus or preclude transmission. Because HCMV is among the largest and most complex of known viruses, the T cell resources committed to maintaining this balance have never been characterized completely. Here, using cytokine flow cytometry and 13,687 overlapping 15mer peptides comprising 213 HCMV open reading frames (ORFs), we found that 151 HCMV ORFs were immunogenic for CD4+ and/or CD8+ T cells, and that ORF immunogenicity was influenced only modestly by ORF expression kinetics and function. We further documented that total HCMV-specific T cell responses in seropositive subjects were enormous, comprising on average ∼10% of both the CD4+ and CD8+ memory compartments in blood, whereas cross-reactive recognition of HCMV proteins in seronegative individuals was limited to CD8+ T cells and was rare. These data provide the first glimpse of the total human T cell response to a complex infectious agent and will provide insight into the rules governing immunodominance and cross-reactivity in complex viral infections of humans. PMID:16147978

The pan-ErbB tyrosine kinase inhibitor canertinib induces caspase-mediated cell death in human T-cell leukemia (Jurkat) cells

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

Trinks, Cecilia, E-mail: Cecilia.trinks@liu.se; Severinsson, Emelie A., E-mail: Emelie.severinsson@liu.se; Holmlund, Birgitta, E-mail: Birgitta.holmlund@lio.se

2011-07-08

Highlights: {yields} Canertinib induces caspase-mediated apoptosis in T-cell leukemia cells in vitro. {yields} Canertinib mediates activation of the intrinsic apoptotic pathway. {yields} Canertinib induces apoptosis in an ErbB receptor independent manner. {yields} Lymphocyte specific proteins as well as survival kinases are inhibited. {yields} Canertinib may act as a multi-kinase inhibiting drug in human T-cell malignancies. -- Abstract: Canertinib is a novel ErbB-receptor inhibitor currently in clinical development for the treatment of solid tumors overexpressing ErbB-receptors. We have recently demonstrated that canertinib displays anti-proliferative and pro-apoptotic effects in human myeloid leukemia cells devoid of ErbB-receptors. The mechanism mediating these effects aremore » however unknown. In this study, we show that canertinib is able to act as a multi-kinase inhibitor by inhibition of several intracellular kinases involved in T-cell signaling such as Akt, Erk1/2 and Zap-70, and reduced Lck protein expression in the human T-cell leukemia cell line Jurkat. Treatment with canertinib at a concentration of 2 {mu}M caused accumulation of Jurkat cells in the G{sub 1} cell cycle phase and increased doses induced apoptosis in a time-dependent manner. Apoptotic signs of treated cells were detected by Annexin V staining and cleavage of PARP, caspase-3, -8, -9, -10 and Bid. A subset of the pro-apoptotic signals mediated by canertinib could be significantly reduced by specific caspase inhibitors. Taken together, these results demonstrate the dual ability of canertinib to downregulate important signaling pathways and to activate caspase-mediated intrinsic apoptosis pathway in human T-cell leukemia cells.« less

The Viral Transcription Group Determines the HLA Class I Cellular Immune Response Against Human Respiratory Syncytial Virus*

PubMed Central

Johnstone, Carolina; Lorente, Elena; Barriga, Alejandro; Barnea, Eilon; Infantes, Susana; Lemonnier, François A.; David, Chella S.; Admon, Arie; López, Daniel

2015-01-01

The cytotoxic T-lymphocyte-mediated killing of virus-infected cells requires previous recognition of short viral antigenic peptides bound to human leukocyte antigen class I molecules that are exposed on the surface of infected cells. The cytotoxic T-lymphocyte response is critical for the clearance of human respiratory syncytial virus infection. In this study, naturally processed viral human leukocyte antigen class I ligands were identified with mass spectrometry analysis of complex human leukocyte antigen-bound peptide pools isolated from large amounts of human respiratory syncytial virus-infected cells. Acute antiviral T-cell response characterization showed that viral transcription determines both the immunoprevalence and immunodominance of the human leukocyte antigen class I response to human respiratory syncytial virus. These findings have clear implications for antiviral vaccine design. PMID:25635267

Dynamics of human T-cell lymphotropic virus I (HTLV-I) infection of CD4+ T-cells.

PubMed

Katri, Patricia; Ruan, Shigui

2004-11-01

Stilianakis and Seydel (Bull. Math. Biol., 1999) proposed an ODE model that describes the T-cell dynamics of human T-cell lymphotropic virus I (HTLV-I) infection and the development of adult T-cell leukemia (ATL). Their model consists of four components: uninfected healthy CD4+ T-cells, latently infected CD4+ T-cells, actively infected CD4+ T-cells, and ATL cells. Mathematical analysis that completely determines the global dynamics of this model has been done by Wang et al. (Math. Biosci., 2002). In this note, we first modify the parameters of the model to distinguish between contact and infectivity rates. Then we introduce a discrete time delay to the model to describe the time between emission of contagious particles by active CD4+ T-cells and infection of pure cells. Using the results in Culshaw and Ruan (Math. Biosci., 2000) in the analysis of time delay with respect to cell-free viral spread of HIV, we study the effect of time delay on the stability of the endemically infected equilibrium. Numerical simulations are presented to illustrate the results.

T cell ignorance is bliss: T cells are not tolerized by Langerhans cells presenting human papillomavirus antigens in the absence of costimulation.

PubMed

Woodham, Andrew W; Yan, Lisa; Skeate, Joseph G; van der Veen, Daniel; Brand, Heike H; Wong, Michael K; Da Silva, Diane M; Kast, W Martin

2016-12-01

Human papillomavirus type 16 (HPV16) infections are intra-epithelial, and thus, HPV16 is known to interact with Langerhans cells (LCs), the resident epithelial antigen-presenting cells (APCs). The current paradigm for APC-mediated induction of T cell anergy is through delivery of T cell receptor signals via peptides on MHC molecules (signal 1), but without costimulation (signal 2). We previously demonstrated that LCs exposed to HPV16 in vitro present HPV antigens to T cells without costimulation, but it remained uncertain if such T cells would remain ignorant, become anergic, or in the case of CD4+ T cells, differentiate into Tregs. Here we demonstrate that Tregs were not induced by LCs presenting only signal 1, and through a series of in vitro immunizations show that CD8 + T cells receiving signal 1 + 2 from LCs weeks after consistently receiving signal 1 are capable of robust effector functions. Importantly, this indicates that T cells are not tolerized but instead remain ignorant to HPV, and are activated given the proper signals.

From Hayflick to Walford: the role of T cell replicative senescence in human aging.

PubMed

Effros, Rita B

2004-06-01

The immunologic theory of aging, proposed more than 40 years ago by Roy Walford, suggests that the normal process of aging in man and in animals is pathogenetically related to faulty immunological processes. Since that time, research on immunological aging has undergone extraordinary expansion, leading to new information in areas spanning from molecular biology and cell signaling to large-scale clinical studies. Investigation in this area has also provided unexpected insights into HIV disease, many aspects of which represent accelerated immunological aging. This article describes the initial insights and vision of Roy Walford into one particular facet of human immunological aging, namely, the potential relevance of the well-studied human fibroblast replicative senescence model, initially developed by Leonard Hayflick, to cells of the immune system. Extensive research on T cell senescence in cell culture has now documented changes in vitro that closely mirror alterations occurring during in vivo aging in humans, underscoring the biological significance of T cell replicative senescence. Moreover, the inclusion of high proportions of putatively senescent T cells in the 'immune risk phenotype' that is associated with early mortality in octogenarians provides initial clinical confirmation of both the immunologic theory of aging and the role of the T cell Hayflick Limit in human aging, two areas of gerontological research pioneered by Roy Walford.

A new effect of IL-4 on human γδ T cells: promoting regulatory Vδ1 T cells via IL-10 production and inhibiting function of Vδ2 T cells.

PubMed

Mao, Yujia; Yin, Shanshan; Zhang, Jianmin; Hu, Yu; Huang, Bo; Cui, Lianxian; Kang, Ning; He, Wei

2016-03-01

Interleukin 4 (IL-4) has a variety of immune functions, including helper T-cell (Th-cell) differentiation and innate immune-response processes. However, the impact of IL-4 on gamma delta (γδ) T cells remains unclear. In this study, we investigate the effects of IL-4 on the activation and proliferation of γδ T cells and the balance between variable delta 1 (Vδ1) and Vδ2 T cells in humans. The results show that IL-4 inhibits the activation of γδ T cells in the presence of γδ T-cell receptor (TCR) stimulation in a STAT6-dependent manner. IL-4 promoted the growth of activated γδ T cells and increased the levels of Vδ1 T cells, which in turn inhibited Vδ2 T-cell growth via significant IL-10 secretion. Vδ1 T cells secreted significantly less interferon gamma (IFNγ) and more IL-10 relative to Vδ2. Furthermore, Vδ1 T cells showed relatively low levels of Natural Killer Group 2D (NKG2D) expression in the presence of IL-4, suggesting that Vδ1 T cells weaken the γδ T cell-mediated anti-tumor immune response. For the first time, our findings demonstrate a negative regulatory role of IL-4 in γδ T cell-mediated anti-tumor immunity.

Loss of intra-islet heparan sulfate is a highly sensitive marker of type 1 diabetes progression in humans.

PubMed

Simeonovic, Charmaine J; Popp, Sarah K; Starrs, Lora M; Brown, Debra J; Ziolkowski, Andrew F; Ludwig, Barbara; Bornstein, Stefan R; Wilson, J Dennis; Pugliese, Alberto; Kay, Thomas W H; Thomas, Helen E; Loudovaris, Thomas; Choong, Fui Jiun; Freeman, Craig; Parish, Christopher R

2018-01-01

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells in pancreatic islets are progressively destroyed. Clinical trials of immunotherapies in recently diagnosed T1D patients have only transiently and partially impacted the disease course, suggesting that other approaches are required. Our previous studies have demonstrated that heparan sulfate (HS), a glycosaminoglycan conventionally expressed in extracellular matrix, is present at high levels inside normal mouse beta cells. Intracellular HS was shown to be critical for beta cell survival and protection from oxidative damage. T1D development in Non-Obese Diabetic (NOD) mice correlated with loss of islet HS and was prevented by inhibiting HS degradation by the endoglycosidase, heparanase. In this study we investigated the distribution of HS and heparan sulfate proteoglycan (HSPG) core proteins in normal human islets, a role for HS in human beta cell viability and the clinical relevance of intra-islet HS and HSPG levels, compared to insulin, in human T1D. In normal human islets, HS (identified by 10E4 mAb) co-localized with insulin but not glucagon and correlated with the HSPG core proteins for collagen type XVIII (Col18) and syndecan-1 (Sdc1). Insulin-positive islets of T1D pancreases showed significant loss of HS, Col18 and Sdc1 and heparanase was strongly expressed by islet-infiltrating leukocytes. Human beta cells cultured with HS mimetics showed significantly improved survival and protection against hydrogen peroxide-induced death, suggesting that loss of HS could contribute to beta cell death in T1D. We conclude that HS depletion in beta cells, possibly due to heparanase produced by insulitis leukocytes, may function as an important mechanism in the pathogenesis of human T1D. Our findings raise the possibility that intervention therapy with dual activity HS replacers/heparanase inhibitors could help to protect the residual beta cell mass in patients recently diagnosed with T1D.

Immunology of Psoriasis

PubMed Central

Lowes, Michelle A.; Suárez-Fariñas, Mayte; Krueger, James G.

2014-01-01

The skin is the front line of defense against insult and injury and contains many epidermal and immune elements that comprise the skin-associated lymphoid tissue (SALT). The reaction of these components to injury allows an effective cutaneous response to restore homeostasis. Psoriasis vulgaris is the best-understood and most accessible human disease that is mediated by T cells and dendritic cells. Inflammatory myeloid dendritic cells release IL-23 and IL-12 to activate IL-17-producing T cells, Th1 cells, and Th22 cells to produce abundant psoriatic cytokines IL-17, IFN-γ, TNF, and IL-22. These cytokines mediate effects on keratinocytes to amplify psoriatic inflammation. Therapeutic studies with anticytokine antibodies have shown the importance of the key cytokines IL-23, TNF, and IL-17 in this process. We discuss the genetic background of psoriasis and its relationship to immune function, specifically genetic mutations, key PSORS loci, single nucleotide polymorphisms, and the skin transcriptome. The association between comorbidities and psoriasis is reviewed by correlating the skin transcriptome and serum proteins. Psoriasis-related cytokine-response pathways are considered in the context of the transcriptome of different mouse models. This approach offers a model for other inflammatory skin and autoimmune diseases. PMID:24655295

DNA Double-Strand Break Repair Genes and Oxidative Damage in Brain Metastasis of Breast Cancer

PubMed Central

Evans, Lynda; Duchnowska, Renata; Reed, L. Tiffany; Palmieri, Diane; Qian, Yongzhen; Badve, Sunil; Sledge, George; Gril, Brunilde; Aladjem, Mirit I.; Fu, Haiqing; Flores, Natasha M.; Gökmen-Polar, Yesim; Biernat, Wojciech; Szutowicz-Zielińska, Ewa; Mandat, Tomasz; Trojanowski, Tomasz; Och, Waldemar; Czartoryska-Arlukowicz, Bogumiła; Jassem, Jacek; Mitchell, James B.

2014-01-01

Background Breast cancer frequently metastasizes to the brain, colonizing a neuro-inflammatory microenvironment. The molecular pathways facilitating this colonization remain poorly understood. Methods Expression profiling of 23 matched sets of human resected brain metastases and primary breast tumors by two-sided paired t test was performed to identify brain metastasis–specific genes. The implicated DNA repair genes BARD1 and RAD51 were modulated in human (MDA-MB-231-BR) and murine (4T1-BR) brain-tropic breast cancer cell lines by lentiviral transduction of cDNA or short hairpin RNA (shRNA) coding sequences. Their functional contribution to brain metastasis development was evaluated in mouse xenograft models (n = 10 mice per group). Results Human brain metastases overexpressed BARD1 and RAD51 compared with either matched primary tumors (1.74-fold, P < .001; 1.46-fold, P < .001, respectively) or unlinked systemic metastases (1.49-fold, P = .01; 1.44-fold, P = .008, respectively). Overexpression of either gene in MDA-MB-231-BR cells increased brain metastases by threefold to fourfold after intracardiac injections, but not lung metastases upon tail-vein injections. In 4T1-BR cells, shRNA-mediated RAD51 knockdown reduced brain metastases by 2.5-fold without affecting lung metastasis development. In vitro, BARD1- and RAD51-overexpressing cells showed reduced genomic instability but only exhibited growth and colonization phenotypes upon DNA damage induction. Reactive oxygen species were present in tumor cells and elevated in the metastatic neuro-inflammatory microenvironment and could provide an endogenous source of genotoxic stress. Tempol, a brain-permeable oxygen radical scavenger suppressed brain metastasis promotion induced by BARD1 and RAD51 overexpression. Conclusions BARD1 and RAD51 are frequently overexpressed in brain metastases from breast cancer and may constitute a mechanism to overcome reactive oxygen species–mediated genotoxic stress in the metastatic brain. PMID:24948741

Effects of Aloe barbadensis Mill. extract (AVH200®) on human blood T cell activity in vitro.

PubMed

Ahluwalia, Bani; Magnusson, Maria K; Isaksson, Stefan; Larsson, Fredrik; Öhman, Lena

2016-02-17

Aloe barbadensis Mill. (Aloe vera) is a widely used medicinal plant well reputed for its diverse therapeutic applications. It has been used for thousands of years in folk medicine to treat various conditions and the Aloe vera gel has been reported to possess anti-inflammatory as well as immunostimulatory and immunomodulatory properties. However, the mode of action is still unclear. The aim of this study was determine the effects of two well-defined A. barbadensis Mill. extracts AVH200® and AVE200 on human blood T cells in vitro. Peripheral blood mononuclear cells (PBMC) from healthy donors were stimulated polyclonally in the presence or absence of AVH200® and AVE200. The T cell phenotype was investigated by flow cytometry, cell proliferation was determined by CFSE dye and thymidine assay, respectively and cytokine secretion was determined by MSD® Multi-Spot Assay system and ELISA. The presence of AVH200® resulted in a reduced expression of CD25 among CD3(+) T cells and suppression of T cell proliferation in a dose dependent manner. Furthermore, AVH200® reduced the expression of CD28 on CD3(+) T cells. AVH200® also reduced the secretion of IL-2, IFN-γ and IL-17A in PBMC cultures. The AVH200® dose dependent reduction in T cell activation and proliferation recorded in the cell cultures was not due to apoptosis or cell death. Additionally, AVH200® was found to be more effective as compared to AVE200 in reducing T cell activation and proliferation. AVH200® has the potential to reduce the activation, proliferation and cytokine secretion of healthy human blood T cells. Our study suggests that AVH200® has a suppressive effect on human blood T cells in vitro. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Human Milk Oligosaccharides and Synthetic Galactosyloligosaccharides Contain 3′-, 4-, and 6′-Galactosyllactose and Attenuate Inflammation in Human T84, NCM-460, and H4 Cells and Intestinal Tissue Ex Vivo12

PubMed Central

Ko, Jae Sung; Leone, Serena; Nanthakumar, N Nanda

2016-01-01

Background: The immature intestinal mucosa responds excessively to inflammatory insult, but human milk protects infants from intestinal inflammation. The ability of galactosyllactoses [galactosyloligosaccharides (GOS)], newly found in human milk oligosaccharides (HMOS), to suppress inflammation was not known. Objective: The objective was to test whether GOS can directly attenuate inflammation and to explore the components of immune signaling modulated by GOS. Methods: Galactosyllactose composition was measured in sequential human milk samples from days 1 through 21 of lactation and in random colostrum samples from 38 mothers. Immature [human normal fetal intestinal epithelial cell (H4)] and mature [human metastatic colonic epithelial cell (T84) and human normal colon mucosal epithelial cell (NCM-460)] enterocyte cell lines were treated with the pro-inflammatory molecules tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β) or infected with Salmonella or Listeria. The inflammatory response was measured as induction of IL-8, monocyte chemoattractant protein 1 (MCP-1), or macrophage inflammatory protein-3α (MIP-3α) protein by ELISA and mRNA by quantitative reverse transcriptase-polymerase chain reaction. The ability of HMOS or synthetic GOS to attenuate this inflammation was tested in vitro and in immature human intestinal tissue ex vivo. Results: The 3 galactosyllactoses (3′-GL, 4-GL, and 6′-GL) expressed in colostrum rapidly declined over early lactation (P < 0.05). In H4 cells, HMOS attenuated TNF-α– and IL-1β–induced expression of IL-8, MIP-3α, and MCP-1 to 48–51% and pathogen-induced IL-8 and MCP-1 to 26–30% of positive controls (P < 0.001). GOS reduced TNF-α– and IL-1β–induced inflammatory responses to 25–26% and pathogen-induced IL-8 and MCP-1 to 36–39% of positive controls (P < 0.001). GOS and HMOS mitigated nuclear translocation of nuclear transcription factor κB (NF-κB) p65. HMOS quenched the inflammatory response to Salmonella infection by immature human intestinal tissue ex vivo to 26% and by GOS to 50% of infected controls (P < 0.01). Conclusion: Galactosyllactose attenuated NF-κB inflammatory signaling in human intestinal epithelial cells and in human immature intestine. Thus, galactosyllactoses are strong physiologic anti-inflammatory agents in human colostrum and early milk, contributing to innate immune modulation. The potential clinical utility of galactosyllactose warrants investigation. PMID:26701795

Human adipose tissue-derived mesenchymal stem cells inhibit T-cell lymphoma growth in vitro and in vivo.

PubMed

Ahn, Jin-Ok; Chae, Ji-Sang; Coh, Ye-Rin; Jung, Woo-Sung; Lee, Hee-Woo; Shin, Il-Seob; Kang, Sung-Keun; Youn, Hwa-Young

2014-09-01

Human mesenchymal stem cells (hMSCs) are thought to be one of the most reliable stem cell sources for a variety of cell therapies. This study investigated the anti-tumor effect of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) on EL4 murine T-cell lymphoma in vitro and in vivo. The growth-inhibitory effect of hAT-MSCs on EL4 tumor cells was evaluated using a WST-1 cell proliferation assay. Cell-cycle arrest and apoptosis were investigated by flow cytometry and western blot. To evaluate an anti-tumor effect of hAT-MSCs on T-cell lymphoma in vivo, CM-DiI-labeled hAT-MSCs were circumtumorally injected in tumor-bearing nude mice, and tumor size was measured. hAT-MSCs inhibited T-cell lymphoma growth by altering cell-cycle progression and inducing apoptosis in vitro. hAT-MSCs inhibited tumor growth in tumor-bearing nude mice and prolonged survival time. Immunofluorescence analysis showed that hAT-MSCs migrated to tumor sites. hAT-MSCs suppress the growth of T-cell lymphoma, suggesting a therapeutic option for T-cell lymphoma. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Establishment and characterization of five immortalized human scalp dermal papilla cell lines.

PubMed

Kwack, Mi Hee; Yang, Jung Min; Won, Gong Hee; Kim, Moon Kyu; Kim, Jung Chul; Sung, Young Kwan

2018-02-05

Dermal papilla (DP) regulates the growth and cycling of hair follicles. Cultured DP cells are useful for the study of their role in relation to hair growth and regeneration. However, cultivation of human DP cells is tedious and difficult. In addition, cultured DP cells possess a relatively short replicative life span, requiring immortalized human DP cell lines. We previously established an immortalized human DP cell line, SV40T-hTERT-DPC, by introducing human telomerase reverse transcriptase (hTERT) gene into the transformed cell line, SV40T-DPC. In this study, we co-transfected the simian virus 40 large T antigen (SV40T-Ag) and hTERT into DP cells from scalp hair follicles from a male with androgenetic alopecia and established five immortalized DP cell lines and named KNU-101, KNU-102, KNU-103, KNU-201 and KNU-202. We then evaluated tumorigenicity, expression of DP markers, responses to androgen, Wnt3a and BMP4, and expression of DP signature genes. These cell lines displayed early passage morphology and maintained responses to androgen, Wnt and BMP. Furthermore, these cell lines expressed DP markers and DP signature genes. KNU cell lines established in this study are potentially useful sources for hair research. Copyright © 2018 Elsevier Inc. All rights reserved.

