Mouse pancreatic beta cells express MHC class II and stimulate CD4+ T cells to proliferate

Type 1 diabetes results from destruction of pancreatic beta cells by autoreactive T cells. Both CD4⁺ and CD8⁺ T cells have been shown to mediate beta‐cell killing. While CD8⁺ T cells can directly recognize MHC class I on beta cells, the interaction between CD4⁺ T cells and beta cells remains unclear. Genetic association studies have strongly implicated HLA‐DQ alleles in human type 1 diabetes. Here we studied MHC class II expression on beta cells in nonobese diabetic mice that were induced to develop diabetes by diabetogenic CD4⁺ T cells with T‐cell receptors that recognize beta‐cell antigens. Acute infiltration of CD4⁺ T cells in islets occurred with rapid onset of diabetes. Beta cells from islets with immune infiltration expressed MHC class II mRNA and protein. Exposure of beta cells to IFN‐γ increased MHC class II gene expression, and blocking IFN‐γ signaling in beta cells inhibited MHC class II upregulation. IFN‐γ also increased HLA‐DR expression in human islets. MHC class II⁺ beta cells stimulated the proliferation of beta‐cell‐specific CD4⁺ T cells. Our study indicates that MHC class II molecules may play an important role in beta‐cell interaction with CD4⁺ T cells in the development of type 1 diabetes.     

Analysis of islet inflammation in human type 1 diabetes

The immunopathology of type 1 diabetes (T1D) has proved difficult to study in man because of the limited availability of appropriate samples, but we now report a detailed study charting the evolution of insulitis in human T1D. Pancreas samples removed post‐mortem from 29 patients (mean age 11·7 years) with recent‐onset T1D were analysed by immunohistochemistry. The cell types constituting the inflammatory infiltrate within islets (insulitis) were determined in parallel with islet insulin content. CD8⁺ cytotoxic T cells were the most abundant population during insulitis. Macrophages (CD68⁺) were also present during both early and later insulitis, although in fewer numbers. CD20⁺ cells were present in only small numbers in early insulitis but were recruited to islets as beta cell death progressed. CD138⁺ plasma cells were infrequent at all stages of insulitis. CD4⁺ cells were present in the islet infiltrate in all patients but were less abundant than CD8⁺ or CD68⁺ cells. Forkhead box protein P3⁺ regulatory T cells were detected in the islets of only a single patient. Natural killer cells were detected rarely, even in heavily inflamed islets. The results suggest a defined sequence of immune cell recruitment in human T1D. They imply that both CD8⁺ cytotoxic cells and macrophages may contribute to beta cell death during early insulitis. CD20⁺ cells are recruited in greatest numbers during late insulitis, suggesting an increasing role for these cells as insulitis develops. Natural killer cells and forkhead box protein P3⁺ T cells do not appear to be required for beta cell death.     

Tolerogenic dendritic cells impede priming of naïve CD8+ T cells and deplete memory CD8+ T cells

Type 1 diabetes is a T‐cell‐mediated autoimmune disease in which autoreactive CD8⁺ T cells destroy the insulin‐producing pancreatic beta cells. Vitamin D3 and dexamethasone‐modulated dendritic cells (Combi‐DCs) loaded with islet antigens inducing islet‐specific regulatory CD4⁺ T cells may offer a tissue‐specific intervention therapy. The effect of Combi‐DCs on CD8⁺ T cells, however, remains unknown. To investigate the interaction of CD8⁺ T cells with Combi‐DCs presenting epitopes on HLA class I, naive, and memory CD8⁺ T cells were co‐cultured with DCs and proliferation and function of peptide‐specific T cells were analyzed. Antigen‐loaded Combi‐DCs were unable to prime naïve CD8⁺ T cells to proliferate, although a proportion of T cells converted to a memory phenotype. Moreover, expansion of CD8⁺ T cells that had been primed by mature monocyte‐derived DCs (moDCs) was curtailed by Combi‐DCs in co‐cultures. Combi‐DCs expanded memory T cells once, but CD8⁺ T‐cell numbers collapsed by subsequent re‐stimulation with Combi‐DCs. Our data point that (re)activation of CD8⁺ T cells by antigen‐pulsed Combi‐DCs does not promote, but rather deteriorates, CD8⁺ T‐cell immunity. Yet, Combi‐DCs pulsed with CD8⁺ T‐cell epitopes also act as targets of cytotoxicity, which is undesirable for survival of Combi‐DCs infused into patients in therapeutic immune intervention strategies.     

The good turned ugly: immunopathogenic basis for diabetogenic CD8+ T cells in NOD mice

Summary: Type 1 diabetes (T1D) in both humans and nonobese diabetic (NOD) mice is a T‐cell‐mediated autoimmune disease in which the insulin‐producing pancreatic islet β‐cells are selectively eliminated. As a result, glucose metabolism cannot be regulated unless exogenous insulin is administered. Both the CD4⁺ and the CD8⁺ T‐cell subsets are required for T1D development. Approximately 20 years ago, an association between certain class II major histocompatibility complex (MHC) alleles and susceptibility to T1D was reported. This finding led to enormous interest in the CD4⁺ T cells participating in the development of T1D, while the CD8⁺ subset was relatively ignored. However, the isolation of β‐cell‐autoreactive CD8⁺ T‐cell clones from the islets of NOD mice helped to generate interest in the pathogenic role of this subset, as has accumulating evidence that certain class I MHC alleles are additional risk factors for T1D development in humans. Three distinct diabetogenic CD8⁺ T‐cell populations have now been characterized in NOD mice. Here, we review recent investigations exploring their selection, activation, trafficking, and antigenic specificities. As CD8⁺ T cells are suspected contributors to β‐cell demise in humans, continued exploration of these critical areas could very possibly lead to tangible benefits for T1D patients and at‐risk individuals.     

The role of CD8+ T cells in the initiation of insulin-dependent diabetes mellitus

While it is generally accepted that T cells are critical for the development of diabetes in the non-obese diabetic (NOD) mouse, the precise functions of the CD4⁺ and CD8⁺ subsets remain ill-defined. Transfer experiments have provided evidence that CD4⁺ cells are the disease initiators, provoking massive mononuclear leukocyte infiltration into the pancreatic islets, while CD8⁺ cells play an effector role, responsible for the final destruction of islet beta cells. It was surprising, then, to find that NOD mice carrying a null mutation at the β2-microglobulin (β2-μ) locus, and thereby lacking major histocompatibility complex class I molecules and CD8⁺ T cells, developed neither insulitis nor diabetes. Here, we argue that the absence of insulitis in these animals results from their lack of CD8⁺ cells because islet infiltration is also absent when NOD mice are treated with an anti-CD8 monoclonal antibody (mAb) at a young age. Interestingly, the anti-CD8 effect is only observed when the mAb is injected during a discrete age window – 2 to 5 weeks after birth. Transfer experiments indicate that the lack of CD8⁺ cells during this period somehow alters the phenotype of CD4⁺ cells, preventing them from expressing their insulitic potential. This is not because they are generally immuno-incompetent nor because they are generally more prone to differentiating into cells with Th2 characteristics. Given that neither the β2-μ mutation nor anti-CD8 treatment affect insulitis in a T cell receptor transgenic (tg) mouse strain with a CD4⁺ T cell repertoire highly skewed for an anti-islet cell reactivity, the most straight-forward interpretation of these observations is that CD8⁺ cells are required for effective priming and expansion of autoreactive CD4⁺ cells.     