In situ induction of dendritic cell–based T cell tolerance in humanized mice and nonhuman primates

PubMed Central

Jung, Kyeong Cheon; Jeon, Yoon Kyung; Ban, Young Larn; Min, Hye Sook; Kim, Eun Ji; Kim, Ju Hyun; Kang, Byung Hyun; Bae, Youngmee; Yoon, Il-Hee; Kim, Yong-Hee; Lee, Jae-Il; Kim, Jung-Sik; Shin, Jun-Seop; Yang, Jaeseok; Kim, Sung Joo; Rostlund, Emily; Muller, William A.

2011-01-01

Induction of antigen-specific T cell tolerance would aid treatment of diverse immunological disorders and help prevent allograft rejection and graft versus host disease. In this study, we establish a method of inducing antigen-specific T cell tolerance in situ in diabetic humanized mice and Rhesus monkeys receiving porcine islet xenografts. Antigen-specific T cell tolerance is induced by administration of an antibody ligating a particular epitope on ICAM-1 (intercellular adhesion molecule 1). Antibody-mediated ligation of ICAM-1 on dendritic cells (DCs) led to the arrest of DCs in a semimature stage in vitro and in vivo. Ablation of DCs from mice completely abrogated anti–ICAM-1–induced antigen-specific T cell tolerance. T cell responses to unrelated antigens remained unaffected. In situ induction of DC-mediated T cell tolerance using this method may represent a potent therapeutic tool for preventing graft rejection. PMID:22025302

Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance

PubMed Central

Romano Ibarra, Guillermo S; Paul, Biswajit; Sather, Blythe D; Younan, Patrick M; Sommer, Karen; Kowalski, John P; Hale, Malika; Stoddard, Barry; Jarjour, Jordan; Astrakhan, Alexander; Kiem, Hans-Peter; Rawlings, David J

2016-01-01

A naturally occurring 32-base pair deletion of the HIV-1 co-receptor CCR5 has demonstrated protection against HIV infection of human CD4+ T cells. Recent genetic engineering approaches using engineered nucleases to disrupt the gene and mimic this mutation show promise for HIV therapy. We developed a megaTAL nuclease targeting the third extracellular loop of CCR5 that we delivered to primary human T cells by mRNA transfection. The CCR5 megaTAL nuclease established resistance to HIV in cell lines and disrupted the expression of CCR5 on primary human CD4+ T cells with a high efficiency, achieving up to 80% modification of the locus in primary cells as measured by molecular analysis. Gene-modified cells engrafted at levels equivalent to unmodified cells when transplanted into immunodeficient mice. Furthermore, genetically modified CD4+ cells were preferentially expanded during HIV-1 infection in vivo in an immunodeficient mouse model. Our results demonstrate the feasibility of targeting CCR5 in primary T cells using an engineered megaTAL nuclease, and the potential to use gene-modified cells to reconstitute a patient's immune system and provide protection from HIV infection. PMID:27741222

The role of bovine gamma delta T cells and their WC1 co-receptor in response to bacterial pathogens and promoting vaccine efficacy: A model for cattle and humans

USDA-ARS?s Scientific Manuscript database

Gamma deltaT cells are critical to immune surveillance and protection since they are found as resident cells in many organs and tissues, including in humans and ruminants, and circulate at substantial numbers in the blood. It is known that gamma delta T cells contribute to cellular immunity and pro...

Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4+ T cells

PubMed Central

Hepworth, Matthew R.; Fung, Thomas C.; Masur, Samuel H.; Kelsen, Judith R.; McConnell, Fiona M.; Dubrot, Juan; Withers, David R.; Hugues, Stephanie; Farrar, Michael A.; Reith, Walter; Eberl, Gerard; Baldassano, Robert N.; Laufer, Terri M.; Elson, Charles O.; Sonnenberg, Gregory F.

2015-01-01

Inflammatory CD4+ T cell responses to self or commensal bacteria underlie the pathogenesis of autoimmunity and inflammatory bowel disease (IBD), respectively. While selection of self-specific T cells in the thymus limits responses to tissue antigens, the mechanisms that control selection of commensal bacteria-specific T cells remain poorly understood. Here we demonstrate that group 3 innate lymphoid cell (ILC3)-intrinsic expression of major histocompatibility complex class II (MHCII) is regulated similarly to thymic epithelial cells, and that MHCII+ ILC3s directly induce cell death of activated commensal bacteria-specific T cells. Further, MHCII on human colonic ILC3s was reduced in pediatric IBD patients. Collectively, these results define a selection pathway for commensal bacteria-specific CD4+ T cells in the intestine, and suggest that this process is dysregulated in human IBD. PMID:25908663

CD8+CD28- T cells: certainties and uncertainties of a prevalent human T-cell subset.

PubMed

Arosa, Fernando A

2002-02-01

Human peripheral blood CD8+ T cells comprise cells that are in different states of differentiation and under the control of complex homeostatic processes. In a number of situations ranging from chronic inflammatory conditions and infectious diseases to ageing, immunodeficiency, iron overload and heavy alcohol intake, major phenotypic changes, usually associated with an increase in CD8+ T cells lacking CD28 expression, take place. CD8+CD28- T cells are characterized by a low proliferative capacity to conventional stimulation in vitro and by morphological and functional features of activated/memory T cells. Although the nature of the signals that give origin to this T-cell subset is uncertain, growing evidence argues for the existence of an interplay between epithelial cells, molecules with the MHC-class I fold and CD8+ T cells. The possibility that the generation of CD8+CD28- T cells is the combination of TCR/CD3zeta- and regulatory factor-mediated signals as a result of the sensing of modifications of the internal environment is discussed.

The promise of γδ T cells and the γδ T cell receptor for cancer immunotherapy.

PubMed

Legut, Mateusz; Cole, David K; Sewell, Andrew K

2015-11-01

γδ T cells form an important part of adaptive immune responses against infections and malignant transformation. The molecular targets of human γδ T cell receptors (TCRs) remain largely unknown, but recent studies have confirmed the recognition of phosphorylated prenyl metabolites, lipids in complex with CD1 molecules and markers of cellular stress. All of these molecules are upregulated on various cancer types, highlighting the potential importance of the γδ T cell compartment in cancer immunosurveillance and paving the way for the use of γδ TCRs in cancer therapy. Ligand recognition by the γδ TCR often requires accessory/co-stimulatory stress molecules on both T cells and target cells; this cellular stress context therefore provides a failsafe against harmful self-reactivity. Unlike αβ T cells, γδ T cells recognise their targets irrespective of HLA haplotype and therefore offer exciting possibilities for off-the-shelf, pan-population cancer immunotherapies. Here, we present a review of known ligands of human γδ T cells and discuss the promise of harnessing these cells for cancer treatment.

HLA Class I Binding 9mer Peptides from Influenza A Virus Induce CD4+ T Cell Responses

PubMed Central

Wang, Mingjun; Larsen, Mette V.; Nielsen, Morten; Harndahl, Mikkel; Justesen, Sune; Dziegiel, Morten H.; Buus, Søren; Tang, Sheila T.; Lund, Ole; Claesson, Mogens H.

2010-01-01

Background Identification of human leukocyte antigen class I (HLA-I) restricted cytotoxic T cell (CTL) epitopes from influenza virus is of importance for the development of new effective peptide-based vaccines. Methodology/Principal Findings In the present work, bioinformatics was used to predict 9mer peptides derived from available influenza A viral proteins with binding affinity for at least one of the 12 HLA-I supertypes. The predicted peptides were then selected in a way that ensured maximal coverage of the available influenza A strains. One hundred and thirty one peptides were synthesized and their binding affinities for the HLA-I supertypes were measured in a biochemical assay. Influenza-specific T cell responses towards the peptides were quantified using IFNγ ELISPOT assays with peripheral blood mononuclear cells (PBMC) from adult healthy HLA-I typed donors as responder cells. Of the 131 peptides, 21 were found to induce T cell responses in 19 donors. In the ELISPOT assay, five peptides induced responses that could be totally blocked by the pan-specific anti-HLA-I antibody W6/32, whereas 15 peptides induced responses that could be completely blocked in the presence of the pan-specific anti-HLA class II (HLA-II) antibody IVA12. Blocking of HLA-II subtype reactivity revealed that 8 and 6 peptide responses were blocked by anti-HLA-DR and -DP antibodies, respectively. Peptide reactivity of PBMC depleted of CD4+ or CD8+ T cells prior to the ELISPOT culture revealed that effectors are either CD4+ (the majority of reactivities) or CD8+ T cells, never a mixture of these subsets. Three of the peptides, recognized by CD4+ T cells showed binding to recombinant DRA1*0101/DRB1*0401 or DRA1*0101/DRB5*0101 molecules in a recently developed biochemical assay. Conclusions/Significance HLA-I binding 9mer influenza virus-derived peptides induce in many cases CD4+ T cell responses restricted by HLA-II molecules. PMID:20479886

The CD8 T cell in multiple sclerosis: suppressor cell or mediator of neuropathology?

PubMed

Johnson, Aaron J; Suidan, Georgette L; McDole, Jeremiah; Pirko, Istvan

2007-01-01

Multiple sclerosis (MS) is the most common human demyelinating disease of the central nervous system. It is universally accepted that the immune system plays a major role in the pathogenesis of MS. For decades, CD4 T cells have been considered the predominant mediator of neuropathology in MS. This perception was largely due to the similarity between MS and CD4 T-cell-driven experimental allergic encephalomyelitis, the most commonly studied murine model of MS. Over the last decade, several new observations in MS research imply an emerging role for CD8 T cells in neuropathogenesis. In certain experimental autoimmune encephalomyelitis (EAE) models, CD8 T cells are considered suppressors of pathology, whereas in other EAE models, neuropathology can be exacerbated by adoptive transfer of CD8 T cells. Studies using the Theiler's murine encephalomyelitis virus (TMEV) model have demonstrated preservation of motor function and axonal integrity in animals deficient in CD8 T cells or their effector molecules. CD8 T cells have also been demonstrated to be important regulators of blood-brain barrier permeability. There is also an emerging role for CD8 T cells in human MS. Human genetic studies reveal an important role for HLA class I molecules in MS susceptibility. In addition, neuropathologic studies demonstrate that CD8 T cells are the most numerous inflammatory infiltrate in MS lesions at all stages of lesion development. CD8 T cells are also capable of damaging neurons and axons in vitro. In this chapter, we discuss the neuropathologic, genetic, and experimental evidence for a critical role of CD8 T cells in the pathogenesis of MS and its most frequently studied animal models. We also highlight important new avenues for future research.

3,3′-Diindolylmethane Induces G1 Arrest and Apoptosis in Human Acute T-Cell Lymphoblastic Leukemia Cells

PubMed Central

Shorey, Lyndsey E.; Hagman, Amanda M.; Williams, David E.; Ho, Emily; Dashwood, Roderick H.; Benninghoff, Abby D.

2012-01-01

Certain bioactive food components, including indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM) from cruciferous vegetables, have been shown to target cellular pathways regulating carcinogenesis. Previously, our laboratory showed that dietary I3C is an effective transplacental chemopreventive agent in a dibenzo[def,p]chrysene (DBC)-dependent model of murine T-cell lymphoblastic lymphoma. The primary objective of the present study was to extend our chemoprevention studies in mice to an analogous human neoplasm in cell culture. Therefore, we tested the hypothesis that I3C or DIM may be chemotherapeutic in human T-cell acute lymphoblastic leukemia (T-ALL) cells. Treatment of the T-ALL cell lines CCRF-CEM, CCRF-HSB2, SUP-T1 and Jurkat with DIM in vitro significantly reduced cell proliferation and viability at concentrations 8- to 25-fold lower than the parent compound I3C. DIM (7.5 µM) arrested CEM and HSB2 cells at the G1 phase of the cell cycle and 15 µM DIM significantly increased the percentage of apoptotic cells in all T-ALL lines. In CEM cells, DIM reduced protein expression of cyclin dependent kinases 4 and 6 (CDK4, CDK6) and D-type cyclin 3 (CCND3); DIM also significantly altered expression of eight transcripts related to human apoptosis (BCL2L10, CD40LG, HRK, TNF, TNFRSF1A, TNFRSF25, TNFSF8, TRAF4). Similar anticancer effects of DIM were observed in vivo. Dietary exposure to 100 ppm DIM significantly decreased the rate of growth of human CEM xenografts in immunodeficient SCID mice, reduced final tumor size by 44% and increased the apoptotic index compared to control-fed mice. Taken together, our results demonstrate a potential for therapeutic application of DIM in T-ALL. PMID:22514694

Migration of Toxoplasma gondii–Infected Dendritic Cells across Human Retinal Vascular Endothelium

PubMed Central

Furtado, João M.; Bharadwaj, Arpita S.; Ashander, Liam M.; Olivas, Antoinette; Smith, Justine R.

2012-01-01

Purpose. Toxoplasma gondii, the parasite responsible for ocular toxoplasmosis, accesses the retina from the bloodstream. We investigated the dendritic cell as a potential taxi for T. gondii tachyzoites moving across the human retinal endothelium, and examined the participation of adhesion molecules and chemokines in this process. Methods. CD14-positive monocytes were isolated from human peripheral blood by antibody-mediated cell enrichment, and cultured in granulocyte-macrophage colony-stimulating factor and interleukin-4 to generate dendritic cells. Transmigration assays were performed over 18 hours in transwells seeded with human retinal endothelial cells and using dendritic cells exposed to laboratory or natural strains of T. gondii tachyzoites. Parasites were tagged with yellow fluorescent protein to verify infection. In some experiments, endothelial monolayers were preincubated with antibody directed against adhesion molecules, or chemokine was added to lower chambers of transwells. Results. Human monocyte–derived dendritic cell preparations infected with laboratory or natural strain T. gondii tachyzoites transmigrated in larger numbers across simulated human retinal endothelium than uninfected dendritic cells (P ≤ 0.0004 in 5 of 6 experiments). Antibody blockade of intercellular adhesion molecule (ICAM)–1, vascular cell adhesion molecule (VCAM)–1, and activated leukocyte cell adhesion molecule (ALCAM) inhibited transmigration (P ≤ 0.007), and CCL21 or CXCL10 increased transmigration (P ≤ 0.031). Conclusions. Transmigration of human dendritic cells across retinal endothelium is increased following infection with T. gondii. Movement may be impacted by locally produced chemokines and is mediated in part by ICAM-1, VCAM-1, and ALCAM. These findings have implications for development of novel therapeutics aimed at preventing retinal infection by T. gondii. PMID:22952125

Human mesenchymal stem cells suppress donor CD4(+) T cell proliferation and reduce pathology in a humanized mouse model of acute graft-versus-host disease.

PubMed

Tobin, L M; Healy, M E; English, K; Mahon, B P

2013-05-01

Acute graft-versus-host disease (aGVHD) is a life-threatening complication following allogeneic haematopoietic stem cell transplantation (HSCT), occurring in up to 30-50% of patients who receive human leucocyte antigen (HLA)-matched sibling transplants. Current therapies for steroid refractory aGVHD are limited, with the prognosis of patients suboptimal. Mesenchymal stem or stromal cells (MSC), a heterogeneous cell population present in many tissues, display potent immunomodulatory abilities. Autologous and allogeneic ex-vivo expanded human MSC have been utilized to treat aGVHD with promising results, but the mechanisms of therapeutic action remain unclear. Here a robust humanized mouse model of aGVHD based on delivery of human peripheral blood mononuclear cells (PBMC) to non-obese diabetic (NOD)-severe combined immunodeficient (SCID) interleukin (IL)-2rγ(null) (NSG) mice was developed that allowed the exploration of the role of MSC in cell therapy. MSC therapy resulted in the reduction of liver and gut pathology and significantly increased survival. Protection was dependent upon the timing of MSC therapy, with conventional MSC proving effective only after delayed administration. In contrast, interferon (IFN)-γ-stimulated MSC were effective when delivered with PBMC. The beneficial effect of MSC therapy in this model was not due to the inhibition of donor PBMC chimerism, as CD45(+) and T cells engrafted successfully in this model. MSC therapy did not induce donor T cell anergy, FoxP3(+) T regulatory cells or cause PBMC apoptosis in this model; however, it was associated with the direct inhibition of donor CD4(+) T cell proliferation and reduction of human tumour necrosis factor-α in serum. © 2012 British Society for Immunology.

Increased Regulatory T-Cell Percentage Contributes to Poor CD4(+) Lymphocytes Recovery: A 2-Year Prospective Study After Introduction of Antiretroviral Therapy.

PubMed

Saison, Julien; Maucort Boulch, Delphine; Chidiac, Christian; Demaret, Julie; Malcus, Christophe; Cotte, Laurent; Poitevin-Later, Francoise; Miailhes, Patrick; Venet, Fabienne; Trabaud, Mary Anne; Monneret, Guillaume; Ferry, Tristan

2015-04-01

Background.  The primary aim of this study was to determine the impact of regulatory T cells (Tregs) percentage on immune recovery in human immunodeficiency virus (HIV)-infected patients after antiretroviral therapy introduction. Methods.  A 2-year prospective study was conducted in HIV-1 chronically infected naive patients with CD4 count <500 cells/mm(3). Regulatory T cells were identified as CD4(+)CD25(high)CD127(low) cells among CD4(+) lymphocytes. Effect of Treg percentage at inclusion on CD4 evolution overtime was analyzed using a mixed-effect Poisson regression for count data. Results.  Fifty-eight patients were included (median CD4 = 293/mm(3), median Treg percentage = 6.1%). Percentage of Treg at baseline and CD4 nadir were independently related to the evolution of CD4 absolute value according to time: (1) at any given nadir CD4 count, 1% increase of initial Treg was associated with a 1.9% lower CD4 absolute value at month 24; (2) at any given Treg percentage at baseline, 10 cell/mm(3) increase of CD4 nadir was associated with a 2.4% increase of CD4 at month 24; and (3) both effects did not attenuate with time. The effect of Treg at baseline on CD4 evolution was as low as the CD4 nadir was high. Conclusions.  Regulatory T-cell percentage at baseline is a strong independent prognostic factor of immune recovery, particularly among patients with low CD4 nadir.

Local convection-enhanced delivery of an anti-CD40 agonistic monoclonal antibody induces antitumor effects in mouse glioma models

PubMed Central

Shoji, Takuhiro; Saito, Ryuta; Chonan, Masashi; Shibahara, Ichiyo; Sato, Aya; Kanamori, Masayuki; Sonoda, Yukihiko; Kondo, Toru; Ishii, Naoto; Tominaga, Teiji

2016-01-01

Background Glioblastoma is one of the most malignant brain tumors in adults and has a dismal prognosis. In a previous report, we reported that CD40, a TNF-R-related cell surface receptor, and its ligand CD40L were associated with glioma outcomes. Here we attempted to activate CD40 signaling in the tumor and determine if it exerted therapeutic efficacy. Methods CD40 expression was examined in 3 mouse glioma cell lines (GL261, NSCL61, and bRiTs-G3) and 5 human glioma cell lines (U87, U251, U373, T98, and A172). NSCL61 and bRiTs-G3, as glioma stem cells, also expressed the glioma stem cell markers MELK and CD44. In vitro, we demonstrated direct antitumor effects of an anti-CD40 agonistic monoclonal antibody (FGK45) against the cell lines. The efficacy of FGK45 was examined by local convection-enhanced delivery of the monoclonal antibody against each glioma model. Results CD40 was expressed in all mouse and human cell lines tested and was found at the cell membrane of each of the 3 mouse cell lines. FGK45 administration induced significant, direct antitumor effects in vitro. The local delivery of FGK45 significantly prolonged survival compared with controls in the NSCL61 and bRiTs-G3 models, but the effect was not significant in the GL261 model. Increases in apoptosis and CD4+ and CD8+ T cell infiltration were observed in the bRiTs-G3 model after FGK45 treatment. Conclusions Local delivery of FGK45 significantly prolonged survival in glioma stem cell models. Thus, local delivery of this monoclonal antibody is promising for immunotherapy against gliomas. PMID:26917236

Automated Expansion of Primary Human T Cells in Scalable and Cell-Friendly Hydrogel Microtubes for Adoptive Immunotherapy.

PubMed

Lin, Haishuang; Li, Qiang; Wang, Ou; Rauch, Jack; Harm, Braden; Viljoen, Hendrik J; Zhang, Chi; Van Wyk, Erika; Zhang, Chi; Lei, Yuguo

2018-05-11

Adoptive immunotherapy is a highly effective strategy for treating many human cancers, such as melanoma, cervical cancer, lymphoma, and leukemia. Here, a novel cell culture technology is reported for expanding primary human T cells for adoptive immunotherapy. T cells are suspended and cultured in microscale alginate hydrogel tubes (AlgTubes) that are suspended in the cell culture medium in a culture vessel. The hydrogel tubes protect cells from hydrodynamic stresses and confine the cell mass less than 400 µm (in radial diameter) to ensure efficient mass transport, creating a cell-friendly microenvironment for growing T cells. This system is simple, scalable, highly efficient, defined, cost-effective, and compatible with current good manufacturing practices. Under optimized culture conditions, the AlgTubes enable culturing T cells with high cell viability, low DNA damage, high growth rate (≈320-fold expansion over 14 days), high purity (≈98% CD3+), and high yield (≈3.2 × 10 8 cells mL -1 hydrogel). All offer considerable advantages compared to current T cell culturing approaches. This new culture technology can significantly reduce the culture volume, time, and cost, while increasing the production. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Human T-cell leukemia virus type 1 Tax oncoprotein represses the expression of the BCL11B tumor suppressor in T-cells.