Major histocompatibility complex class I molecules are required for the development of insulitis in non-obese diabetic mice

An early step in the development of autoimmune diabetes is lymphocyte infiltration into the islets of Langerhans of the pancreas, or insulitis. The infiltrate contains both CD4⁺ and CD8⁺ T cells and both are required for progression to diabetes in non-obese diabetic (NOD) mice. It has been thought that the CD4⁺ lymphocytes are the initiators of the disease, the islet invaders, while CD8⁺ cells are the effectors, the islet destroyers. We question this interpretation because NOD mice lacking MHC class I molecules, hence CD8⁺ T cells, do not display even insulitis when expected.     

Effect of standard tuberculosis treatment on naive,memory and regulatory T‐cell homeostasis in tuberculosis–diabetes co‐morbidity

Perturbations in CD4⁺ and CD8⁺ T‐cell phenotype and function are hallmarks of tuberculosis–diabetes co‐morbidity. However, their contribution to the pathogenesis of this co‐morbidity and the effect of anti‐tuberculosis treatment on the phenotype of the T‐cell subsets is poorly understood. In this study, we examined the frequency of different T‐cell subsets in individuals with pulmonary tuberculosis (PTB) with diabetes mellitus (DM) or without coincident diabetes mellitus (NDM) before, during and after completion of anti‐tuberculosis chemotherapy. PTB‐DM is characterized by heightened frequencies of central memory CD4⁺ and CD8⁺ T cells and diminished frequencies of naive, effector memory and/or effector CD4⁺ and CD8⁺ T cells at baseline and after 2 months of treatment but not following treatment completion in comparison with PTB‐NDM. Central memory CD4⁺ and CD8⁺ T‐cell frequencies exhibited a positive correlation with fasting blood glucose and glycated haemoglobin A1c levels, whereas the frequencies of naive and effector memory or effector CD4⁺ and CD8⁺ T cells exhibited a negative correlation. However, the frequencies of CD4⁺ and CD8⁺ T‐cell subsets in individuals with PTB exhibited no significant relationship with bacterial burdens. Finally, although minor alterations in the T‐cell subset compartment were observed at 2 months of treatment, significantly decreased frequencies of central memory and significantly enhanced frequencies of naive CD4⁺ and CD8⁺ T cells were observed at the completion of treatment. Our data reveal a profound effect of coexistent diabetes on the altered frequencies of central memory, effector memory and naive T cells and its normalization following therapy.     

Detection of vasostatin‐1‐specific CD8+ T cells in non‐obese diabetic mice that contribute to diabetes pathogenesis

Chromogranin A (ChgA) is an antigenic target of pathogenic CD4⁺ T cells in a non‐obese diabetic (NOD) mouse model of type 1 diabetes (T1D). Vasostatin‐1 is a naturally processed fragment of ChgA. We have now identified a novel H2‐K^d‐restricted epitope of vasostatin‐1, ChgA 36‐44, which elicits CD8⁺ T cell responses in NOD mice. By using ChgA 36‐44/K^d tetramers we have determined the frequency of vasostatin‐1‐specific CD8⁺ T cells in pancreatic islets and draining lymph nodes of NOD mice. We also demonstrate that vasostatin‐1‐specific CD4⁺ and CD8⁺ T cells constitute a significant fraction of islet‐infiltrating T cells in diabetic NOD mice. Adoptive transfer of T cells from ChgA 36‐44 peptide‐primed NOD mice into NOD/severe combined immunodeficiency (SCID) mice led to T1D development. These findings indicate that vasostatin‐1‐specific CD8⁺ T cells contribute to the pathogenesis of type 1 diabetes in NOD mice.     