PubMed

Takachi, Takayuki; Takahashi, Masahiko; Takahashi-Yoshita, Manami; Higuchi, Masaya; Obata, Miki; Mishima, Yukio; Okuda, Shujiro; Tanaka, Yuetsu; Matsuoka, Masao; Saitoh, Akihiko; Green, Patrick L; Fujii, Masahiro

2015-04-01

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia (ATL), which is an aggressive form of T-cell malignancy. HTLV-1 oncoproteins, Tax and HBZ, play crucial roles in the immortalization of T-cells and/or leukemogenesis by dysregulating the cellular functions in the host. Recent studies show that HTLV-1-infected T-cells have reduced expression of the BCL11B tumor suppressor protein. In the present study, we explored whether Tax and/or HBZ play a role in downregulating BCL11B in HTLV-1-infected T-cells. Lentiviral transduction of Tax in a human T-cell line repressed the expression of BCL11B at both the protein and mRNA levels, whereas the transduction of HBZ had little effect on the expression. Tax mutants with a decreased activity for the NF-κB, CREB or PDZ protein pathways still showed a reduced expression of the BCL11B protein, thereby implicating a different function of Tax in BCL11B downregulation. In addition, the HTLV-2 Tax2 protein reduced the BCL11B protein expression in T-cells. Seven HTLV-1-infected T-cell lines, including three ATL-derived cell lines, showed reduced BCL11B mRNA and protein expression relative to an uninfected T-cell line, and the greatest reductions were in the cells expressing Tax. Collectively, these results indicate that Tax is responsible for suppressing BCL11B protein expression in HTLV-1-infected T-cells; Tax-mediated repression of BCL11B is another mechanism that Tax uses to promote oncogenesis of HTLV-1-infected T-cells. © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Compartmentalized, functional role of angiogenin during spotted fever group rickettsia-induced endothelial barrier dysfunction: evidence of possible mediation by host tRNA-derived small noncoding RNAs

PubMed Central

2013-01-01

Background Microvascular endothelial barrier dysfunction is the central enigma in spotted fever group (SFG) rickettsioses. Angiogenin (ANG) is one of the earliest identified angiogenic factors, of which some are relevant to the phosphorylation of VE-cadherins that serve as endothelial adherens proteins. Although exogenous ANG is known to translocate into the nucleus of growing endothelial cells (ECs) where it plays a functional role, nuclear ANG is not detected in quiescent ECs. Besides its nuclear role, ANG is thought to play a cytoplasmic role, owing to its RNase activity that cleaves tRNA to produce small RNAs. Recently, such tRNA-derived RNA fragments (tRFs) have been shown to be induced under stress conditions. All these observations raise an intriguing hypothesis about a novel cytoplasmic role of ANG, which is induced upon infection with Rickettsia and generates tRFs that may play roles in SFG rickettsioses. Methods C3H/HeN mice were infected intravenously with a sublethal dose of R. conorii. At days 1, 3, and 5 post infection (p.i.), liver, lung and brain were collected for immunofluorescence (IF) studies of R. conorii and angiogenin (ANG). Human umbilical vein endothelial cells (HUVECs) were infected with R. conorii for 24, 48, and 72 hrs before incubation with 1μg/ml recombinant human ANG (rANG) in normal medium for 2 hrs. HUVEC samples were subjected to IF, exogenous ANG translocation, endothelial permeability, and immunoprecipitation phosphorylation assays. To identify small non-coding RNAs (sncRNAs) upon rickettsial infection, RNAs from pulverized mouse lung tissues and HUVECs were subjected to library preparation and deep sequencing analysis using an Illumina 2000 instrument. Identified sncRNAs were confirmed by Northern hybridization, and their target mRNAs were predicted in silico using BLAST and RNA hybrid programs. Results In the present study, we have demonstrated endothelial up-regulation of ANG, co-localized with SFG rickettsial infection in vivo. We also have provided direct evidence that rickettsial infection sensitizes human ECs to the translocation of exogenous ANG in a compartmentalized pattern at different times post-infection. Typically, exogenous ANG translocates into the nucleus at 24 hrs and to the cytoplasm at 72 hrs post-infection. The ANG cytoplasmic translocation enhances phosphorylation and destabilization of VE-cadherin and attenuates endothelial barrier function. Of note, deep sequencing analysis detected tRFs, mostly derived from the 5'-halves of host tRNAs, that are induced by ANG. Northern hybridization validates the two most abundantly cloned tRFs derived from tRNA-ValGTG and tRNA-GlyGCC, in both mouse tissues and human cells. Bioinformatics analysis predicted that these tRFs may interact with transcripts associated with the endothelial barrier, the host cell inflammatory response, and autophagy. Conclusions Our data provide new insight into the role of compartmentalized ANG during SFG rickettsioses, and highlight its possible mediation through tRFs. PMID:23800282

Antigen-Conjugated Human IgE Induces Antigen-Specific T Cell Tolerance in a Humanized Mouse Model

PubMed Central

Baravalle, Günther; Greer, Alexandra M.; LaFlam, Taylor N.; Shin, Jeoung-Sook

2015-01-01

Dendritic cells (DCs) play an important role in immune homeostasis through their ability to present Ags at steady state and mediate T cell tolerance. This characteristic renders DCs an attractive therapeutic target for the induction of tolerance against auto-antigens or allergens. Accordingly, Ag-conjugated DC–specific Abs have been proposed to be an excellent vehicle to deliver Ags to DCs for presentation and tolerance induction. However, this approach requires laborious reagent generation procedures and entails unpredictable side effects resulting from Ab-induced crosslinking of DC surface molecules. In this study, we examined whether IgE, a high-affinity, non–cross-linking natural ligand of FcεRI, could be used to target Ags to DCs and to induce Ag-specific T cell tolerance. We found that Ag-conjugated human IgE Fc domain (Fcε) effectively delivered Ags to DCs and enhanced Ag presentation by 1000- to 2500-fold in human FcεRIα-transgenic mice. Importantly, this presentation resulted in a systemic deletion of Ag-specific T cells and prevented these mice from developing delayed-type hypersensitivity, which is critically dependent on Ag-specific T cell immunity. Thus, targeting FcεRI on DCs via Ag-Fcε fusion protein may serve an alternative method to induce Ag-specific T cell tolerance in humans. PMID:24610015

A20 Haploinsufficiency Aggravates Transplant Arteriosclerosis in Mouse Vascular Allografts: Implications for Clinical Transplantation

PubMed Central

Cervantes, Jesus Revuelta; Wojcik, Brandon M.; Parulkhar, Anshul; Mele, Alessandra; LoGerfo, Philip J.; Siracuse, Jeffrey J.; Csizmadia, Eva; da Silva, Cleide G.; Ferran, Christiane

2016-01-01

Background Inflammation is central to the pathogenesis of transplant arteriosclerosis (TA). We questioned whether physiologic levels of anti-inflammatory A20 influence TA severity. Methods We performed major histocompatibility complex (MHC) mismatched aorta to carotid artery interposition grafts, using wild type (WT) or A20 heterozygote (HET) C57BL/6 (H-2b) donors and BALB/c (H-2d) recipients, and conversely BALB/c donors and WT/HET recipients. We analyzed aortic allografts by histology, immunohistochemistry, immunofluorescence, and gene profiling (qPCR). We validated select in vivo A20 targets in human and mouse smooth muscle cell (SMC) cultures. Results We noted significantly greater intimal hyperplasia in HET vs. WT allografts, indicating aggravated TA. Inadequate upregulation of A20 in HET allografts after transplantation was associated with excessive NF-κB activation, gauged by higher levels of IκBα, p65, VCAM-1, ICAM-1, CXCL10, CCL2, TNF, and IL-6 (mostly localized to SMC). Correspondingly, cytokine-induced upregulation of TNF and IL-6 in human and mouse SMC cultures inversely correlated with A20 expression. Aggravated TA in HET vs. WT allografts correlated with increased intimal SMC proliferation, and a higher number of infiltrating IFNγ+ and Granzyme B+ CD4+ T cells and natural killer cells, and lower number of FoxP3+ regulatory T cells. A20 haploinsufficiency in allograft recipients did not influence TA. Conclusions A20 haploinsufficiency in vascular allografts aggravates lesions of TA by exacerbating inflammation, SMC proliferation, and infiltration of pathogenic T cells. A20 single nucleotide polymorphisms (SNPs) associating with lower A20 expression or function in donors of vascularized allografts may inform risk and severity of TA, highlighting the clinical implications of our findings. PMID:27495763

Herpesvirus Saimiri Transforms Human T-Cell Clones to Stable Growth without Inducing Resistance to Apoptosis

PubMed Central

Kraft, Michael S.; Henning, Golo; Fickenscher, Helmut; Lengenfelder, Doris; Tschopp, Jürg; Fleckenstein, Bernhard; Meinl, Edgar

1998-01-01

Herpesvirus saimiri (HVS) transforms human T cells to stable growth in vitro. Since HVS codes for two different antiapoptotic proteins, growth transformation by HVS might be expected to confer resistance to apoptosis. We found that the expression of both viral antiapoptotic genes was restricted to cultures with viral replication and absent in growth-transformed human T cells. A comparative examination of HVS-transformed T-cell clones and their native parental clones revealed that the expression of Bcl-2, Bcl-XL, Bax, and members of the tumor necrosis factor receptor (TNF-R) superfamily with a death domain, namely, TNF-RI, CD95, and TRAMP, were not modulated by HVS. Expression of CD30 was induced in HVS-transformed T cells, and these cells also expressed the CD30 ligand. Uninfected and transformed T cells were sensitive to CD95 ligation but resistant to apoptosis mediated by TRAIL or soluble TNF-α. CD95 ligand was constitutively expressed on transformed but not uninfected parental T cells. Both cell types showed similar sensitivity to cell death induction or inhibition of T-cell activation mediated by irradiation, oxygen radicals, dexamethasone, cyclosporine, and prostaglandin E2. Altogether, this study strongly suggests that growth transformation by HVS is based not on resistance to apoptosis but, rather, on utilization of normal cellular activation pathways. PMID:9525639

Immunotherapy for osteosarcoma: genetic modification of T cells overcomes low levels of tumor antigen expression.

PubMed

Ahmed, Nabil; Salsman, Vita S; Yvon, Eric; Louis, Chrystal U; Perlaky, Laszlo; Wels, Winfried S; Dishop, Meghan K; Kleinerman, Eugenie E; Pule, Martin; Rooney, Cliona M; Heslop, Helen E; Gottschalk, Stephen

2009-10-01

Human epidermal growth factor receptor 2 (HER2) is expressed by the majority of human osteosarcomas and is a risk factor for poor outcome. Unlike breast cancer, osteosarcoma cells express HER2 at too low, a level for patients to benefit from HER2 monoclonal antibodies. We reasoned that this limitation might be overcome by genetically modifying T cells with HER2-specific chimeric antigen receptors (CARs), because even a low frequency of receptor engagement could be sufficient to induce effector cell killing of the tumor. HER2-specific T cells were generated by retroviral transduction with a HER2-specific CAR containing a CD28.zeta signaling domain. HER2-specific T cells recognized HER2-positive osteosarcoma cells as judged by their ability to proliferate, produce immunostimulatory T helper 1 cytokines, and kill HER2-positive osteosarcoma cell lines in vitro. The adoptive transfer of HER2-specific T cells caused regression of established osteosarcoma xenografts in locoregional as well as metastatic mouse models. In contrast, delivery of nontransduced (NT) T cells did not change the tumor growth pattern. Genetic modification of T cells with CARs specific for target antigens, expressed at too low a level to be effectively recognized by monoclonal antibodies, may allow immunotherapy to be more broadly applicable for human cancer therapy.

Boswellia sacra essential oil induces tumor cell-specific apoptosis and suppresses tumor aggressiveness in cultured human breast cancer cells

PubMed Central

2011-01-01

Background Gum resins obtained from trees of the Burseraceae family (Boswellia sp.) are important ingredients in incense and perfumes. Extracts prepared from Boswellia sp. gum resins have been shown to possess anti-inflammatory and anti-neoplastic effects. Essential oil prepared by distillation of the gum resin traditionally used for aromatic therapy has also been shown to have tumor cell-specific anti-proliferative and pro-apoptotic activities. The objective of this study was to optimize conditions for preparing Boswellea sacra essential oil with the highest biological activity in inducing tumor cell-specific cytotoxicity and suppressing aggressive tumor phenotypes in human breast cancer cells. Methods Boswellia sacra essential oil was prepared from Omani Hougari grade resins through hydrodistillation at 78 or 100 oC for 12 hours. Chemical compositions were identified by gas chromatography-mass spectrometry; and total boswellic acids contents were quantified by high-performance liquid chromatography. Boswellia sacra essential oil-mediated cell viability and death were studied in established human breast cancer cell lines (T47D, MCF7, MDA-MB-231) and an immortalized normal human breast cell line (MCF10-2A). Apoptosis was assayed by genomic DNA fragmentation. Anti-invasive and anti-multicellular tumor properties were evaluated by cellular network and spheroid formation models, respectively. Western blot analysis was performed to study Boswellia sacra essential oil-regulated proteins involved in apoptosis, signaling pathways, and cell cycle regulation. Results More abundant high molecular weight compounds, including boswellic acids, were present in Boswellia sacra essential oil prepared at 100 oC hydrodistillation. All three human breast cancer cell lines were sensitive to essential oil treatment with reduced cell viability and elevated cell death, whereas the immortalized normal human breast cell line was more resistant to essential oil treatment. Boswellia sacra essential oil hydrodistilled at 100 oC was more potent than the essential oil prepared at 78 oC in inducing cancer cell death, preventing the cellular network formation (MDA-MB-231) cells on Matrigel, causing the breakdown of multicellular tumor spheroids (T47D cells), and regulating molecules involved in apoptosis, signal transduction, and cell cycle progression. Conclusions Similar to our previous observations in human bladder cancer cells, Boswellia sacra essential oil induces breast cancer cell-specific cytotoxicity. Suppression of cellular network formation and disruption of spheroid development of breast cancer cells by Boswellia sacra essential oil suggest that the essential oil may be effective for advanced breast cancer. Consistently, the essential oil represses signaling pathways and cell cycle regulators that have been proposed as therapeutic targets for breast cancer. Future pre-clinical and clinical studies are urgently needed to evaluate the safety and efficacy of Boswellia sacra essential oil as a therapeutic agent for treating breast cancer. PMID:22171782

Immunological alterations and associated diseases in mandrills (Mandrillus sphinx) naturally co-infected with SIV and STLV.

PubMed

Souquière, Sandrine; Makuwa, Maria; Sallé, Bettina; Lepelletier, Yves; Mortreux, Franck; Hermine, Olivier; Kazanji, Mirdad

2014-04-01

Mandrills are naturally infected with simian T-cell leukaemia virus type 1 (STLV-1) and simian immunodeficiency virus (SIV)mnd. In humans, dual infection with human immunodeficiency virus (HIV) and human T-cell lymphotropic virus type 1 (HTLV-1) may worsen their clinical outcome. We evaluated the effect of co-infection in mandrills on viral burden, changes in T-cell subsets and clinical outcome. The SIV viral load was higher in SIV-infected mandrills than in co-infected animals, whereas the STLV-1 proviral load was higher in co-infected than in mono-infected groups. Dually infected mandrills had a statistically significantly lower CD4+ T-cell count, a lower proportion of naive CD8+ T cells and a higher proportion of central memory cells. CD4(+) and CD8(+) T cells from SIV-infected animals had a lower percentage of Ki67 than those from the other groups. Co-infected monkeys had higher percentages of activated CD4(+) and CD8(+) T cells. Two co-infected mandrills with high immune activation and clonal integration of STLV provirus showed pathological manifestations (infective dermatitis and generalised scabies) rarely encountered in nonhuman primates. Copyright © 2014 Elsevier Inc. All rights reserved.

Iodine-131 induces apoptosis in human cardiac muscle cells through the p53/Bax/caspase-3 and PIDD/caspase-2/ t‑BID/cytochrome c/caspase-3 signaling pathway.

PubMed

Wang, Yansheng; Liu, Changqing; Wang, Jianchun; Zhang, Yang; Chen, Linlin

2017-09-01

The aim of this study was to elucidate the effects of iodine-131 on the induction of apoptosis in human cardiac muscle cells and the underlying molecular mechanisms. We found that iodine-131 reduced cell proliferation, induced apoptosis, induced p53, PIDD, t-BID (mitochondria) protein expression, suppressed cytochrome c (mitochondria) protein expression, and increased Bax protein expression, and promoted caspase-2, -3 and -9 expression levels in human cardiac muscle cells. Meanwhile, si-p53 inhibited the effects of iodine-131 on the reduction in cell proliferation and induction of apoptosis in human cardiac muscle cells through regulation of Bax/cytochrome c/caspase-3 and PIDD/caspase‑2/t-BID/cytochrome c/caspase-3 signaling pathway. After si-Bax reduced the effects of iodine-131, it reduced cell proliferation and induced apoptosis in human cardiac muscle cells through the cytochrome c/caspase-3 signaling pathway. However, si-caspase-2 also reduced the effects of iodine-131 on the reduction of cell proliferation and induction of apoptosis in human cardiac muscle cells through the t-BID/cytochrome c/caspase-3 signaling pathway. These findings demonstrated that iodine-131 induces apoptosis in human cardiac muscle cells through the p53/Bax/caspase-3 and PIDD/caspase-2/t-BID/cytochrome c/caspase-3 signaling pathway.

Gut microbiota modulate T cell trafficking into human colorectal cancer.

PubMed

Cremonesi, Eleonora; Governa, Valeria; Garzon, Jesus Francisco Glaus; Mele, Valentina; Amicarella, Francesca; Muraro, Manuele Giuseppe; Trella, Emanuele; Galati-Fournier, Virginie; Oertli, Daniel; Däster, Silvio Raffael; Droeser, Raoul A; Weixler, Benjamin; Bolli, Martin; Rosso, Raffaele; Nitsche, Ulrich; Khanna, Nina; Egli, Adrian; Keck, Simone; Slotta-Huspenina, Julia; Terracciano, Luigi M; Zajac, Paul; Spagnoli, Giulio Cesare; Eppenberger-Castori, Serenella; Janssen, Klaus-Peter; Borsig, Lubor; Iezzi, Giandomenica

2018-02-06

Tumour-infiltrating lymphocytes (TILs) favour survival in human colorectal cancer (CRC). Chemotactic factors underlying their recruitment remain undefined. We investigated chemokines attracting T cells into human CRCs, their cellular sources and microenvironmental triggers. Expression of genes encoding immune cell markers, chemokines and bacterial 16S ribosomal RNA (16SrRNA) was assessed by quantitative reverse transcription-PCR in fresh CRC samples and corresponding tumour-free tissues. Chemokine receptor expression on TILs was evaluated by flow cytometry on cell suspensions from digested tissues. Chemokine production by CRC cells was evaluated in vitro and in vivo, on generation of intraperitoneal or intracecal tumour xenografts in immune-deficient mice. T cell trafficking was assessed on adoptive transfer of human TILs into tumour-bearing mice. Gut flora composition was analysed by 16SrRNA sequencing. CRC infiltration by distinct T cell subsets was associated with defined chemokine gene signatures, including CCL5, CXCL9 and CXCL10 for cytotoxic T lymphocytes and T-helper (Th)1 cells; CCL17, CCL22 and CXCL12 for Th1 and regulatory T cells; CXCL13 for follicular Th cells; and CCL20 and CCL17 for interleukin (IL)-17-producing Th cells. These chemokines were expressed by tumour cells on exposure to gut bacteria in vitro and in vivo. Their expression was significantly higher in intracecal than in intraperitoneal xenografts and was dramatically reduced by antibiotic treatment of tumour-bearing mice. In clinical samples, abundance of defined bacteria correlated with high chemokine expression, enhanced T cell infiltration and improved survival. Gut microbiota stimulate chemokine production by CRC cells, thus favouring recruitment of beneficial T cells into tumour tissues. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Increased numbers of CD4+ and CD8+ T cells in lesional skin of cats with allergic dermatitis.

PubMed

Roosje, P J; van Kooten, P J; Thepen, T; Bihari, I C; Rutten, V P; Koeman, J P; Willemse, T

1998-07-01

The aim of this study was to characterize T cells in the skin of cats with an allergic dermatitis histologically compatible with atopic dermatitis, since T cells play an important role in the pathogenesis of atopic dermatitis in humans. We observed a significantly greater number of T cells in lesional skin of domestic short-haired cats with allergic dermatitis (n = 10; median age 5.8 years) than in the skin of healthy control animals (n = 10; median age 5.0 years). In the skin of the healthy control animals, one or two CD4+ cells and no CD8+ cells were found. A predominant increase of CD4+ T cells and a CD4+/CD8+ ratio (mean +/- SD: 3.9 +/- 2.0) was found in the lesional skin of 10 cats with allergic dermatitis. The CD4+/CD8+ cell ratio in the skin of healthy control animals could not be determined because of the absence of CD8+ cells. The CD4+/CD8+ cell ratio in the peripheral blood of 10 cats with allergic dermatitis (mean +/- SD: 1.9 +/- 0.4) did not differ significantly from that in 10 healthy control animals (2.2 +/- 0.4). The CD4+/CD8+ cell ratio and predominance of CD4+ T cells in the lesional skin of cats with allergic dermatitis is comparable to that found in atopic dermatitis in humans. In addition, the observed increase of CD4+ T cells in the nonlesional skin of cats with allergic dermatitis compared to the skin of healthy cats is similar to what is seen in humans. Cytokines produced by T cells and antigen-specific T cells are important mediators in the inflammatory cascade resulting in atopic dermatitis in humans. This study is a first step to investigate their role in feline allergic dermatitis.

Humanized NOG mice as a model for tuberculosis vaccine-induced immunity: a comparative analysis with the mouse and guinea pig models of tuberculosis.

PubMed

Grover, Ajay; Troy, Amber; Rowe, Jenny; Troudt, JoLynn M; Creissen, Elizabeth; McLean, Jennifer; Banerjee, Prabal; Feuer, Gerold; Izzo, Angelo A

2017-09-01

The humanized mouse model has been developed as a model to identify and characterize human immune responses to human pathogens and has been used to better identify vaccine candidates. In the current studies, the humanized mouse was used to determine the ability of a vaccine to affect the immune response to infection with Mycobacterium tuberculosis. Both human CD4 + and CD8 + T cells responded to infection in humanized mice as a result of infection. In humanized mice vaccinated with either BCG or with CpG-C, a liposome-based formulation containing the M. tuberculosis antigen ESAT-6, both CD4 and CD8 T cells secreted cytokines that are known to be required for induction of protective immunity. In comparison to the C57BL/6 mouse model and Hartley guinea pig model of tuberculosis, data obtained from humanized mice complemented the data observed in the former models and provided further evidence that a vaccine can induce a human T-cell response. Humanized mice provide a crucial pre-clinical platform for evaluating human T-cell immune responses in vaccine development against M. tuberculosis. © 2017 John Wiley & Sons Ltd.

Ex Vivo Expanded Human Regulatory T Cells Delay Islet Allograft Rejection via Inhibiting Islet-Derived Monocyte Chemoattractant Protein-1 Production in CD34+ Stem Cells-Reconstituted NOD-scid IL2rγnull Mice

PubMed Central

Xiao, Fang; Ma, Liang; Zhao, Min; Huang, Guocai; Mirenda, Vincenzo; Dorling, Anthony

2014-01-01

Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by immune-mediated destruction of insulin-secreting β cells of the pancreas. Near complete dependence on exogenous insulin makes T1DM very difficult to control, with the result that patients are exposed to high blood glucose and risk of diabetic complications and/or intermittent low blood glucose that can cause unconsciousness, fits and even death. Allograft transplantation of pancreatic islets restores normoglycemia with a low risk of surgical complications. However, although successful immediately after transplantation, islets are progressively lost, with most of the patients requiring exogenous insulin within 2 years post-transplant. Therefore, there is an urgent requirement for the development of new strategies to prevent islet rejection. In this study, we explored the importance of human regulatory T cells in the control of islets allograft rejection. We developed a pre-clinical model of human islet transplantation by reconstituting NOD-scid IL2rγnull mice with cord blood-derived human CD34+ stem cells and demonstrated that although the engrafted human immune system mediated the rejection of human islets, their survival was significantly prolonged following adoptive transfer of ex vivo expanded human Tregs. Mechanistically, Tregs inhibited the infiltration of innate immune cells and CD4+ T cells into the graft by down-regulating the islet graft-derived monocyte chemoattractant protein-1. Our findings might contribute to the development of clinical strategies for Treg therapy to control human islet rejection. We also show for the first time that CD34+ cells-reconstituted NOD-scid IL2rγnull mouse model could be beneficial for investigating human innate immunity in vivo. PMID:24594640

Mapping of NKp46+ Cells in Healthy Human Lymphoid and Non-Lymphoid Tissues

PubMed Central

Tomasello, Elena; Yessaad, Nadia; Gregoire, Emilie; Hudspeth, Kelly; Luci, Carmelo; Mavilio, Domenico; Hardwigsen, Jean; Vivier, Eric

2012-01-01

Understanding Natural Killer (NK) cell anatomical distribution is key to dissect the role of these unconventional lymphocytes in physiological and disease conditions. In mouse, NK cells have been detected in various lymphoid and non-lymphoid organs, while in humans the current knowledge of NK cell distribution at steady state is mainly restricted to lymphoid tissues. The translation to humans of findings obtained in mice is facilitated by the identification of NK cell markers conserved between these two species. The Natural Cytotoxicity Receptor (NCR) NKp46 is a marker of the NK cell lineage evolutionary conserved in mammals. In mice, NKp46 is also present on rare T cell subsets and on a subset of gut Innate Lymphoid Cells (ILCs) expressing the retinoic acid receptor-related orphan receptor γt (RORγt) transcription factor. Here, we documented the distribution and the phenotype of human NKp46+ cells in lymphoid and non-lymphoid tissues isolated from healthy donors. Human NKp46+ cells were found in splenic red pulp, in lymph nodes, in lungs, and gut lamina propria, thus mirroring mouse NKp46+ cell distribution. We also identified a novel cell subset of CD56dimNKp46low cells that includes RORγt+ ILCs with a lineage−CD94−CD117brightCD127bright phenotype. The use of NKp46 thus contributes to establish the basis for analyzing quantitative and qualitative changes of NK cell and ILC subsets in human diseases. PMID:23181063

Protection by universal influenza vaccine is mediated by memory CD4 T cells.

PubMed

Valkenburg, Sophie A; Li, Olive T W; Li, Athena; Bull, Maireid; Waldmann, Thomas A; Perera, Liyanage P; Peiris, Malik; Poon, Leo L M

2018-07-05

There is a diverse array of influenza viruses which circulate between different species, reassort and drift over time. Current seasonal influenza vaccines are ineffective in controlling these viruses. We have developed a novel universal vaccine which elicits robust T cell responses and protection against diverse influenza viruses in mouse and human models. Vaccine mediated protection was dependent on influenza-specific CD4 + T cells, whereby depletion of CD4 + T cells at either vaccination or challenge time points significantly reduced survival in mice. Vaccine memory CD4 + T cells were needed for early antibody production and CD8 + T cell recall responses. Furthermore, influenza-specific CD4 + T cells from vaccination manifested primarily Tfh and Th1 profiles with anti-viral cytokine production. The vaccine boosted H5-specific T cells from human PBMCs, specifically CD4 + and CD8 + T effector memory type, ensuring the vaccine was truly universal for its future application. These findings have implications for the development and optimization of T cell activating vaccines for universal immunity against influenza. Copyright © 2018 Elsevier Ltd. All rights reserved.

Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition

PubMed Central

Cherkassky, Leonid; Morello, Aurore; Villena-Vargas, Jonathan; Feng, Yang; Dimitrov, Dimiter S.; Jones, David R.; Sadelain, Michel; Adusumilli, Prasad S.

2016-01-01

Following immune attack, solid tumors upregulate coinhibitory ligands that bind to inhibitory receptors on T cells. This adaptive resistance compromises the efficacy of chimeric antigen receptor (CAR) T cell therapies, which redirect T cells to solid tumors. Here, we investigated whether programmed death-1–mediated (PD-1–mediated) T cell exhaustion affects mesothelin-targeted CAR T cells and explored cell-intrinsic strategies to overcome inhibition of CAR T cells. Using an orthotopic mouse model of pleural mesothelioma, we determined that relatively high doses of both CD28- and 4-1BB–based second-generation CAR T cells achieved tumor eradication. CAR-mediated CD28 and 4-1BB costimulation resulted in similar levels of T cell persistence in animals treated with low T cell doses; however, PD-1 upregulation within the tumor microenvironment inhibited T cell function. At lower doses, 4-1BB CAR T cells retained their cytotoxic and cytokine secretion functions longer than CD28 CAR T cells. The prolonged function of 4-1BB CAR T cells correlated with improved survival. PD-1/PD-1 ligand [PD-L1] pathway interference, through PD-1 antibody checkpoint blockade, cell-intrinsic PD-1 shRNA blockade, or a PD-1 dominant negative receptor, restored the effector function of CD28 CAR T cells. These findings provide mechanistic insights into human CAR T cell exhaustion in solid tumors and suggest that PD-1/PD-L1 blockade may be an effective strategy for improving the potency of CAR T cell therapies. PMID:27454297

Age-Dependent Metabolic and Immunosuppressive Effects of Tacrolimus.

PubMed

Krenzien, F; Quante, M; Heinbokel, T; Seyda, M; Minami, K; Uehara, H; Biefer, H R C; Schuitenmaker, J M; Gabardi, S; Splith, K; Schmelzle, M; Petrides, A K; Azuma, H; Pratschke, J; Li, X C; ElKhal, A; Tullius, S G

2017-05-01

Immunosuppression in elderly recipients has been underappreciated in clinical trials. Here, we assessed age-specific effects of the calcineurin inhibitor tacrolimus (TAC) in a murine transplant model and assessed its clinical relevance on human T cells. Old recipient mice exhibited prolonged skin graft survival compared with young animals after TAC administration. More important, half of the TAC dose was sufficient in old mice to achieve comparable systemic trough levels. TAC administration was able to reduce proinflammatory interferon-γ cytokine production and promote interleukin-10 production in old CD4 + T cells. In addition, TAC administration decreased interleukin-2 secretion in old CD4 + T cells more effectively while inhibiting the proliferation of CD4 + T cells in old mice. Both TAC-treated murine and human CD4 + T cells demonstrated an age-specific suppression of intracellular calcineurin levels and Ca 2+ influx, two critical pathways in T cell activation. Of note, depletion of CD8 + T cells did not alter allograft survival outcome in old TAC-treated mice, suggesting that TAC age-specific effects were mainly CD4 + T cell mediated. Collectively, our study demonstrates age-specific immunosuppressive capacities of TAC that are CD4 + T cell mediated. The suppression of calcineurin levels and Ca 2+ influx in both old murine and human T cells emphasizes the clinical relevance of age-specific effects when using TAC. © 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor.

PubMed

Zhao, Yangbing; Moon, Edmund; Carpenito, Carmine; Paulos, Chrystal M; Liu, Xiaojun; Brennan, Andrea L; Chew, Anne; Carroll, Richard G; Scholler, John; Levine, Bruce L; Albelda, Steven M; June, Carl H

2010-11-15

Redirecting T lymphocyte antigen specificity by gene transfer can provide large numbers of tumor-reactive T lymphocytes for adoptive immunotherapy. However, safety concerns associated with viral vector production have limited clinical application of T cells expressing chimeric antigen receptors (CAR). T lymphocytes can be gene modified by RNA electroporation without integration-associated safety concerns. To establish a safe platform for adoptive immunotherapy, we first optimized the vector backbone for RNA in vitro transcription to achieve high-level transgene expression. CAR expression and function of RNA-electroporated T cells could be detected up to a week after electroporation. Multiple injections of RNA CAR-electroporated T cells mediated regression of large vascularized flank mesothelioma tumors in NOD/scid/γc(-/-) mice. Dramatic tumor reduction also occurred when the preexisting intraperitoneal human-derived tumors, which had been growing in vivo for >50 days, were treated by multiple injections of autologous human T cells electroporated with anti-mesothelin CAR mRNA. This is the first report using matched patient tumor and lymphocytes showing that autologous T cells from cancer patients can be engineered to provide an effective therapy for a disseminated tumor in a robust preclinical model. Multiple injections of RNA-engineered T cells are a novel approach for adoptive cell transfer, providing flexible platform for the treatment of cancer that may complement the use of retroviral and lentiviral engineered T cells. This approach may increase the therapeutic index of T cells engineered to express powerful activation domains without the associated safety concerns of integrating viral vectors. Copyright © 2010 AACR.

Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor

PubMed Central

Zhao, Yangbing; Moon, Edmund; Carpenito, Carmine; Paulos, Chrystal M.; Liu, Xiaojun; Brennan, Andrea L; Chew, Anne; Carroll, Richard G.; Scholler, John; Levine, Bruce L.; Albelda, Steven M.; June, Carl H.

2010-01-01

Redirecting T lymphocyte antigen specificity by gene transfer can provide large numbers of tumor reactive T lymphocytes for adoptive immunotherapy. However, safety concerns associated with viral vector production have limited clinical application of T cells expressing chimeric antigen receptors (CARs). T lymphocytes can be gene modified by RNA electroporation without integration-associated safety concerns. To establish a safe platform for adoptive immunotherapy, we first optimized the vector backbone for RNA in vitro transcription to achieve high level transgene expression. CAR expression and function of RNA-electroporated T cells could be detected up to a week post electroporation. Multiple injections of RNA CAR electroporated T cells mediated regression of large vascularized flank mesothelioma tumors in NOD/scid/γc(−/−) mice. Dramatic tumor reduction also occurred when the pre-existing intraperitoneal human-derived tumors, that had been growing in vivo for over 50 days, were treated by multiple injections of autologous human T cells electroporated with anti-mesothelin CAR mRNA. This is the first report using matched patient tumor and lymphocytes demonstrating that autologous T cells from cancer patients can be engineered to provide an effective therapy for a disseminated tumor in a robust preclinical model. Multiple injections of RNA engineered T cells are a novel approach for adoptive cell transfer, providing flexible platform for the treatment of cancer that may complement the use of retroviral and lentiviral engineered T cells. This approach may increase the therapeutic index of T cells engineered to express powerful activation domains without the associated safety concerns of integrating viral vectors. PMID:20926399

Glycolysis controls the induction of human regulatory T cells by modulating the expression of FOXP3 exon 2 splicing variants

PubMed Central

De Rosa, Veronica; Galgani, Mario; Porcellini, Antonio; Colamatteo, Alessandra; Santopaolo, Marianna; Zuchegna, Candida; Romano, Antonella; De Simone, Salvatore; Procaccini, Claudio; La Rocca, Claudia; Carrieri, Pietro Biagio; Maniscalco, Giorgia Teresa; Salvetti, Marco; Buscarinu, Maria Chiara; Franzese, Adriana; Mozzillo, Enza; La Cava, Antonio; Matarese, Giuseppe

2016-01-01

Human regulatory T cells (Treg cells) that develop from conventional T cells (Tconv cells) following suboptimal stimulation via the T cell antigen receptor (TCR) (induced Treg cells (iTreg cells)) express the transcription factor Foxp3, are suppressive, and display an active proliferative and metabolic state. Here we found that the induction and suppressive function of iTreg cells tightly depended on glycolysis, which controlled Foxp3 splicing variants containing exon 2 (Foxp3-E2) through the glycolytic enzyme enolase-1. The Foxp3-E2–related suppressive activity of iTreg cells was altered in human autoimmune diseases, including multiple sclerosis and type 1 diabetes, and was associated with impaired glycolysis and signaling via interleukin 2. This link between glycolysis and Foxp3-E2 variants via enolase-1 shows a previously unknown mechanism for controlling the induction and function of Treg cells in health and in autoimmunity. PMID:26414764

Single-Cell RNA Sequencing of Human T Cells.

PubMed

Villani, Alexandra-Chloé; Shekhar, Karthik

2017-01-01

Understanding how populations of human T cells leverage cellular heterogeneity, plasticity, and diversity to achieve a wide range of functional flexibility, particularly during dynamic processes such as development, differentiation, and antigenic response, is a core challenge that is well suited for single-cell analysis. Hypothesis-free evaluation of cellular states and subpopulations by transcriptional profiling of single T cells can identify relationships that may be obscured by targeted approaches such as FACS sorting on cell-surface antigens, or bulk expression analysis. While this approach is relevant to all cell types, it is of particular interest in the study of T cells for which classical phenotypic criteria are now viewed as insufficient for distinguishing different T cell subtypes and transitional states, and defining the changes associated with dysfunctional T cell states in autoimmunity and tumor-related exhaustion. This unit describes a protocol to generate single-cell transcriptomic libraries of human blood CD4 + and CD8 + T cells, and also introduces the basic bioinformatic steps to process the resulting sequence data for further computational analysis. We show how cellular subpopulations can be identified from transcriptional data, and derive characteristic gene expression signatures that distinguish these states. We believe single-cell RNA-seq is a powerful technique to study the cellular heterogeneity in complex tissues, a paradigm that will be of great value for the immune system.

The viral transcription group determines the HLA class I cellular immune response against human respiratory syncytial virus.

PubMed

Johnstone, Carolina; Lorente, Elena; Barriga, Alejandro; Barnea, Eilon; Infantes, Susana; Lemonnier, François A; David, Chella S; Admon, Arie; López, Daniel

2015-04-01

The cytotoxic T-lymphocyte-mediated killing of virus-infected cells requires previous recognition of short viral antigenic peptides bound to human leukocyte antigen class I molecules that are exposed on the surface of infected cells. The cytotoxic T-lymphocyte response is critical for the clearance of human respiratory syncytial virus infection. In this study, naturally processed viral human leukocyte antigen class I ligands were identified with mass spectrometry analysis of complex human leukocyte antigen-bound peptide pools isolated from large amounts of human respiratory syncytial virus-infected cells. Acute antiviral T-cell response characterization showed that viral transcription determines both the immunoprevalence and immunodominance of the human leukocyte antigen class I response to human respiratory syncytial virus. These findings have clear implications for antiviral vaccine design. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Tim-3-expressing macrophages are functionally suppressed and expanded in oral squamous cell carcinoma due to virus-induced Gal-9 expression.

PubMed

Dong, Jianfeng; Cheng, Lijun; Zhao, Minchao; Pan, Xiangfeng; Feng, Zhiqiang; Wang, Dawei

2017-05-01

Oropharyngeal head and neck squamous cell carcinoma is a common malignant tumor in the oral cavity. High-risk human papillomavirus 16 infection is a major cause of oropharyngeal head and neck squamous cell carcinoma development. Strong antitumor immune responses, especially CD8 + T cell responses, are thought to be essential to effective cancer treatment and are associated with better prognosis in oropharyngeal head and neck squamous cell carcinoma. In this study, we examined the role of the Tim-3/Gal-9 pathway in oropharyngeal head and neck squamous cell carcinoma patients. We found that Gal-9 expression by CD4 + T cells was increased in human papillomavirus-positive oropharyngeal head and neck squamous cell carcinoma patients, but not in human papillomavirus-negative oropharyngeal head and neck squamous cell carcinoma patients. Increased Gal-9 secretion by CD4 + T cells presented multiple immunosuppressive effects. Coculturing monocytes with high Gal-9-expressing CD4 + T cells resulted in the expansion of Tim-3 + monocytes, which suppressed interferon gamma production by activated CD8 + T cells. Subsequently, total monocytes incubated with exogenous Gal-9, or high Gal-9-expressing CD4 + T cells, suppressed the expression of interferon gamma by CD8 + T cells. Exogenous Gal-9 and high Gal-9-expressing CD4 + T cells also suppressed the secretion of both interleukin 10 and interleukin 12 by monocytes. These effects are Tim-3/Gal-9-dependent because blocking Tim-3 and/or Gal-9 could enhance the support of CD8 + T cell interferon gamma production and the interleukin 10 and interleukin 12 secretion by monocytes. Together, these data suggest that the high Tim-3 expression in monocytes could be utilized by tumor-promoting Gal-9 expression on CD4 + T cells. Immunotherapy in human papillomavirus-positive oropharyngeal head and neck squamous cell carcinoma patients therefore faces an additional challenge posed by Tim-3 and Gal-9 and likely requires the blockade of these molecules.

Downregulation of proapoptotic Bim augments IL-2-independent T-cell transformation by human T-cell leukemia virus type-1 Tax.

PubMed

Higuchi, Masaya; Takahashi, Masahiko; Tanaka, Yuetsu; Fujii, Masahiro

2014-12-01

Human T-cell leukemia virus type 1 (HTLV-1), an etiological agent of adult T-cell leukemia, immortalizes and transforms primary human T cells in vitro in both an interleukin (IL)-2-dependent and IL-2-independent manner. Expression of the HTLV-1 oncoprotein Tax transforms the growth of the mouse T-cell line CTLL-2 from being IL-2-dependent to IL-2-independent. Withdrawal of IL-2 from normal activated T cells induces apoptosis, which is mediated through the inducible expression of several proapoptotic proteins, including Bim. In this study, we found that Tax protects IL-2-depleted T cells against Bim-induced apoptosis. Withdrawal of IL-2 from CTLL-2 cells induced a prominent increase in the level of Bim protein in CTLL-2 cells, but not in Tax-transformed CTLL-2 cells. This inhibition of Bim in Tax-transformed CTLL-2 cells was mediated by two mechanisms: downregulation of Bim mRNA and posttranscriptional reduction of Bim protein. Transient expression of Tax in CTLL-2 cells also inhibited IL-2 depletion-induced expression of Bim, however, this decrease in Bim protein expression was not due to downregulation of Bim mRNA, thus indicating that Bim mRNA downregulation in Tax-transformed CTLL-2 occurs only after long-term expression of Tax. Transient expression of Tax in CTLL-2 cells also induced Erk activation, however, this was not involved in the reduction of Bim protein. Knockdown of Bim expression in CTLL-2 cells augmented Tax-induced IL-2-independent transformation. HTLV-1 infection of human T cells also reduced their levels of Bim protein, and restoring Bim expression in HTLV-1-infected cells reduced their proliferation by inducing apoptosis. Taken together, these results indicate that Tax-induced downregulation of Bim in HTLV-1-infected T cells promotes their IL-2-independent growth, thereby supporting the persistence of HTLV-1 infection in vivo. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

ICAM-3 influences human immunodeficiency virus type 1 replication in CD4+ T-cells independent of DC-SIGN-mediated transmission

PubMed Central

Biggins, Julia E.; Biesinger, Tasha; Yu Kimata, Monica T.; Arora, Reetakshi; Kimata, Jason T.

2007-01-01

We investigated the role of ICAM-3 in DC-SIGN-mediated human immunodeficiency virus (HIV) infection of CD4+ T cells. Our results demonstrate that ICAM-3 does not appear to play a role in DC-SIGN-mediated infection of CD4+ T cells as virus is transmitted equally to ICAM-3+ or ICAM-3− Jurkat T cells. However, HIV-1 replication is enhanced in ICAM-3− cells, suggesting that ICAM-3 may limit HIV-1 replication. Similar results were obtained when SIV replication was examined in ICAM-3+ and ICAM-3− CEMx174 cells. Furthermore, while ICAM-3 has been proposed to play a co-stimulatory role in T cell activation, DC-SIGN expression on antigen presenting cells did not enhance antigen-dependent activation of T cells. Together, these data indicate that while ICAM-3 may influence HIV-1 replication, it does so independent of DC-SIGN mediated virus transmission or activation of CD4+ T cells. PMID:17434553

Xenogeneic graft-versus-host-disease in NOD-scid IL-2Rγnull mice display a T-effector memory phenotype.

PubMed

Ali, Niwa; Flutter, Barry; Sanchez Rodriguez, Robert; Sharif-Paghaleh, Ehsan; Barber, Linda D; Lombardi, Giovanna; Nestle, Frank O

2012-01-01

The occurrence of Graft-versus-Host Disease (GvHD) is a prevalent and potentially lethal complication that develops following hematopoietic stem cell transplantation. Humanized mouse models of xenogeneic-GvHD based upon immunodeficient strains injected with human peripheral blood mononuclear cells (PBMC; "Hu-PBMC mice") are important tools to study human immune function in vivo. The recent introduction of targeted deletions at the interleukin-2 common gamma chain (IL-2Rγ(null)), notably the NOD-scid IL-2Rγ(null) (NSG) and BALB/c-Rag2(null) IL-2Rγ(null) (BRG) mice, has led to improved human cell engraftment. Despite their widespread use, a comprehensive characterisation of engraftment and GvHD development in the Hu-PBMC NSG and BRG models has never been performed in parallel. We compared engrafted human lymphocyte populations in the peripheral blood, spleens, lymph nodes and bone marrow of these mice. Kinetics of engraftment differed between the two strains, in particular a significantly faster expansion of the human CD45(+) compartment and higher engraftment levels of CD3(+) T-cells were observed in NSG mice, which may explain the faster rate of GvHD development in this model. The pathogenesis of human GvHD involves anti-host effector cell reactivity and cutaneous tissue infiltration. Despite this, the presence of T-cell subsets and tissue homing markers has only recently been characterised in the peripheral blood of patients and has never been properly defined in Hu-PBMC models of GvHD. Engrafted human cells in NSG mice shows a prevalence of tissue homing cells with a T-effector memory (T(EM)) phenotype and high levels of cutaneous lymphocyte antigen (CLA) expression. Characterization of Hu-PBMC mice provides a strong preclinical platform for the application of novel immunotherapies targeting T(EM)-cell driven GvHD.

Exposure of Human CD8+ T Cells to Type-2 Cytokines Impairs Division and Differentiation and Induces Limited Polarization.

PubMed

Fox, Annette; Harland, Kim L; Kedzierska, Katherine; Kelso, Anne

2018-01-01

Effector CD8 + T cells generally produce type-1 cytokines and mediators of the perforin/granzyme cytolytic pathway, yet type-2-polarized CD8 + cells (Tc2) are detected in type-2 (T2) cytokine-driven diseases such as asthma. It is unclear whether T2 cytokine exposure during activation is sufficient to polarize human CD8 + T cells. To address this question, a protocol was developed for high-efficiency activation of human CD8 + T cells in which purified single cells or populations were stimulated with plate-bound anti-CD3 and anti-CD11a mAb for up to 8 days in T2 polarizing or neutral conditions, before functional analysis. Activation of CD8 + naïve T cells (T N ) in T2 compared with neutral conditions decreased the size of single-cell clones, although early division kinetics were equivalent, indicating an effect on overall division number. Activation of T N in T2 conditions followed by brief anti-CD3 mAb restimulation favored expression of T2 cytokines, GATA3 and Eomes , and lowered expression of type-1 cytokines, Prf1 , Gzmb, T-BET, and Prdm1 . However, IL-4 was only weakly expressed, and PMA and ionomycin restimulation favored IFN-γ over IL-4 expression. Activation of T N in T2 compared with neutral conditions prevented downregulation of costimulatory (CD27, CD28) and lymph-node homing receptors (CCR7) and CD95 acquisition, which typically occur during differentiation into effector phenotypes. CD3 was rapidly and substantially induced after activation in neutral, but not T2 conditions, potentially contributing to greater division and differentiation in neutral conditions. CD8 + central memory T cells (T CM ) were less able to enter division upon reactivation in T2 compared with neutral conditions, and were more refractory to modulating IFN-γ and IL-4 production than CD8 + T N. In summary, while activation of T N in T2 conditions can generate T2 cytokine-biased cells, IL-4 expression is weak, T2 bias is lost upon strong restimulation, differentiation, and division are arrested, and reactivation of T CM is reduced in T2 conditions. Taken together, this suggests that exposure to T2 cytokines during activation may not be sufficient to generate and retain human Tc2 cells.

Regulation of IFN regulatory factor 4 expression in human T cell leukemia virus-I-transformed T cells.

PubMed

Sharma, Sonia; Grandvaux, Nathalie; Mamane, Yael; Genin, Pierre; Azimi, Nazli; Waldmann, Thomas; Hiscott, John

2002-09-15

IFN regulatory factor (IRF)-4 is a lymphoid/myeloid-restricted member of the IRF transcription factor family that plays an essential role in the homeostasis and function of mature lymphocytes. IRF-4 expression is tightly regulated in resting primary T cells and is transiently induced at the mRNA and protein levels after activation by Ag-mimetic stimuli such as TCR cross-linking or treatment with phorbol ester and calcium ionophore (PMA/ionomycin). However, IRF-4 is constitutively upregulated in human T cell leukemia virus type I (HTLV-I) infected T cells as a direct gene target for the HTLV-I Tax oncoprotein. In this study we demonstrate that chronic IRF-4 expression in HTLV-I-infected T lymphocytes is associated with a leukemic phenotype, and we examine the mechanisms by which continuous production of IRF-4 is achieved in HTLV-I-transformed T cells. IRF-4 expression in HTLV-1-infected cells is driven through activation of the NF-kappaB and NF-AT pathways, resulting in the binding of p50, p65, and c-Rel to the kappaB1 element and p50, c-Rel, and NF-ATp to the CD28RE element within the -617 to -209 region of the IRF-4 promoter. Furthermore, mutation of either the kappaB1 or CD28RE sites blocks Tax-mediated transactivation of the human IRF-4 promoter in T cells. These experiments constitute the first detailed analysis of human IRF-4 transcriptional regulation within the context of HTLV-I infection and transformation of CD4(+) T lymphocytes.

Impact of alemtuzumab treatment on the survival and function of human regulatory T cells in vitro

PubMed Central

Havari, Evis; Turner, Michael J; Campos-Rivera, Juanita; Shankara, Srinivas; Nguyen, Tri-Hung; Roberts, Bruce; Siders, William; Kaplan, Johanne M

2014-01-01

Alemtuzumab is a humanized monoclonal antibody specific for the CD52 protein present at high levels on the surface of B and T lymphocytes. In clinical trials, alemtuzumab has shown a clinical benefit superior to that of interferon-β in relapsing–remitting multiple sclerosis patients. Treatment with alemtuzumab leads to the depletion of circulating lymphocytes followed by a repopulation process characterized by alterations in the number, proportions and properties of lymphocyte subsets. Of particular interest, an increase in the percentage of T cells with a regulatory phenotype (Treg cells) has been observed in multiple sclerosis patients after alemtuzumab. Since Treg cells play an important role in the control of autoimmune responses, the effect of alemtuzumab on Treg cells was further studied in vitro. Alemtuzumab effectively mediated complement-dependent cytolysis of human T lymphocytes and the remaining population was enriched in T cells with a regulatory phenotype. The alemtuzumab-exposed T cells displayed functional regulatory characteristics including anergy to stimulation with allogeneic dendritic cells and ability to suppress the allogeneic response of autologous T cells. Consistent with the observed increase in Treg cell frequency, the CD25hi T-cell population was necessary for the suppressive activity of alemtuzumab-exposed T cells. The mechanism of this suppression was found to be dependent on both cell–cell contact and interleukin-2 consumption. These findings suggest that an alemtuzumab-mediated increase in the proportion of Treg cells may play a role in promoting the long-term efficacy of alemtuzumab in patients with multiple sclerosis. PMID:24116901

Effect of irradiation on human T-cell proliferation: low dose irradiation stimulates mitogen-induced proliferation and function of the suppressor/cytotoxic T-cell subset.

PubMed

Gualde, N; Goodwin, J S

1984-04-01

Unfractionated human T cells exposed to 10-50 rad of X irradiation incorporated less [3H]thymidine than nonirradiated T cells when subsequently cultured with PHA or Con A. The cytotoxic/suppressor T-cell subset, isolated as either OKT8(+) or OKT4(-) cells, demonstrated significantly enhanced [3H]thymidine incorporation in PHA- or Con A-stimulated cultures after exposure to 10-50 rad, compared to unirradiated cells, while the proliferation of the OKT4(+) helper/inducer subset was inhibited by low dose irradiation. It has been previously reported that approximately 30% of the cytotoxic/suppressor subset also stains with OKM1. When the cytotoxic/suppressor subset was further subdivided into OKT4(-), OKM1(+), and OKT4(-), OKM1(-) cells, proliferation of the OKT4(-), OKM1(+) population was inhibited by exposure to 25 rad while proliferation of the OKT4(-), OKM1(-) population was stimulated. The increase in proliferation of the cytotoxic/suppressor T-cell subset after low dose irradiation is paralleled by an increase in suppressor activity of these cells. T cells exposed to 25 rad and then cultured with Con A for 48 hr caused greater inhibition of IgG production when added to fresh autologous lymphocytes stimulated by pokeweed mitogen than did unirradiated cells. Thus, low dose irradiation enhances both the proliferation and function of the human suppressor T-cell subset.

Effect of irradiation on human T-cell proliferation: low dose irradiation stimulates mitogen-induced proliferation and function of the suppressor/cytotoxic T-cell subset

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

Gualde, N.; Goodwin, J.S.

1984-04-01

Unfractionated human T cells exposed to 10-50 rad of X irradiation incorporated less (/sup 3/H)thymidine than nonirradiated T cells when subsequently cultured with PHA or Con A. The cytotoxic/suppressor T-cell subset, isolated as either OKT8(+) or OKT4(-) cells, demonstrated significantly enhanced (/sup 3/H)thymidine incorporation in PHA- or Con A-stimulated cultures after exposure to 10-50 rad, compared to unirradiated cells, while the proliferation of the OKT4(+) helper/inducer subset was inhibited by low dose irradiation. It has been previously reported that approximately 30% of the cytotoxic/suppressor subset also stains with OKM1. When the cytotoxic/suppressor subset was further subdivided into OKT4(-), OKM1(+), andmore » OKT4(-), OKM1(-) cells, proliferation of the OKT4(-), OKM1(+) population was inhibited by exposure to 25 rad while proliferation of the OKT4(-), OKM1(-) population was stimulated. The increase in proliferation of the cytotoxic/suppressor T-cell subset after low dose irradiation is paralleled by an increase in suppressor activity of these cells. T cells exposed to 25 rad and then cultured with Con A for 48 hr caused greater inhibition of IgG production when added to fresh autologous lymphocytes stimulated by pokeweed mitogen than did unirradiated cells. Thus, low dose irradiation enhances both the proliferation and function of the human suppressor T-cell subset.« less

Asymptomatic memory CD8+ T cells

PubMed Central

Khan, Arif Azam; Srivastava, Ruchi; Lopes, Patricia Prado; Wang, Christine; Pham, Thanh T; Cochrane, Justin; Thai, Nhi Thi Uyen; Gutierrez, Lucas; BenMohamed, Lbachir

2014-01-01

Generation and maintenance of high quantity and quality memory CD8+ T cells determine the level of protection from viral, bacterial, and parasitic re-infections, and hence constitutes a primary goal for T cell epitope-based human vaccines and immunotherapeutics. Phenotypically and functionally characterizing memory CD8+ T cells that provide protection against herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) infections, which cause blinding ocular herpes, genital herpes, and oro-facial herpes, is critical for better vaccine design. We have recently categorized 2 new major sub-populations of memory symptomatic and asymptomatic CD8+ T cells based on their phenotype, protective vs. pathogenic function, and anatomical locations. In this report we are discussing a new direction in developing T cell-based human herpes vaccines and immunotherapeutics based on the emerging new concept of “symptomatic and asymptomatic memory CD8+ T cells.” PMID:24499824

The metabolic perturbators metformin, phenformin and AICAR interfere with the growth and survival of murine PTEN-deficient T cell lymphomas and human T-ALL/T-LL cancer cells.

PubMed

Rosilio, Célia; Lounnas, Nadia; Nebout, Marielle; Imbert, Véronique; Hagenbeek, Thijs; Spits, Hergen; Asnafi, Vahid; Pontier-Bres, Rodolphe; Reverso, Julie; Michiels, Jean-François; Sahra, Issam Ben; Bost, Fréderic; Peyron, Jean-François

2013-08-09

We show here that the antidiabetic agents metformin and phenformin and the AMPK activator AICAR exert strong anti-tumoural effects on tPTEN-/- lymphoma cells and on human T-ALL cell lines and primary samples. The compounds act by inhibiting tumour metabolism and proliferation and by inducing apoptosis in parallel with an activation of AMPK and an inhibition of constitutive mTOR. In tPTEN-/- cells, the drugs potentiated the anti-leukaemic effects of dexamethasone, and metformin and phenformin synergised with 2-deoxyglucose (2DG) to impair tumour cell survival. In vivo, metformin and AICAR strongly decreased the growth of luciferase-expressing tPTEN-/- cells xenografted in Nude mice, demonstrating that metabolism targeting could be a potent adjuvant strategy for lymphoma/leukaemia treatment. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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

PubMed Central

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

2016-01-01

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

Curcumin induces apoptotic cell death of activated human CD4+ T cells via increasing endoplasmic reticulum stress and mitochondrial dysfunction.

PubMed

Zheng, Min; Zhang, Qinggao; Joe, Yeonsoo; Lee, Bong Hee; Ryu, Do Gon; Kwon, Kang Beom; Ryter, Stefan W; Chung, Hun Taeg

2013-03-01

Curcumin, a natural polyphenolic antioxidant compound, exerts well-known anti-inflammatory and immunomodulatory effects, the latter which can influence the activation of immune cells including T cells. Furthermore, curcumin can inhibit the expression of pro-inflammatory cytokines and chemokines, through suppression of the NF-κB signaling pathway. The beneficial effects of curcumin in diseases such as arthritis, allergy, asthma, atherosclerosis, diabetes and cancer may be due to its immunomodulatory properties. We studied the potential of curcumin to modulate CD4+ T cells-mediated autoimmune disease, by examining the effects of this compound on human CD4+ lymphocyte activation. Stimulation of human T cells with PHA or CD3/CD28 induced IL-2 mRNA expression and activated the endoplasmic reticulum (ER) stress response. The treatment of T cells with curcumin induced the unfolded protein response (UPR) signaling pathway, initiated by the phosphorylation of PERK and IRE1. Furthermore, curcumin increased the expression of the ER stress associated transcriptional factors XBP-1, cleaved p50ATF6α and C/EBP homologous protein (CHOP) in human CD4+ and Jurkat T cells. In PHA-activated T cells, curcumin further enhanced PHA-induced CHOP expression and reduced the expression of the anti-apoptotic protein Bcl-2. Finally, curcumin treatment induced apoptotic cell death in activated T cells via eliciting an excessive ER stress response, which was reversed by the ER-stress inhibitor 4-phenylbutyric acid or transfection with CHOP-specific siRNA. These results suggest that curcumin can impact both ER stress and mitochondria functional pathways, and thereby could be used as a promising therapy in the context of Th1-mediated autoimmune diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

A hybrid of B and T lymphoblastic cell line could potentially substitute dendritic cells to efficiently expand out Her-2/neu-specific cytotoxic T lymphocytes from advanced breast cancer patients in vitro.

PubMed

Chen, Sheng; Gu, Feifei; Li, Kang; Zhang, Kai; Liu, Yangyang; Liang, Jinyan; Gao, Wei; Wu, Gang; Liu, Li

2017-02-28

Adoptive transfer of cytotoxic T lymphocytes (CTLs) holds promises to cure cancer. However, this treatment is hindered by lacking a robust way to specifically expand out CTLs. Here, we developed a hybrid of B lymphoblastic cell line and T lymphoblastic cell line (T2 cells) as a substitute of dendritic cells, together with irradiated autologous peripheral blood mononuclear cell (PBMC) as feeder cells and rhIL-2, to activate and expand Her-2/neu-specific CD8 + T cells from human epidermal growth factor receptor 2 (Her-2/neu) and human leukocyte antigen (HLA)-A2 double positive advanced breast cancer patients in vitro. These Her-2/neu-loaded T2 cells reproducibly activated and expanded out Her-2/neu-specific CD8 + T cells to 10 7 in 8 weeks. Furthermore, these Her-2/neu-specific CD8 + T cells had good sensitivity of recognition and killing Her-2/neu-overexpressed breast cancer cell line SK.BR.3. This technique gives us another insight on how to rapidly obtain sufficient CTLs for adoptive cancer immunotherapy.

CHARACTERIZATION OF NORMAL HUMAN LUNG LYMPHOCYTES AND INTERLEUKIN-2-INDUCED LUNG T CELL LINES

EPA Science Inventory

Lymphocytes from the lower respiratory tract were obtained by bronchoalveolar lavage of healthy, non-smoking individuals. arious monoclonal antibodies characterizing activated T cells, helper-inducer and suppressor-inducer T cell subsets, and naive versus memory cells were used t...

Induction of suppressor cells from peripheral blood T cells by 15-hydroperoxyeicosatetraenoic acid (15-HPETE).

PubMed

Gualde, N; Rigaud, M; Goodwin, J S

1985-11-01

15-hydroperoxyeicosetetraenoic acid (15-HPETE), a lipoxygenase metabolite of arachidonic acid, inhibited polyclonal IgG and IgM production in pokeweed mitogen (PWM)-stimulated cultures of human peripheral blood mononuclear cells, whereas 15-hydroxyeicosetetraenoic acid (15-HETE) had little effect in this system. T cells preincubated for 18 hr with 15-HPETE caused substantial inhibition of IgG and IgM production of fresh, autologous B and T cells stimulated by PWM. The suppressive effect of the 15-HPETE-treated cells was lost if the cells were irradiated before the PWM culture, but not by treatment with mitomycin C. The suppressive effect was also lost if OKT8+ T cells were removed after, but not before, preincubation of the T cells with 15-HPETE. OKT8- T cells incubated with 15-HPETE for 18 hr showed a large increase in the percentage of cells staining with directly fluoresceinated Leu-2, another marker for suppressor cells. Thus, 15-HPETE induces functional and phenotypic suppressor cells from resting human peripheral blood T cells.

Vaccination for Disease

NASA Astrophysics Data System (ADS)

Oehen, Stephan; Hengartner, Hans; Zinkernagel, Rolf M.

1991-01-01

Recombinant virus vaccines that express a limited number of epitopes are currently being developed to prevent disease by changing the relative balance between viral spread and the immune response. Some circumstances, however, were found in infections with a noncytopathic virus in which vaccination caused disease; sensitive parameters included the genetic background of the host, the time or dose of infection, and the constituents of the vaccine. Thus, immunopathologic damage by T cells may be an unwanted consequence of vaccination with the new types of peptide or recombinant vaccines that are being investigated for the human immunodeficiency viruses and other pathogens.

Human Stem Cell-like Memory T Cells Are Maintained in a State of Dynamic Flux.

PubMed

Ahmed, Raya; Roger, Laureline; Costa Del Amo, Pedro; Miners, Kelly L; Jones, Rhiannon E; Boelen, Lies; Fali, Tinhinane; Elemans, Marjet; Zhang, Yan; Appay, Victor; Baird, Duncan M; Asquith, Becca; Price, David A; Macallan, Derek C; Ladell, Kristin

2016-12-13

Adaptive immunity requires the generation of memory T cells from naive precursors selected in the thymus. The key intermediaries in this process are stem cell-like memory T (T SCM ) cells, multipotent progenitors that can both self-renew and replenish more differentiated subsets of memory T cells. In theory, antigen specificity within the T SCM pool may be imprinted statically as a function of largely dormant cells and/or retained dynamically by more transitory subpopulations. To explore the origins of immunological memory, we measured the turnover of T SCM cells in vivo using stable isotope labeling with heavy water. The data indicate that T SCM cells in both young and elderly subjects are maintained by ongoing proliferation. In line with this finding, T SCM cells displayed limited telomere length erosion coupled with high expression levels of active telomerase and Ki67. Collectively, these observations show that T SCM cells exist in a state of perpetual flux throughout the human lifespan. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Therapeutic PD-L1 and LAG-3 blockade rapidly clears established blood-stage Plasmodium infection

PubMed Central

Butler, Noah S.; Moebius, Jacqueline; Pewe, Lecia L.; Traore, Boubacar; Doumbo, Ogobara K.; Tygrett, Lorraine T.; Waldschmidt, Thomas J.; Crompton, Peter D.; Harty, John T.

2011-01-01

Plasmodium infection of erythrocytes induces clinical malaria. Parasite-specific CD4+ T cells correlate with reduced parasite burdens and severity of human malaria, and are required to control blood-stage infection in mice. However, the characteristics of CD4+ T cells that determine protection or parasite persistence remain unknown. Here we show that P. falciparum infection of humans increased expression of an inhibitory receptor (PD-1) associated with T cell dysfunction. In vivo blockade of PD-L1 and LAG-3 restored CD4+ T cell function, amplified T follicular helper cell and germinal center B cell and plasmablast numbers, enhanced protective antibodies and rapidly cleared blood-stage malaria in mice. Thus, chronic malaria drives specific T cell dysfunction, which can be rescued to enhance parasite control using inhibitory therapies. PMID:22157630

Human MAIT-cell responses to Escherichia coli: activation, cytokine production, proliferation, and cytotoxicity.

PubMed

Dias, Joana; Sobkowiak, Michał J; Sandberg, Johan K; Leeansyah, Edwin

2016-07-01

Mucosa-associated invariant T cells are a large and relatively recently described innate-like antimicrobial T-cell subset in humans. These cells recognize riboflavin metabolites from a range of microbes presented by evolutionarily conserved major histocompatibility complex, class I-related molecules. Given the innate-like characteristics of mucosa-associated invariant T cells and the novel type of antigens they recognize, new methodology must be developed and existing methods refined to allow comprehensive studies of their role in human immune defense against microbial infection. In this study, we established protocols to examine a range of mucosa-associated invariant T-cell functions as they respond to antigen produced by Escherichia coli These improved and dose- and time-optimized experimental protocols allow detailed studies of MR1-dependent mucosa-associated invariant T-cell responses to Escherichia coli pulsed antigen-presenting cells, as assessed by expression of activation markers and cytokines, by proliferation, and by induction of apoptosis and death in major histocompatibility complex, class I-related-expressing target cells. The novel and optimized protocols establish a framework of methods and open new possibilities to study mucosa-associated invariant T-cell immunobiology, using Escherichia coli as a model antigen. Furthermore, we propose that these robust experimental systems can also be adapted to study mucosa-associated invariant T-cell responses to other microbes and types of antigen-presenting cells. © The Author(s).

Cellular immune responses to HIV

NASA Astrophysics Data System (ADS)

McMichael, Andrew J.; Rowland-Jones, Sarah L.

2001-04-01

The cellular immune response to the human immunodeficiency virus, mediated by T lymphocytes, seems strong but fails to control the infection completely. In most virus infections, T cells either eliminate the virus or suppress it indefinitely as a harmless, persisting infection. But the human immunodeficiency virus undermines this control by infecting key immune cells, thereby impairing the response of both the infected CD4+ T cells and the uninfected CD8+ T cells. The failure of the latter to function efficiently facilitates the escape of virus from immune control and the collapse of the whole immune system.

Two alternate strategies for innate immunity to Epstein-Barr virus: One using NK cells and the other NK cells and γδ T cells

PubMed Central

Horowitz, Amir; Nemat-Gorgani, Neda; Münz, Christian

2017-01-01

Most humans become infected with Epstein–Barr virus (EBV), which then persists for life. Infrequently, EBV infection causes infectious mononucleosis (IM) or Burkitt lymphoma (BL). Type I EBV infection, particularly type I BL, stimulates strong responses of innate immune cells. Humans respond to EBV in two alternative ways. Of 24 individuals studied, 13 made strong NK and γδ T cell responses, whereas 11 made feeble γδ T cell responses but stronger NK cell responses. The difference does not correlate with sex, HLA type, or previous exposure to EBV or cytomegalovirus. Cohorts of EBV+ children and pediatric IM patients include both group 1 individuals, with high numbers of γδ T cells, and group 2 individuals, with low numbers. The even balance of groups 1 and 2 in the human population points to both forms of innate immune response to EBV having benefit for human survival. Correlating these distinctive responses with the progress of EBV infection might facilitate the management of EBV-mediated disease. PMID:28468758

Bradykinin regulates human colonic ion transport in vitro

PubMed Central

Baird, A W; Skelly, M M; O'Donoghue, D P; Barrett, K E; Keely, S J

2008-01-01

Background and purpose: Kinins are acknowledged as important regulators of intestinal function during inflammation; however, their effects on human intestinal ion transport have not been reported. Here, we used muscle-stripped human colonic tissue and cultured T84-cell monolayers to study bradykinin (BK) actions on human intestinal ion transport. Experimental approach: Ion transport was measured as changes in short-circuit current (Isc) across colonic epithelia mounted in Ussing chambers. Key results: In intact tissue, there was a distinct polarity to BK-elicited Isc responses. Whereas basolateral BK stimulated sustained responses (EC50=0.5±0.1 μM), those to apical BK were more rapid and transient (EC50=4.1±1.2 nM). In T84 cells, responses to both apical and basolateral BK were similar to those seen upon apical addition to intact tissues. Cross-desensitization between apical and basolateral domains was not observed. BK-induced responses were largely due to Cl− secretion as shown by their sensitivity to bumetanide and removal of Cl− from the bathing solution. Studies using selective agonists and antagonists indicate responses to BK are mediated by B2 receptors. Finally, responses to basolateral BK in intact tissues were inhibited by tetrodotoxin (1 μM), atropine (1 μM), capsaicin (100 μM) and piroxicam (10 μM). BK-stimulated prostaglandin (PG)E2 release from colonic tissue. Conclusions: BK stimulates human colonic Cl− secretion by activation of apical and basolateral B2 receptors. Responses to apical BK reflect a direct action on epithelial cells, whereas those to basolateral BK are amplified by stimulation of enteric nerves and PG synthesis. PMID:18604228

p53 functional impairment and high p21waf1/cip1 expression in human T-cell lymphotropic/leukemia virus type I-transformed T cells.

PubMed

Cereseto, A; Diella, F; Mulloy, J C; Cara, A; Michieli, P; Grassmann, R; Franchini, G; Klotman, M E

1996-09-01

Human T-cell lymphotropic/leukemia virus type I (HTLV-I) is associated with T-cell transformation both in vivo and in vitro. Although some of the mechanisms responsible for transformation remain unknown, increasing evidence supports a direct role of viral as well as dysregulated cellular proteins in transformation. We investigated the potential role of the tumor suppressor gene p53 and of the p53-regulated gene, p21waf1/cip1 (wild-type p53 activated fragment 1/cycling dependent kinases [cdks] interacting protein 1), in HTLV-I-infected T cells. We have found that the majority of HTLV-I-infected T cells have the wild-type p53 gene. However, its function in HTLV-I-transformed cells appears to be impaired, as shown by the lack of appropriate p53-mediated responses to ionizing radiation (IR). Interestingly, the expression of the p53 inducible gene, p21waf1/cip1, is elevated at the messenger ribonucleic acid and protein levels in all HTLV-I-infected T-cell lines examined as well as in Taxl-1, a human T-cell line stably expressing Tax. Additionally, Tax induces upregulation of a p21waf1/cip1 promoter-driven luciferase gene in p53 null cells, and increases p21waf1/cip1 expression in Jurkat T cells. These findings suggest that the Tax protein is at least partially responsible for the p53-independent expression of p21waf1/cip1 in HTLV-I-infected cells. Dysregulation of p53 and p21waf1/cip1 proteins regulating cell-cycle progression, may represent an important step in HTLV-I-induced T-cell transformation.

Thyroid hormone stimulates progesterone release from human luteal cells by generating a proteinaceous factor.

PubMed

Datta, M; Roy, P; Banerjee, J; Bhattacharya, S

1998-09-01

Blood samples collected from 29 women (aged between 19 and 35 years) during the luteal phase of the menstrual cycle (between days 18 and 23 of the cycle) showed that deficiency in thyroid hormone level is related to a decrease in progesterone (P4) secretion. To observe the effect of thyroid hormone on human ovarian luteal cells, 3,5,3'-triiodothyronine (T3; 125 ng/ml) was added to luteal cells in vitro. T3 significantly stimulated progesterone release (P < 0.01) from luteal cells and this could be blocked by cycloheximide, indicating a protein mediator for the T3 effect. The T3 stimulatory effect was inhibited by anti-T3 antibody suggesting specificity of T3 action. Addition of T3 caused a more than threefold increase in cellular protein synthesis which was inhibited by cycloheximide. Preparation of partially purified thyroid hormone-induced factor (TIF) (from peak II of Sephadex G 100 chromatography of T3-incubated cells), and its addition to luteal cell incubations caused a significant increase in P4 release (P < 0.05). Incubation with trypsin or treatment with heat destroyed the stimulatory effect of TIF on P4 release, indicating the proteinaceous nature of TIF. Purified thyroid hormone-induced protein. (TIP) from rat granulosa cells and fish ovarian follicles greatly stimulated P4 release from human luteal cells. These results suggest that T3 stimulation of P4 release from human luteal cells is not direct, but is mediated through a putative protein factor, which appears to be a protein conserved through evolution as far as its biological activity is concerned.

Abnormal Interactions between Perifollicular Mast Cells and CD8+ T-Cells May Contribute to the Pathogenesis of Alopecia Areata

PubMed Central

Bertolini, Marta; Zilio, Federica; Rossi, Alfredo; Gilhar, Amos; Keren, Aviad; Meyer, Katja C.; Wang, Eddy; Funk, Wolfgang; McElwee, Kevin; Paus, Ralf

2014-01-01

Alopecia areata (AA) is a CD8+ T-cell dependent autoimmune disease of the hair follicle (HF) in which the collapse of HF immune privilege (IP) plays a key role. Mast cells (MCs) are crucial immunomodulatory cells implicated in the regulation of T cell-dependent immunity, IP, and hair growth. Therefore, we explored the role of MCs in AA pathogenesis, focusing on MC interactions with CD8+ T-cells in vivo, in both human and mouse skin with AA lesions. Quantitative (immuno-)histomorphometry revealed that the number, degranulation and proliferation of perifollicular MCs are significantly increased in human AA lesions compared to healthy or non-lesional control skin, most prominently in subacute AA. In AA patients, perifollicular MCs showed decreased TGFβ1 and IL-10 but increased tryptase immunoreactivity, suggesting that MCs switch from an immuno-inhibitory to a pro-inflammatory phenotype. This concept was supported by a decreased number of IL-10+ and PD-L1+ MCs, while OX40L+, CD30L+, 4–1BBL+ or ICAM-1+ MCs were increased in AA. Lesional AA-HFs also displayed significantly more peri- and intrafollicular- CD8+ T-cells as well as more physical MC/CD8+ T-cell contacts than healthy or non-lesional human control skin. During the interaction with CD8+ T-cells, AA MCs prominently expressed MHC class I and OX40L, and sometimes 4–1BBL or ICAM-1, suggesting that MC may present autoantigens to CD8+ T-cells and/or co-stimulatory signals. Abnormal MC numbers, activities, and interactions with CD8+ T-cells were also seen in the grafted C3H/HeJ mouse model of AA and in a new humanized mouse model for AA. These phenomenological in vivo data suggest the novel AA pathobiology concept that perifollicular MCs are skewed towards pro-inflammatory activities that facilitate cross-talk with CD8+ T-cells in this disease, thus contributing to triggering HF-IP collapse in AA. If confirmed, MCs and their CD8+ T-cell interactions could become a promising new therapeutic target in the future management of AA. PMID:24832234

Cytokine-mediated activation of human ex vivo-expanded Vγ9Vδ2 T cells

PubMed Central

Domae, Eisuke; Hirai, Yuya; Ikeo, Takashi; Goda, Seiji; Shimizu, Yoji

2017-01-01

Vγ9Vδ2 T cells, the major subset of the human peripheral blood γδ T-cell, respond to microbial infection and stressed cells through the recognition of phosphoantigens. In contrast to the growing knowledge of antigen-mediated activation mechanisms, the antigen-independent and cytokine-mediated activation mechanisms of Vγ9Vδ2 T cells are poorly understood. Here, we show that interleukin (IL) -12 and IL-18 synergize to activate human ex vivo-expanded Vγ9Vδ2 T cells. Vγ9Vδ2 T cells treated with IL-12 and IL-18 enhanced effector functions, including the expression of IFN-γ and granzyme B, and cytotoxicity. These enhanced effector responses following IL-12 and IL-18 treatment were associated with homotypic aggregation, enhanced expression of ICAM-1 and decreased expression of the B- and T-lymphocyte attenuator (BTLA), a co-inhibitory receptor. IL-12 and IL-18 also induced the antigen-independent proliferation of Vγ9Vδ2 T cells. Increased expression of IκBζ, IL-12Rβ2 and IL-18Rα following IL-12 and IL-18 stimulation resulted in sustained activation of STAT4 and NF-κB. The enhanced production of IFN-γ and cytotoxic activity are critical for cancer immunotherapy using Vγ9Vδ2 T cells. Thus, the combined treatment of ex vivo-expanded Vγ9Vδ2 T cells with IL-12 and IL-18 may serve as a new strategy for the therapeutic activation of these cells. PMID:28521284

Advances in direct T-cell alloreactivity: function, avidity, biophysics and structure.

PubMed

Smith, C; Miles, J J; Khanna, R

2012-01-01

Although T-cell-based adaptive immunity plays a crucial role in protection against infectious pathogens and uncontrolled outgrowth of malignant cells, a large portion of these T cells are also capable of responding to allogeneic HLA molecules, violating the paradigm of self-major histocompatibility complex (MHC) restriction. Recent studies have provided insights into the mechanisms by which these T cells recognize allogeneic targets. The role of antiviral T cells in direct alloreactivity through peptide-dependent molecular mimicry and alternate peptide-MHC docking modes has emerged as major models for the human alloresponse. Here, we review in depth recent advances in this field and discuss how molecular interactions between T cells and HLA molecules drive the activation of these effector cells and its potential implications for alloreactivity in human transplantation. ©Copyright 2011 American Society of Transplantation and the American Society of Transplant Surgeons.

Physical association and functional interaction between beta1 integrin and CD98 on human T lymphocytes

NASA Technical Reports Server (NTRS)

Miyamoto, Yuko J.; Mitchell, Jason S.; McIntyre, Bradley W.

2003-01-01

CD98 is a cell surface protein previously characterized as a cell activation marker, an amino acid transporter, and has recently been implicated in integrin-related functions. Integrins are cell surface proteins, important for homotypic cell aggregation, cell adhesion, and coactivation of T lymphocytes. We have previously shown that the anti-CD98 mAb 80A10, when coimmobilized with anti-CD3 mAb OKT3, is able to mediate human T cell coactivation that is inhibited by anti-beta1 integrin specific mAb 18D3. These results indicated a functional association of CD98 and beta1 integrin signaling but left open the question of a physical association. We now show the induction of homotypic aggregation through CD98 among human T cells and this aggregation was inhibited by anti-beta1 integrin mAb. Therefore, CD98-dependent lymphocyte proliferation and adhesion may involve integrins. Competitive binding assays and fluorescence colocalization analysis suggested that CD98 and beta1 integrin were physically associated. Differential extraction techniques and immunoprecipitations provided the first evidence that the alpha4beta1 integrin and CD98 are specifically associated on human T lymphocytes.

Functional and genomic analyses of FOXP3-transduced Jurkat-T cells as regulatory T (Treg)-like cells

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

Kim, Joon-Young; Kim, Han-Jong; Hurt, Elaine M.

2007-10-12

FOXP3, a forkhead transcription factor is essential for the development and function of CD4{sup +}CD25{sup +} regulatory T cells (Tregs). In contrast to conversion of murine naive T cells to Tregs by transduction of Foxp3, it is controversial whether ectopic expression of FOXP3 in human CD4{sup +} T cells is sufficient for acquisition of suppressive activity. Here, we show that retroviral transduction of FOXP3 induces a Treg phenotype in human leukemic CD4{sup +} Jurkat-T cells, evidenced by increased expression of Treg-associated cell surface markers as well as inhibition of cytokine production. Furthermore, FOXP3-transduced Jurkat-T cells suppress the proliferation of humanmore » CD4{sup +}CD25{sup -} T cells. Additionally, DNA microarray analysis identifies Treg-related genes regulated by FOXP3. Our study demonstrates that enforced expression of FOXP3 confers Treg-like properties on Jurkat-T cells, which can be a convenient and efficient Treg-like cell model for further study to identify Treg cell surface markers and target genes regulated by FOXP3.« less

TGF-β induces surface LAP expression on murine CD4 T cells independent of Foxp3 induction.

PubMed

Oida, Takatoku; Weiner, Howard L

2010-11-24

It has been reported that human FOXP3(+) CD4 Tregs express GARP-anchored surface latency-associated peptide (LAP) after activation, based on the use of an anti-human LAP mAb. Murine CD4 Foxp3(+) Tregs have also been reported to express surface LAP, but these studies have been hampered by the lack of suitable anti-mouse LAP mAbs. We generated anti-mouse LAP mAbs by immunizing TGF-β(-/-) animals with a mouse Tgfb1-transduced P3U1 cell line. Using these antibodies, we demonstrated that murine Foxp3(+) CD4 Tregs express LAP on their surface. In addition, retroviral transduction of Foxp3 into mouse CD4(+)CD25(-) T cells induced surface LAP expression. We then examined surface LAP expression after treating CD4(+)CD25(-) T cells with TGF-β and found that TGF-β induced surface LAP not only on T cells that became Foxp3(+) but also on T cells that remained Foxp3(-) after TGF-β treatment. GARP expression correlated with the surface LAP expression, suggesting that surface LAP is GARP-anchored also in murine T cells. Unlike human CD4 T cells, surface LAP expression on mouse CD4 T cells is controlled by Foxp3 and TGF-β. Our newly described anti-mouse LAP mAbs will provide a useful tool for the investigation and functional analysis of T cells that express LAP on their surface.

Rapid alterations of cell cycle control proteins in human T lymphocytes in microgravity

PubMed Central

2012-01-01

In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response. In a combination of experiments using "functional weightlessness" provided by 2D-clinostats and real microgravity provided by several parabolic flight campaigns and compared to in-flight-1g-controls, we identified rapid gravity-responsive reactions inside the cell cycle regulatory machinery of human T lymphocytes. In response to 2D clinorotation, we detected an enhanced expression of p21 Waf1/Cip1 protein within minutes, less cdc25C protein expression and enhanced Ser147-phosphorylation of cyclinB1 after CD3/CD28 stimulation. Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls. In CD3/CD28-stimulated primary human T cells, mRNA expression of the cell cycle arrest protein p21 increased 4.1-fold after 20s real microgravity in primary CD4+ T cells and 2.9-fold in Jurkat T cells, compared to 1 g in-flight controls after CD3/CD28 stimulation. The histone acetyltransferase (HAT) inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC) inhibitor. Therefore, we suppose that cell cycle progression in human T lymphocytes requires Earth gravity and that the disturbed expression of cell cycle regulatory proteins could contribute to the breakdown of the human immune system in space. PMID:22273506

Expression of NK cell receptors on decidual T cells in human pregnancy.

PubMed

Tilburgs, Tamara; van der Mast, Barbara J; Nagtzaam, Nicole M A; Roelen, Dave L; Scherjon, Sicco A; Claas, Frans H J

2009-06-01

Specific receptors enable NK cells to discriminate between cells with normal expression of MHC class I and cells that have low or absent expression of MHC class I molecules. In addition to NK cells, these receptors can be expressed on T cell subsets, mainly on CD8+ T cells but also on gammadeltaTCR+ T cells and CD4+ T cells. Although the function of NK cell receptor expression on T cells is not completely understood, various studies have shown that they are involved in down regulation of T cell receptor (TCR)-mediated activation and influence effector functions, like cytotoxicity and cytokine production. The aim of this study was to analyze expression of NK cell receptors on peripheral blood and decidual T cells during human pregnancy using flow cytometry. We demonstrate that a proportion of decidual T cells express HLA-C specific killer immunoglobulin-like receptors (KIRs). Furthermore, a small proportion of decidual T cells express the HLA-E specific CD94-NKG2A inhibitory and CD94-NKG2C activating receptors. Decidual KIR+ and CD94-NKG2+ T cells mainly display a CD3+CD4-CD8- phenotype. However, decidual tissue also contains higher percentages of KIR and CD94-NKG2 expressing CD4+ and CD8+ T cells compared to peripheral blood. So far, the functional capacities of decidual T cells expressing the NK cell receptors are unknown but NK cell receptor expression on decidual T cells may provide an alternative means by which decidual T cells distinguish self (maternal) cells from allogeneic fetal cells, and act to modulate the decidual immune response.

An anti-tumor protein produced by Trichinella spiralis and identified by screening a T7 phage display library, induces apoptosis in human hepatoma H7402 cells

USDA-ARS?s Scientific Manuscript database

Trichinella spiralis infection confers effective resistance to tumor cell expansion. In this study, a T7 phage cDNA display library was constructed to express genes encoded by T. spiralis. Organic phase multi-cell screening was used to sort through candidate proteins in a transfected human chronic m...

Cynomolgus macaques naturally infected with Trypanosoma cruzi-I exhibit an overall mixed pro-inflammatory/modulated cytokine signature characteristic of human Chagas disease.

PubMed

Vitelli-Avelar, Danielle Marquete; Sathler-Avelar, Renato; Mattoso-Barbosa, Armanda Moreira; Gouin, Nicolas; Perdigão-de-Oliveira, Marcelo; Valério-Dos-Reis, Leydiane; Costa, Ronaldo Peres; Elói-Santos, Silvana Maria; Gomes, Matheus de Souza; Amaral, Laurence Rodrigues do; Teixeira-Carvalho, Andréa; Martins-Filho, Olindo Assis; Dick, Edward J; Hubbard, Gene B; VandeBerg, Jane F; VandeBerg, John L

2017-02-01

Non-human primates have been shown to be useful models for Chagas disease. We previously reported that natural T. cruzi infection of cynomolgus macaques triggers clinical features and immunophenotypic changes of peripheral blood leukocytes resembling those observed in human Chagas disease. In the present study, we further characterize the cytokine-mediated microenvironment to provide supportive evidence of the utility of cynomolgus macaques as a model for drug development for human Chagas disease. In this cross-sectional study design, flow cytometry and systems biology approaches were used to characterize the ex vivo and in vitro T. cruzi-specific functional cytokine signature of circulating leukocytes from TcI-T. cruzi naturally infected cynomolgus macaques (CH). Results showed that CH presented an overall CD4+-derived IFN-γ pattern regulated by IL-10-derived from CD4+ T-cells and B-cells, contrasting with the baseline profile observed in non-infected hosts (NI). Homologous TcI-T. cruzi-antigen recall in vitro induced a broad pro-inflammatory cytokine response in CH, mediated by TNF from innate/adaptive cells, counterbalanced by monocyte/B-cell-derived IL-10. TcIV-antigen triggered a more selective cytokine signature mediated by NK and T-cell-derived IFN-γ with modest regulation by IL-10 from T-cells. While NI presented a cytokine network comprised of small number of neighborhood connections, CH displayed a complex cross-talk amongst network elements. Noteworthy, was the ability of TcI-antigen to drive a complex global pro-inflammatory network mediated by TNF and IFN-γ from NK-cells, CD4+ and CD8+ T-cells, regulated by IL-10+CD8+ T-cells, in contrast to the TcIV-antigens that trigger a modest network, with moderate connecting edges. Altogether, our findings demonstrated that CH present a pro-inflammatory/regulatory cytokine signature similar to that observed in human Chagas disease. These data bring additional insights that further validate these non-human primates as experimental models for Chagas disease.

Flow cytometry crossmatch reactivity with pronase-treated T cells induced by non-HLA autoantibodies in human immunodeficiency virus-infected patients.

PubMed

Szewczyk, Katarzyna; Barrios, Kelly; Magas, Daniel; Sieg, Kristin; Labuda, Bozena; Jendrisak, Martin D; Jaramillo, Andrés

2016-06-01

Pronase treatment is used in the flow cytometry crossmatch (FCXM) to prevent nonspecific antibody binding on B cells. However, we have observed unexpected positive results with pronase-treated T cells in human immunodeficiency virus (HIV)-infected patients. In this study, 25 HIV-infected patients without HLA antibodies were tested with pronase-treated and nontreated cells. HIV-positive sera were pretreated with reducing agents and preabsorbed with pronase-treated and nontreated T or B cells before crossmatching. All patients displayed FCXM reactivity with pronase-treated T cells but not with nontreated T cells. None of the patients exhibited FCXM reactivity with pronase-treated and nontreated B cells. These patients displayed FCXM reactivity with pronase-treated CD4+ and CD8+ T cells but not with their nontreated counterparts. Preabsorption with pronase-treated T cells reduced the T cell FCXM reactivity. Preabsorption with pronase-treated B cells or nontreated T and B cells did not have any effect on the T cell FCXM reactivity. Pretreatment with reducing agents did not affect the T cell FCXM reactivity. 15 of 21 HIV-infected kidney allograft recipients with pronase-treated T cell FCXM reactivity display long-term graft survival (1193±631days). These data indicate that HIV-infected patients have nondeleterious autoantibodies recognizing cryptic epitopes exposed by pronase on T cells. Copyright © 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

CD147 (Basigin/Emmprin) identifies FoxP3+CD45RO+CTLA4+-activated human regulatory T cells.

PubMed

Solstad, Therese; Bains, Simer Jit; Landskron, Johannes; Aandahl, Einar Martin; Thiede, Bernd; Taskén, Kjetil; Torgersen, Knut Martin

2011-11-10

Human CD4(+)FoxP3(+) T cells are functionally and phenotypically heterogeneous providing plasticity to immune activation and regulation. To better understand the functional dynamics within this subset, we first used a combined strategy of subcellular fractionation and proteomics to describe differences at the protein level between highly purified human CD4(+)CD25(+) and CD4(+)CD25(-) T-cell populations. This identified a set of membrane proteins highly expressed on the cell surface of human regulatory T cells (Tregs), including CD71, CD95, CD147, and CD148. CD147 (Basigin or Emmprin) divided CD4(+)CD25(+) cells into distinct subsets. Furthermore, CD147, CD25, FoxP3, and in particular CTLA-4 expression correlated. Phenotypical and functional analyses suggested that CD147 marks the switch between resting (CD45RA(+)) and activated (CD45RO(+)) subsets within the FoxP3(+) T-cell population. Sorting of regulatory T cells into CD147(-) and CD147(+) populations demonstrated that CD147 identifies an activated and highly suppressive CD45RO(+) Treg subset. When analyzing CD4(+) T cells for their cytokine producing potential, CD147 levels grouped the FoxP3(+) subset into 3 categories with different ability to produce IL-2, TNF-α, IFN-γ, and IL-17. Together, this suggests that CD147 is a direct marker for activated Tregs within the CD4(+)FoxP3(+) subset and may provide means to manipulate cells important for immune homeostasis.

The impact of ex vivo clinical grade activation protocols on human T-cell phenotype and function for the generation of genetically modified cells for adoptive cell transfer therapy.

PubMed

Tumeh, Paul C; Koya, Richard C; Chodon, Thinle; Graham, Nicholas A; Graeber, Thomas G; Comin-Anduix, Begoña; Ribas, Antoni

2010-10-01

Optimized conditions for the ex vivo activation, genetic manipulation, and expansion of human lymphocytes for adoptive cell therapy may lead to protocols that maximize their in vivo function. We analyzed the effects of 4 clinical grade activation and expansion protocols over 3 weeks on cell proliferative rate, immunophenotype, cell metabolism, and transduction efficiency of human peripheral blood mononuclear cells (PBMCs). Peak lentiviral transduction efficiency was early (days 2 to 4), at a time when cells showed a larger size, maximal uptake of metabolic substrates, and the highest level of proximal T-cell receptor signaling engagement. Anti-CD2/3/28 activation beads induced greater proliferation rate and skewed PBMCs early on to a CD4 phenotype when compared with the cells cultured in OKT3. Multicolor surface phenotyping demonstrated that changes in T-cell surface markers that define T-cell functional phenotypes were dependent on the time spent in culture as opposed to the particular activation protocol. In conclusion, ex vivo activation of human PBMCs for adoptive cell therapy demonstrate defined immunophenotypic and functional signatures over time, with cells early on showing larger sizes, higher transduction efficiency, maximal metabolic activity, and zeta-chain-associated protein-70 activation.

The role of human chorionic gonadotropin in regulation of naïve and memory T cells activity in vitro.

PubMed

Zamorina, S A; Litvinova, L S; Yurova, K A; Khaziakhmatova, O G; Timganova, V P; Bochkova, M S; Khramtsov, P V; Rayev, M B

2018-01-01

The role of human chorionic gonadotropin (hCG) in the regulation of molecular genetics factors determining the functional activity of human naïve and memory T cells in vitro was studied. It was found that hCG (10 and 100IU/ml) inhibited CD28 and CD25 expression on the naïve T cells (CD45RA+) and CD25 expression on the memory T cells (CD45R0+). hCG didn't affect the CD71 proliferation marker expression in total. Nevertheless, hCG reduced the percentage of proliferating memory T cells with simultaneous suppression of CD71 expression on proliferating CD45R0+cells. In parallel, expression of U2af1l4, Gfi1, and hnRNPLL genes, which are Ptprc gene alternative splicing regulators was evaluated. It was established that hCG stimulated the expression of U2af1l4 and hnRNPLL genes, responsible for the assembly of CD45R0 in memory T cells, but reduced the expression of Gfi1 in these cells. In general, hCG promotes the differentiation of memory T cells by increasing of CD45R0 expression, but inhibits proliferation and CD25 expression which reflects their functional activity. Copyright © 2017 Elsevier B.V. All rights reserved.

Enhancing adoptive cancer immunotherapy with Vγ2Vδ2 T cells through pulse zoledronate stimulation.

PubMed

Nada, Mohanad H; Wang, Hong; Workalemahu, Grefachew; Tanaka, Yoshimasa; Morita, Craig T

2017-01-01

Human γδ T cells expressing Vγ2Vδ2 T cell receptors monitor foreign- and self-prenyl pyrophosphate metabolites in isoprenoid biosynthesis to mediate immunity to microbes and tumors. Adoptive immunotherapy with Vγ2Vδ2 T cells has been used to treat cancer patients with partial and complete remissions. Most clinical trials and preclinical studies have used continuous zoledronate exposure to expand Vγ2Vδ2 cells where zoledronate is slowly diluted over the course of the culture. Zoledronate inhibits farnesyl diphosphate synthase (FDPS) in monocytes causing isopentenyl pyrophosphate to accumulate that then stimulates Vγ2Vδ2 cells. Because zoledronate inhibition of FDPS is also toxic for T cells, we hypothesized that a short period of exposure would reduce T cell toxicity but still be sufficient for monocytes uptake. Additionally, IL-15 increases the anti-tumor activity of murine αβ T cells in mice but its effect on the in vivo anti-tumor activity of human Vγ2Vδ2 cells has not been assessed. Human Vγ2Vδ2 T cells were expanded by pulse or continuous zoledronate stimulation with IL-2 or IL-15. Expanded Vγ2Vδ2 cells were tested for their expression of effector molecules and killing of tumor cells as well as their in vivo control of human prostate cancer tumors in immunodeficient NSG mice. Pulse zoledronate stimulation with either IL-2 or IL-15 resulted in more uniform expansion of Vγ2Vδ2 cells with higher purity and cell numbers as compared with continuous exposure. The Vγ2Vδ2 cells had higher levels of CD107a and perforin and increased tumor cytotoxicity. Adoptive immunotherapy with Vγ2Vδ2 cells derived by pulse stimulation controlled human PC-3 prostate cancer tumors in NSG mice significantly better than those derived by continuous stimulation, halting tumor growth. Although pulse zoledronate stimulation with IL-15 preserved early memory subsets, adoptive immunotherapy with IL-15-derived Vγ2Vδ2 cells equally inhibited PC-3 tumor growth as those derived with IL-2. Pulse zoledronate stimulation maximizes the purity, quantity, and quality of expanded Vγ2Vδ2 cells for adoptive immunotherapy but there is no advantage to using IL-15 over IL-2 in our humanized mouse model. Pulse zoledronate stimulation is a simple modification to existing protocols that will enhance the effectiveness of adoptively transferred Vγ2Vδ2 cells by increasing their numbers and anti-tumor activity.

Alpharetroviral self-inactivating vectors produced by a superinfection-resistant stable packaging cell line allow genetic modification of primary human T lymphocytes.

PubMed

Labenski, Verena; Suerth, Julia D; Barczak, Elke; Heckl, Dirk; Levy, Camille; Bernadin, Ornellie; Charpentier, Emmanuelle; Williams, David A; Fehse, Boris; Verhoeyen, Els; Schambach, Axel

2016-08-01

Primary human T lymphocytes represent an important cell population for adoptive immunotherapies, including chimeric-antigen and T-cell receptor applications, as they have the capability to eliminate non-self, virus-infected and tumor cells. Given the increasing numbers of clinical immunotherapy applications, the development of an optimal vector platform for genetic T lymphocyte engineering, which allows cost-effective high-quality vector productions, remains a critical goal. Alpharetroviral self-inactivating vectors (ARV) have several advantages compared to other vector platforms, including a more random genomic integration pattern and reduced likelihood for inducing aberrant splicing of integrated proviruses. We developed an ARV platform for the transduction of primary human T lymphocytes. We demonstrated functional transgene transfer using the clinically relevant herpes-simplex-virus thymidine kinase variant TK.007. Proof-of-concept of alpharetroviral-mediated T-lymphocyte engineering was shown in vitro and in a humanized transplantation model in vivo. Furthermore, we established a stable, human alpharetroviral packaging cell line in which we deleted the entry receptor (SLC1A5) for RD114/TR-pseudotyped ARVs to prevent superinfection and enhance genomic integrity of the packaging cell line and viral particles. We showed that superinfection can be entirely prevented, while maintaining high recombinant virus titers. Taken together, this resulted in an improved production platform representing an economic strategy for translating the promising features of ARVs for therapeutic T-lymphocyte engineering. Copyright © 2016 Elsevier Ltd. All rights reserved.

Human dendritic cells produce TGF-beta 1 under the influence of lung carcinoma cells and prime the differentiation of CD4+CD25+Foxp3+ regulatory T cells.

PubMed

Dumitriu, Ingrid E; Dunbar, Donald R; Howie, Sarah E; Sethi, Tariq; Gregory, Christopher D

2009-03-01

Dendritic cells (DCs) have a central role in the development of adaptive immune responses, including antitumor immunity. Factors present in the tumor milieu can alter the maturation of DCs and inhibit their capacity to activate T cells. Using gene expression analysis, we found that human DCs increased the expression of TGF-beta1 transcripts following culture with human lung carcinoma cells (LCCs). These DCs produced increased amounts of TGF-beta1 protein compared with DCs not exposed to tumor cells. LCCs also decreased the expression of CD86 and HLA-DR by immature DCs. Furthermore, LCCs decreased CD86 expression and the production of TNF-alpha and IL-12 p70 by mature DCs. Moreover, LCCs also converted mature DCs into cells producing TGF-beta1. These TGF-beta1-producing DCs were poor at eliciting the activation of naive CD4(+) T cells and sustaining their proliferation and differentiation into Th1 (IFN-gamma(+)) effectors. Instead, TGF-beta1-producing DCs demonstrated an increased ability to generate CD4(+)CD25(+)Foxp3(+) regulatory T cells that suppress the proliferation of T lymphocytes. These results identify a novel mechanism by which the function of human DCs is altered by tumor cells and contributes to the evasion of the immune response.

A Herpes Simplex Virus Type 2 Deleted for Glycoprotein D Enables Dendritic Cells to Activate CD4+ and CD8+ T Cells

PubMed Central

Retamal-Díaz, Angello R.; Kalergis, Alexis M.; Bueno, Susan M.; González, Pablo A.

2017-01-01

Herpes simplex virus type 2 (HSV-2) is highly prevalent in the human population producing significant morbidity, mainly because of the generation of genital ulcers and neonatal encephalitis. Additionally, HSV-2 infection significantly increases the susceptibility of the host to acquire HIV and promotes the shedding of the latter in the coinfected. Despite numerous efforts to create a vaccine against HSV-2, no licensed vaccines are currently available. A long-standing strategy, based on few viral glycoproteins combined with adjuvants, recently displayed poor results in a Phase III clinical study fueling exploration on the development of mutant HSV viruses that are attenuated in vivo and elicit protective adaptive immune components, such as antiviral antibodies and T cells. Importantly, such specialized antiviral immune components are likely induced and modulated by dendritic cells, professional antigen presenting cells that process viral antigens and present them to T cells. However, HSV interferes with several functions of DCs and ultimately induces their death. Here, we propose that for an attenuated mutant virus to confer protective immunity against HSV in vivo based on adaptive immune components, such virus should also be attenuated in dendritic cells to promote a robust and effective antiviral response. We provide a background framework for this idea, considerations, as well as the means to assess this hypothesis. Addressing this hypothesis may provide valuable insights for the development of novel, safe, and effective vaccines against herpes simplex viruses. PMID:28848543

Comparative phenotypic and functional analysis of migratory dendritic cell subsets from human oral mucosa and skin

PubMed Central

van de Ven, Rieneke; Thon, Maria; Gibbs, Susan; de Gruijl, Tanja D.

2017-01-01

Antigen exposure to oral mucosa is generally thought to lead to immune tolerance induction. However, very little is known about the subset composition and function of dendritic cells (DC) migrating from human oral mucosa. Here we show that migratory DC from healthy human gingival explants consist of the same phenotypic subsets in the same frequency distribution as DC migrating from human skin. The gingival CD1a+ Langerhans cell and interstitial DC subsets lacked CXCR4 expression in contrast to their cutaneous counterparts, pointing to different migration mechanisms, consistent with previous observations in constructed skin and gingival equivalents. Remarkably, without any exogenous conditioning, gingival explants released higher levels of inflammatory cytokines than human skin explants, resulting in higher DC migration rates and a superior ability of migrated DC to prime allogeneic T cells and to induce type-1 effector T cell differentiation. From these observations we conclude that rather than an intrinsic ability to induce T cell tolerance, DC migrating from oral mucosa may have a propensity to induce effector T cell immunity and maintain a high state of alert against possible pathogenic intruders in the steady state. These findings may have implications for oral immunization strategies. PMID:28704477

Simultaneous Vascular Targeting and Tumor Targeting of Cerebral Breast Cancer Metastases Using a T-Cell Receptor Mimic Antibody

DTIC Science & Technology

2014-05-01

in May 2013, the difference between nude mice (which lack T- cells , but still have a partially functional adaptive and innate immune system) and NSG...Mangada J, Greiner DL, Handgretinger R. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human...Targeting of Cerebral Breast Cancer Metastases Using a T- Cell Receptor Mimic Antibody PRINCIPAL INVESTIGATOR: Ulrich Bickel

Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template.

PubMed

Sather, Blythe D; Romano Ibarra, Guillermo S; Sommer, Karen; Curinga, Gabrielle; Hale, Malika; Khan, Iram F; Singh, Swati; Song, Yumei; Gwiazda, Kamila; Sahni, Jaya; Jarjour, Jordan; Astrakhan, Alexander; Wagner, Thor A; Scharenberg, Andrew M; Rawlings, David J

2015-09-30

Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4(+) T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV)-mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP-modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34(+) cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties. Copyright © 2015, American Association for the Advancement of Science.

Regulation of Mitochondria Function by TRAF3 in B Lymphocytes and B Cell Malignancies

DTIC Science & Technology

2014-08-01

PARP1, PHB2 4 Background B cell neoplasms account for over 90% of lymphoid tumors worldwide, and comprise >50% of blood cancers. Despite recent... cells examined include common lymphoid progenitor, pre-pro-B, pro-B, pre-B, newly-formed B, and transitional (T1, T2 and T3) B cells . The data in...factor 3 is a critical regulator of B cell homeostasis in secondary lymphoid organs. Immunity 2007, 27:253-267. 13. Moore CR, Liu Y, Shao CS, Covey LR

Comparative proteomics of exosomes secreted by tumoral Jurkat T cells and normal human T cell blasts unravels a potential tumorigenic role for valosin-containing protein.

PubMed

Bosque, Alberto; Dietz, Lisa; Gallego-Lleyda, Ana; Sanclemente, Manuel; Iturralde, María; Naval, Javier; Alava, María Angeles; Martínez-Lostao, Luis; Thierse, Hermann-Josef; Anel, Alberto

2016-05-17

We have previously characterized that FasL and Apo2L/TRAIL are stored in their bioactive form inside human T cell blasts in intraluminal vesicles present in multivesicular bodies. These vesicles are rapidly released to the supernatant in the form of exosomes upon re-activation of T cells. In this study we have compared for the first time proteomics of exosomes produced by normal human T cell blasts with those produced by tumoral Jurkat cells, with the objective of identify proteins associated with tumoral exosomes that could have a previously unrecognized role in malignancy. We have identified 359 and 418 proteins in exosomes from T cell blasts and Jurkat cells, respectively. Interestingly, only 145 (around a 40%) are common. The major proteins in both cases are actin and tubulin isoforms and the common interaction nodes correspond to these cytoskeleton and related proteins, as well as to ribosomal and mRNA granule proteins. We detected 14 membrane proteins that were especially enriched in exosomes from Jurkat cells as compared with T cell blasts. The most abundant of these proteins was valosin-containing protein (VCP), a membrane ATPase involved in ER homeostasis and ubiquitination. In this work, we also show that leukemic cells are more sensitive to cell death induced by the VCP inhibitor DBeQ than normal T cells. Furthermore, VCP inhibition prevents functional exosome secretion only in Jurkat cells, but not in T cell blasts. These results suggest VCP targeting as a new selective pathway to exploit in cancer treatment to prevent tumoral exosome secretion.

Epigenomic analysis of primary human T cells reveals enhancers associated with TH2 memory cell differentiation and asthma susceptibility

PubMed Central

Seumois, Grégory; Chavez, Lukas; Gerasimova, Anna; Lienhard, Matthias; Omran, Nada; Kalinke, Lukas; Vedanayagam, Maria; Ganesan, Asha Purnima V; Chawla, Ashu; Djukanović, Ratko; Ansel, K Mark; Peters, Bjoern; Rao, Anjana; Vijayanand, Pandurangan

2014-01-01

A characteristic feature of asthma is the aberrant accumulation, differentiation or function of memory CD4+ T cells that produce type 2 cytokines (TH2 cells). By mapping genome-wide histone modification profiles for subsets of T cells isolated from peripheral blood of healthy and asthmatic individuals, we identified enhancers with known and potential roles in the normal differentiation of human TH1 cells and TH2 cells. We discovered disease-specific enhancers in T cells that differ between healthy and asthmatic individuals. Enhancers that gained the histone H3 Lys4 dimethyl (H3K4me2) mark during TH2 cell development showed the highest enrichment for asthma-associated single nucleotide polymorphisms (SNPs), which supported a pathogenic role for TH2 cells in asthma. In silico analysis of cell-specific enhancers revealed transcription factors, microRNAs and genes potentially linked to human TH2 cell differentiation. Our results establish the feasibility and utility of enhancer profiling in well-defined populations of specialized cell types involved in disease pathogenesis. PMID:24997565

Epigenomic analysis of primary human T cells reveals enhancers associated with TH2 memory cell differentiation and asthma susceptibility.

PubMed

Seumois, Grégory; Chavez, Lukas; Gerasimova, Anna; Lienhard, Matthias; Omran, Nada; Kalinke, Lukas; Vedanayagam, Maria; Ganesan, Asha Purnima V; Chawla, Ashu; Djukanović, Ratko; Ansel, K Mark; Peters, Bjoern; Rao, Anjana; Vijayanand, Pandurangan

2014-08-01

A characteristic feature of asthma is the aberrant accumulation, differentiation or function of memory CD4(+) T cells that produce type 2 cytokines (TH2 cells). By mapping genome-wide histone modification profiles for subsets of T cells isolated from peripheral blood of healthy and asthmatic individuals, we identified enhancers with known and potential roles in the normal differentiation of human TH1 cells and TH2 cells. We discovered disease-specific enhancers in T cells that differ between healthy and asthmatic individuals. Enhancers that gained the histone H3 Lys4 dimethyl (H3K4me2) mark during TH2 cell development showed the highest enrichment for asthma-associated single nucleotide polymorphisms (SNPs), which supported a pathogenic role for TH2 cells in asthma. In silico analysis of cell-specific enhancers revealed transcription factors, microRNAs and genes potentially linked to human TH2 cell differentiation. Our results establish the feasibility and utility of enhancer profiling in well-defined populations of specialized cell types involved in disease pathogenesis.

Fetal liver contains committed NK progenitors, but is not a site for development of CD34+ cells into T cells.

PubMed

Jaleco, A C; Blom, B; Res, P; Weijer, K; Lanier, L L; Phillips, J H; Spits, H

1997-07-15

The presence of T and NK cells in the human fetal liver and the fact that fetal liver hemopoietic progenitor cells develop into T and NK cells suggest a role for the fetal liver compartment in T and NK cell development. In this work, we show that the capacity of fetal liver progenitors to develop into T cells, in a human/mouse fetal thymic organ culture system, is restricted to an immature subset of CD34+ CD38- cells. No T cell-committed precursors are contained within the more differentiated CD34+ CD38+ population. This conclusion is supported by the observations that no TCR-delta gene rearrangements and no pre-TCR-alpha expression can be detected in this population. However, NK cells were derived from CD34+ CD38- and CD34+ CD38+ fetal liver cells cultured in the presence of IL-15, IL-7, and Flt-3 ligand. Eighty to ninety percent of cells arising from the CD34+ CD38+ population expressed the NK cell-associated markers CD56, CD16, CD94, and NKR-P1A. Several subpopulations of NK cell precursors were identified by differential expression of these receptors. Based on the detection of populations with a similar antigenic profile in freshly isolated fetal liver cells, we propose a model of NK cell differentiation. Collectively, our findings suggest that CD34+ cells differentiate into NK cells, but not into mature T cells, in the human fetal liver.

Extracellular MicroRNA Signature of Human Helper T Cell Subsets in Health and Autoimmunity.

PubMed

Torri, Anna; Carpi, Donatella; Bulgheroni, Elisabetta; Crosti, Maria-Cristina; Moro, Monica; Gruarin, Paola; Rossi, Riccardo L; Rossetti, Grazisa; Di Vizio, Dolores; Hoxha, Mirjam; Bollati, Valentina; Gagliani, Cristina; Tacchetti, Carlo; Paroni, Moira; Geginat, Jens; Corti, Laura; Venegoni, Luigia; Berti, Emilio; Pagani, Massimiliano; Matarese, Giuseppe; Abrignani, Sergio; de Candia, Paola

2017-02-17

Upon T cell receptor stimulation, CD4 + T helper (Th) lymphocytes release extracellular vesicles (EVs) containing microRNAs. However, no data are available on whether human CD4 + T cell subsets release EVs containing different pattern of microRNAs. The present work aimed at filling this gap by assessing the microRNA content in EVs released upon in vitro T cell receptor stimulation of Th1, Th17, and T regulatory (Treg) cells. Our results indicate that EVs released by Treg cells are significantly different compared with those released by the other subsets. In particular, miR-146a-5p, miR-150-5p, and miR-21-5p are enriched, whereas miR-106a-5p, miR-155-5p, and miR-19a-3p are depleted in Treg-derived EVs. The in vitro identified EV-associated microRNA signature was increased in serum of autoimmune patients with psoriasis and returned to healthy levels upon effective treatment with etanercept, a biological drug targeting the TNF pathway and suppressing inflammation. Moreover, Gene Set Enrichment Analysis showed an over-representation of genes relevant for T cell activation, such as CD40L, IRAK1, IRAK2, STAT1, and c-Myb in the list of validated targets of Treg-derived EV miRNAs. At functional level, Treg-derived (but not Th1/Th17-derived) EVs inhibited CD4 + T cell proliferation and suppressed two relevant targets of miR-146a-5p: STAT1 and IRAK2. In conclusion, our work identified the miRNAs specifically released by different human CD4 + T cell subsets and started to unveil the potential use of their quantity in human serum to mark the pathological elicitation of these cells in vivo and their biological effect in cell to cell communication during the adaptive immune response. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Extracellular MicroRNA Signature of Human Helper T Cell Subsets in Health and Autoimmunity*

PubMed Central

Torri, Anna; Carpi, Donatella; Bulgheroni, Elisabetta; Crosti, Maria-Cristina; Moro, Monica; Gruarin, Paola; Rossi, Riccardo L.; Rossetti, Grazisa; Di Vizio, Dolores; Hoxha, Mirjam; Bollati, Valentina; Gagliani, Cristina; Tacchetti, Carlo; Paroni, Moira; Geginat, Jens; Corti, Laura; Venegoni, Luigia; Berti, Emilio; Pagani, Massimiliano; Matarese, Giuseppe; Abrignani, Sergio; de Candia, Paola

2017-01-01

Upon T cell receptor stimulation, CD4+ T helper (Th) lymphocytes release extracellular vesicles (EVs) containing microRNAs. However, no data are available on whether human CD4+ T cell subsets release EVs containing different pattern of microRNAs. The present work aimed at filling this gap by assessing the microRNA content in EVs released upon in vitro T cell receptor stimulation of Th1, Th17, and T regulatory (Treg) cells. Our results indicate that EVs released by Treg cells are significantly different compared with those released by the other subsets. In particular, miR-146a-5p, miR-150-5p, and miR-21-5p are enriched, whereas miR-106a-5p, miR-155-5p, and miR-19a-3p are depleted in Treg-derived EVs. The in vitro identified EV-associated microRNA signature was increased in serum of autoimmune patients with psoriasis and returned to healthy levels upon effective treatment with etanercept, a biological drug targeting the TNF pathway and suppressing inflammation. Moreover, Gene Set Enrichment Analysis showed an over-representation of genes relevant for T cell activation, such as CD40L, IRAK1, IRAK2, STAT1, and c-Myb in the list of validated targets of Treg-derived EV miRNAs. At functional level, Treg-derived (but not Th1/Th17-derived) EVs inhibited CD4+ T cell proliferation and suppressed two relevant targets of miR-146a-5p: STAT1 and IRAK2. In conclusion, our work identified the miRNAs specifically released by different human CD4+ T cell subsets and started to unveil the potential use of their quantity in human serum to mark the pathological elicitation of these cells in vivo and their biological effect in cell to cell communication during the adaptive immune response. PMID:28077577

In vitro effects of 4-hydroperoxycyclophosphamide on human immunoregulatory T subset function. I. Selective effects on lymphocyte function in T-B cell collaboration.

PubMed

Ozer, H; Cowens, J W; Colvin, M; Nussbaum-Blumenson, A; Sheedy, D

1982-01-01

The alkylating agent cyclophosphamide may suppress or enhance immune responses in vivo but is inactive in vitro unless metabolized by microsomal enzyme activation. 4-hydroperoxycyclophosphamide (4-HC) is a synthetic compound that is spontaneously converted in aqueous solution to the active metabolites. In this report, we examined the in vitro sensitivity of functional human T cell subsets to 4-HC in a polyclonal B cell differentiation assay and in the generation of mitogen-induced suppressor cells for effector B cell function. Con A-induced T suppression of B cell differentiation is completely abrogated by a 1-h pretreatment of T cells at very low concentrations of between 10(-2) and 20 nmol/ml, whereas inducer T cell function is sensitive only to concentrations in greater than 40 nmol/ml. The effects of 4-HC on suppressor T cells appear to occur at concentrations that do not result in DNA cross-linking or decreased blastogenesis. Con A-induced T suppressors are generated from within the OKT4+, OKT8- subset and are sensitive to low-dose 4-HC only before activation, whereas differentiated suppressor cells are resistant to concentrations in greater than 80 nmol/ml. Low-dose 4-HC pretreatment of the B cell population results in abrogation of immunoglobulin secretion when treated B cells are cocultured with unfractionated T cells, however, this effect is completely reversible if pretreated B cells are cocultured with T cells devoid of suppressor activity. These results demonstrate that human presuppressor cells for B-effector function differentiate in response to Con A from the OKT4+, OKT8- subset and are exquisitely sensitive to low concentrations of CYP whereas mature suppressor and inducer functions are resistant to all but very high concentrations in vitro. The differential sensitivity of functional T and B cell subsets to 4-HC in vitro can be a very useful probe in dissecting immunoregulatory interactions with man.

Regulated expression of telomerase activity in human T lymphocyte development and activation

PubMed Central

1996-01-01

Telomerase, a ribonucleoprotein that is capable of synthesizing telomeric repeats, is expressed in germline and malignant cells, and is absent in most normal human somatic cells. The selective expression of telomerase has thus been proposed to be a basis for the immortality of the germline and of malignant cells. In the present study, telomerase activity was analyzed in normal human T lymphocytes. It was found that telomerase is expressed at a high level in thymocyte subpopulations, at an intermediate level in tonsil T lymphocytes, and at a low to undetectable level in peripheral blood T lymphocytes. Moreover, telomerase activity is highly inducible in peripheral T lymphocytes by activation through CD3 with or without CD28 costimulation, or by stimulation with phorbol myristate acetate (PMA)/ionomycin. The induction of telomerase by anti-CD3 plus anti-CD28 (anti-CD3/CD28) stimulation required RNA and protein synthesis, and was blocked by herbimycin A, an inhibitor of S pi protein tyrosine kinases. The immunosuppressive drug cyclosporin A selectively inhibited telomerase induction by PMA/ionomycin and by anti-CD3, but not by anti-CD3/CD28. Although telomerase activity in peripheral T lymphocytes was activation dependent and correlated with cell proliferation, it was not cell cycle phase restricted. These results indicate that the expression of telomerase in normal human T lymphocytes is both developmentally regulated and activation induced. Telomerase may thus play a permissive role in T cell development and in determining the capacity of lymphoid cells for cell division and clonal expansion. PMID:8676067

Animals are key to human toxoplasmosis.

PubMed

Schlüter, Dirk; Däubener, Walter; Schares, Gereon; Groß, Uwe; Pleyer, Uwe; Lüder, Carsten

2014-10-01

Toxoplasma gondii is an extremely sucessfull protozoal parasite which infects almost all mamalian species including humans. Approximately 30% of the human population worldwide is chronically infected with T. gondii. In general, human infection is asymptomatic but the parasite may induce severe disease in fetuses and immunocompromised patients. In addition, T. gondii may cause sight-threatening posterior uveitis in immunocompetent patients. Apart from few exceptions, humans acquire T. gondii from animals. Both, the oral uptake of T. gondii oocysts released by specific hosts, i.e. felidae, and of cysts persisting in muscle cells of animals result in human toxoplasmosis. In the present review, we discuss recent new data on the cell biology of T. gondii and parasite diversity in animals. In addition, we focus on the impact of these various parasite strains and their different virulence on the clinical outcome of human congenital toxoplasmosis and T. gondii uveitis. Copyright © 2014 Elsevier GmbH. All rights reserved.

Altered Mitochondrial Membrane Potential, Mass, and Morphology in the Mononuclear Cells of Humans with Type 2 Diabetes

PubMed Central

Widlansky, Michael E.; Wang, Jingli; Shenouda, Sherene M.; Hagen, Tory M.; Smith, Anthony R.; Kizhakekuttu, Tinoy J.; Kluge, Matthew A.; Weihrauch, Dorothee; Gutterman, David D.; Vita, Joseph A.

2010-01-01

Mitochondrial membrane hyperpolarization and morphological changes are important in inflammatory cell activation. Despite the pathophysiological relevance, no valid and reproducible method for measuring mitochondrial homeostasis in human inflammatory cells is currently available. This study's purpose was to define and validate reproducible methods for measuring relevant mitochondrial perturbations and to determine whether these methods could discern mitochondrial perturbations in type 2 diabetes mellitus (T2DM), a condition associated with altered mitochondrial homeostasis. We employed 5,5',6,6'-tetrachloro-1,1'3,3'-tetraethylbenzamidazol-carboncyanine (JC-1) to estimate mitochondrial membrane potential (ψm) and acridine orange 10-nonyl bromide (NAO) to assess mitochondrial mass in human mononuclear cells isolated from blood. Both assays were reproducible. We validated our findings by electron microscopy and pharmacological manipulation of ψm. We measured JC-1 and NAO fluorescence in the mononuclear cells of 27 T2DM patients and 32 controls. Mitochondria were more polarized (P=0.02) and mitochondrial mass was lower in T2DM (P=0.008). Electron microscopy demonstrated diabetic mitochondria were smaller, more spherical, and occupied less cellular area in T2DM. Mitochondrial superoxide production was higher in T2DM (P=0.01). Valid and reproducible measurements of mitochondrial homeostasis can be made in human mononuclear cells using these fluorophores. Further, potential clinically relevant perturbations in mitochondrial homeostasis in T2DM human mononuclear cells can be detected. PMID:20621033

Human Beta Defensin 2 Selectively Inhibits HIV-1 in Highly Permissive CCR6⁺CD4⁺ T Cells.

PubMed

Lafferty, Mark K; Sun, Lingling; Christensen-Quick, Aaron; Lu, Wuyuan; Garzino-Demo, Alfredo

2017-05-16

Chemokine receptor type 6 (CCR6)⁺CD4⁺ T cells are preferentially infected and depleted during HIV disease progression, but are preserved in non-progressors. CCR6 is expressed on a heterogeneous population of memory CD4⁺ T cells that are critical to mucosal immunity. Preferential infection of these cells is associated, in part, with high surface expression of CCR5, CXCR4, and α4β7. In addition, CCR6⁺CD4⁺ T cells harbor elevated levels of integrated viral DNA and high levels of proliferation markers. We have previously shown that the CCR6 ligands MIP-3α and human beta defensins inhibit HIV replication. The inhibition required CCR6 and the induction of APOBEC3G. Here, we further characterize the induction of apolipoprotein B mRNA editing enzyme (APOBEC3G) by human beta defensin 2. Human beta defensin 2 rapidly induces transcriptional induction of APOBEC3G that involves extracellular signal-regulated kinases 1/2 (ERK1/2) activation and the transcription factors NFATc2, NFATc1, and IRF4. We demonstrate that human beta defensin 2 selectively protects primary CCR6⁺CD4⁺ T cells infected with HIV-1. The selective protection of CCR6⁺CD4⁺ T cell subsets may be critical in maintaining mucosal immune function and preventing disease progression.

Human T-cell leukemia virus type 1 infects multiple lineage hematopoietic cells in vivo

PubMed Central

Sugata, Kenji; Ueno, Takaharu; Koh, Ki-Ryang; Higuchi, Yusuke; Matsuda, Fumihiko; Melamed, Anat; Bangham, Charles R.

2017-01-01

Human T-cell leukemia virus type 1 (HTLV-1) infects mainly CD4+CCR4+ effector/memory T cells in vivo. However, it remains unknown whether HTLV-1 preferentially infects these T cells or this virus converts infected precursor cells to specialized T cells. Expression of viral genes in vivo is critical to study viral replication and proliferation of infected cells. Therefore, we first analyzed viral gene expression in non-human primates naturally infected with simian T-cell leukemia virus type 1 (STLV-1), whose virological attributes closely resemble those of HTLV-1. Although the tax transcript was detected only in certain tissues, Tax expression was much higher in the bone marrow, indicating the possibility of de novo infection. Furthermore, Tax expression of non-T cells was suspected in bone marrow. These data suggest that HTLV-1 infects hematopoietic cells in the bone marrow. To explore the possibility that HTLV-1 infects hematopoietic stem cells (HSCs), we analyzed integration sites of HTLV-1 provirus in various lineages of hematopoietic cells in patients with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and a HTLV-1 carrier using the high-throughput sequencing method. Identical integration sites were detected in neutrophils, monocytes, B cells, CD8+ T cells and CD4+ T cells, indicating that HTLV-1 infects HSCs in vivo. We also detected Tax protein in myeloperoxidase positive neutrophils. Furthermore, dendritic cells differentiated from HTLV-1 infected monocytes caused de novo infection to T cells, indicating that infected monocytes are implicated in viral spreading in vivo. Certain integration sites were re-detected in neutrophils from HAM/TSP patients at different time points, indicating that infected HSCs persist and differentiate in vivo. This study demonstrates that HTLV-1 infects HSCs, and infected stem cells differentiate into diverse cell lineages. These data indicate that infection of HSCs can contribute to the persistence and spread of HTLV-1 in vivo. PMID:29186194

Working in "NK Mode": Natural Killer Group 2 Member D and Natural Cytotoxicity Receptors in Stress-Surveillance by γδ T Cells.

PubMed

Silva-Santos, Bruno; Strid, Jessica

2018-01-01

Natural killer cell receptors (NKRs) are germline-encoded transmembrane proteins that regulate the activation and homeostasis of NK cells as well as other lymphocytes. For γδ T cells, NKRs play critical roles in discriminating stressed (transformed or infected) cells from their healthy counterparts, as proposed in the "lymphoid stress-surveillance" theory. Whereas the main physiologic role is seemingly fulfilled by natural killer group 2 member D, constitutively expressed by γδ T cells, enhancement of their therapeutic potential may rely on natural cytotoxicity receptors (NCRs), like NKp30 or NKp44, that can be induced selectively on human Vδ1 + T cells. Here, we review the contributions of NCRs, NKG2D, and their multiple ligands, to γδ T cell biology in mouse and human.

Single-cell heterogeneity and cell-cycle-related viral gene bursts in the human leukaemia virus HTLV-1

PubMed Central

Billman, Martin R; Rueda, David; Bangham, Charles R M

2017-01-01

Background: The human leukaemia virus HTLV-1 expresses essential accessory genes that manipulate the expression, splicing and transport of viral mRNAs.  Two of these genes, tax and hbz, also promote proliferation of the infected cell, and both genes are thought to contribute to oncogenesis in adult T-cell leukaemia/lymphoma.  The regulation of HTLV-1 proviral latency is not understood.  tax, on the proviral plus strand, is usually silent in freshly-isolated cells, whereas the minus-strand-encoded hbz gene is persistently expressed at a low level.  However, the persistently activated host immune response to Tax indicates frequent expression of tax in vivo.  Methods: We used single-molecule RNA-FISH to quantify the expression of HTLV-1 transcripts at the single-cell level in a total of >19,000 cells from five T-cell clones, naturally infected with HTLV-1, isolated by limiting dilution from peripheral blood of HTLV-1-infected subjects.  Results: We found strong heterogeneity both within and between clones in the expression of the proviral plus-strand (detected by hybridization to the tax gene) and the minus-strand ( hbz gene). Both genes are transcribed in bursts; tax expression is enhanced in the absence of hbz, while hbz expression increased in cells with high tax expression. Surprisingly, we found that hbz expression is strongly associated with the S and G 2/M phases of the cell cycle, independent of tax expression.  Contrary to current belief, hbz is not expressed in all cells at all times, even within one clone.  In hbz-positive cells, the abundance of hbz transcripts showed a very strong positive linear correlation with nuclear volume. Conclusions: The occurrence of intense, intermittent plus-strand gene bursts in independent primary HTLV-1-infected T-cell clones from unrelated individuals strongly suggests that the HTLV-1 plus-strand is expressed in bursts in vivo.  Our results offer an explanation for the paradoxical correlations observed between the host immune response and HTLV-1 transcription. PMID:29062917

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

PubMed Central

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

2015-01-01

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

Elimination of human T cell leukemia virus type-1-infected cells by neutralizing and antibody-dependent cellular cytotoxicity-inducing antibodies against human t cell leukemia virus type-1 envelope gp46.

PubMed

Tanaka, Yuetsu; Takahashi, Yoshiaki; Tanaka, Reiko; Kodama, Akira; Fujii, Hideki; Hasegawa, Atsuhiko; Kannagi, Mari; Ansari, Aftab A; Saito, Mineki

2014-06-01

Human T cell leukemia virus type-1 (HTLV-1) is prevalent worldwide with foci of high prevalence. However, to date no effective vaccine or drug against HTLV-1 infection has been developed. In efforts to define the role of antibodies in the control of HTLV-1 infection, we capitalized on the use of our previously defined anti-gp46 neutralizing monoclonal antibody (mAb) (clone LAT-27) and high titers of human anti-HTLV-1 IgG purified from HAM/TSP patients (HAM-IgG). LAT-27 and HAM-IgG completely blocked syncytium formation and T cell immortalization mediated by HTLV-1 in vitro. The addition of these antibodies to cultures of CD8(+) T cell-depleted peripheral blood mononuclear cells (PBMCs) from HAM/TSP patients at the initiation of culture not only decreased the numbers of Tax-expressing cells and the production of HTLV-1 p24 but also inhibited the spontaneous immortalization of T cells. Coculture of in vitro-HTLV-1-immortalized T cell lines with autologous PBMCs in the presence of LAT-27 or HAM-IgG, but not an F(ab')2 fragment of LAT-27 or nonneutralizing anti-gp46 mAbs, resulted in depletion of HTLV-1-infected cells. A 24-h (51)Cr release assay showed the presence of significant antibody-dependent cellular cytotoxicity (ADCC) activity in LAT-27 and HAM-IgG, but not F(ab')2 of LAT-27, resulting in the depletion of HTLV-1-infected T cells by autologous PBMCs. The depletion of natural killer (NK) cells from the effector PBMCs reduced this ADCC activity. Altogether, the present data demonstrate that the neutralizing and ADCC-inducing activities of anti-HTLV-1 antibodies are capable of reducing infection and eliminating HTLV-1-infected cells in the presence of autologous PBMCs.

Characterization of a human antigen specific helper factor

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

Richardson, B.

1986-03-01

While antigen (Ag) specific helper factors have been characterized in mice, similar molecules have not been identified in humans. To characterize human antigen specific helper molecules, an IL-2 dependent tetanus toxoid (T.T.) reactive T cell line was fused with a 6-thioguanine resistant CEM line, and hybrids selected in medium containing hypoxanthine and azaserine. Hybrids were screened by culturing the cells with /sup 35/S-Met then reacting the supernatants with T.T. or hepatitis vaccine immobilized on nitrocellulose. One hybrid, TT6BA-O, was identified which secreted a Met-containing molecule which bound T.T. but not hepatitis vaccine. Supernatants from TT6BA-O, but not the parent CEMmore » line, when added to autologous peripheral blood mononuclear cells (PBMC's) stimulated secretion of T.T. specific antibodies (Abs). Specificity controls demonstrated that TT6BA-O supernatant did not induce antibodies to diphtheria toxoid, hepatitis vaccine or pneumococcal polysaccharide, and total immunoglobulin (lg) synthesis was minimally increased. In contrast, pokeweed mitogen stimulated significant lg synthesis as well as Ab's to pneumococcal polysaccharide and T.T. TT6BA-O supernatant induced anti-T.T.Ab's in autologous PBMC's but not PBMC's from 3 unrelated donors, suggesting that the activity of the helper factor is restricted, possibly by the MHC. The molecular weight of the helper factor was estimated at 100,000-150,000 by Sephacryl S-300 chromatography. Finally, the helper factor could be demonstrated to bind and elute from sephorose-immobilized T.T. and anti-DR antisera, but not anti-lg antisera or the T40/25 monoclonal antibody, which binds a nonpolymorphic determinant on the human T cell receptor. These results demonstrate that human Ag specific helper factors exist, bind antigen and bear class II MHC determinants.« less

Infection with human herpesvirus type 8 and human T-cell leukaemia virus type 1 among individuals participating in a case–control study in Havana City, Cuba

PubMed Central

Fernandez, L; Serraino, D; Rezza, G; Lence, J; Ortiz, R M; Cruz, T; Vaccarella, S; Sarmati, L; Andreoni, M; Franceschi, S

2002-01-01

Infection with human herpesvirus type 8 and with human T-cell leukaemia virus type-1 shows strong geographic variations. We conducted this study to assess prevalence and risk factors for human herpesvirus type 8 infection in Havana City, Cuba. Information and residual serum samples already collected for a hospital based case–control study were used. A total of 379 individuals (267 males and 112 females; median age=63 years) were evaluated. Antibodies to the lytic antigen of human herpesvirus type 8 were detected by using an immunofluorescence assay, while human T-cell leukaemia virus type-1 serology was performed by means of an ELISA test (alpha Biotech). Overall, 64 subjects (16.9%, 95% confidence interval: 13.1–20.0) were positive for human herpesvirus type 8 antibodies. Human herpesvirus type 8 seroprevalence significantly increased with age (odds ratio=1.9 for ⩾65 vs <55 years), and was twice as frequent in blacks than in whites. No association emerged with gender, socio-economic indicators, family size, history of sexually transmitted disease, sexual behaviour. Overall, 16 persons had anti-human T-cell leukaemia virus type-1 antibodies (4.2%, 95% confidence interval: 2.2–6.4). No relationship emerged between human T-cell leukaemia virus type-1 and human herpesvirus type 8 serostatus. The study findings indicate that human herpesvirus type 8 infection is relatively common in Havana City, Cuba, suggesting that Cuba may represent an intermediate endemical area. Sexual transmission does not seem to play a major role in the spread human herpesvirus type 8 infection. British Journal of Cancer (2002) 87, 1253–1256. doi:10.1038/sj.bjc.6600613 www.bjcancer.com © 2002 Cancer Research UK PMID:12439714

Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma.

PubMed

Johnson, Laura A; Scholler, John; Ohkuri, Takayuki; Kosaka, Akemi; Patel, Prachi R; McGettigan, Shannon E; Nace, Arben K; Dentchev, Tzvete; Thekkat, Pramod; Loew, Andreas; Boesteanu, Alina C; Cogdill, Alexandria P; Chen, Taylor; Fraietta, Joseph A; Kloss, Christopher C; Posey, Avery D; Engels, Boris; Singh, Reshma; Ezell, Tucker; Idamakanti, Neeraja; Ramones, Melissa H; Li, Na; Zhou, Li; Plesa, Gabriela; Seykora, John T; Okada, Hideho; June, Carl H; Brogdon, Jennifer L; Maus, Marcela V

2015-02-18

Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII(+) glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376). Copyright © 2015, American Association for the Advancement of Science.