Analysis of the functional WT1‐specific T‐cell repertoire in healthy donors reveals a discrepancy between CD4+ and CD8+ memory formation

The Wilms’ tumour‐1 (WT1) protein is considered a prime target for cancer immunotherapy based on its presumptive immunogenicity and widespread expression across a variety of malignancies. However, little is known about the naturally occurring WT1‐specific T‐cell repertoire because self‐derived antigens typically elicit low frequency responses that challenge the sensitivity limits of current detection techniques. In this study, we used highly efficient cell enrichment procedures based on CD137, CD154, and pHLA class I tetramer staining to conduct a detailed analysis of WT1‐specific T cells from the peripheral blood. Remarkably, we detected WT1‐specific CD4⁺ and CD8⁺ T‐cell populations in the vast majority of healthy individuals. Memory responses specific for WT1 were commonly present in the CD4⁺ T‐cell compartment, whereas WT1‐specific CD8⁺ T cells almost universally displayed a naive phenotype. Moreover, memory CD4⁺ and naive CD8⁺ T cells with specificity for WT1 were found to coexist in some individuals. Collectively, these findings suggest a natural discrepancy between the CD4⁺ and CD8⁺ T‐cell lineages with respect to memory formation in response to a self‐derived antigen. Nonetheless, WT1‐specific T cells from both lineages were readily activated ex vivo and expanded in vitro, supporting the use of strategies designed to exploit this expansive reservoir of self‐reactive T cells for immunotherapeutic purposes.     

Influenza infection results in local expansion of memory CD8+ T cells with antigen non‐specific phenotype and function

Primary viral infections induce activation of CD8⁺ T cells responsible for effective resistance. We sought to characterize the nature of the CD8⁺ T cell expansion observed after primary viral infection with influenza. Infection of naive mice with different strains of influenza resulted in the rapid expansion of memory CD8⁺ T cells exhibiting a unique bystander phenotype with significant up‐regulation of natural killer group 2D (NKG2D), but not CD25, on the CD44^highCD8⁺ T cells, suggesting an antigen non‐specific phenotype. We further confirmed the non‐specificity of this phenotype on ovalbumin‐specific (OT‐I) CD8⁺ T cells, which are not specific to influenza. These non‐specific CD8⁺ T cells also displayed increased lytic capabilities and were observed primarily in the lung. Thus, influenza infection was shown to induce a rapid, antigen non‐specific memory T cell expansion which is restricted to the specific site of inflammation. In contrast, CD8⁺ T cells of a similar phenotype could be observed in other organs following administration of systemic agonistic anti‐CD40 and interleukin‐2 immunotherapy, demonstrating that bystander expansion in multiple sites is possible depending on whether the nature of activation is either acute or systemic. Finally, intranasal blockade of NKG2D resulted in a significant increase in viral replication early during the course of infection, suggesting that NKG2D is a critical mediator of anti‐influenza responses prior to the initiation of adaptive immunity. These results characterize further the local bystander expansion of tissue‐resident, memory CD8⁺ T cells which, due to their early induction, may play an important NKG2D‐mediated, antigen non‐specific role during the early stages of viral infection.     

β‐cell‐specific IL‐35 therapy suppresses ongoing autoimmune diabetes in NOD mice

IL‐35 is a recently identified cytokine exhibiting potent immunosuppressive properties. The therapeutic potential and effects of IL‐35 on pathogenic T effector cells (Teff) and Foxp3⁺ Treg, however, are ill defined. We tested the capacity of IL‐35 to suppress ongoing autoimmunity in NOD mice. For this purpose, an adeno‐associated virus vector in which IL‐35 transgene expression is selectively targeted to β cells via an insulin promoter (AAV8mIP‐IL35) was used. AAV8mIP‐IL35 vaccination of NOD mice at a late preclinical stage of type 1 diabetes (T1D) suppressed β‐cell autoimmunity and prevented diabetes onset. Numbers of islet‐resident conventional CD4⁺ and CD8⁺ T cells, and DCs were reduced within 4 weeks of AAV8mIP‐IL35 treatment. The diminished islet T‐cell pool correlated with suppressed proliferation, and a decreased frequency of IFN‐γ‐expressing Teff. Ectopic IL‐35 also reduced islet Foxp3⁺ Treg numbers and proliferation, and protection was independent of induction/expansion of adaptive islet immunoregulatory T cells. These findings demonstrate that IL‐35‐mediated suppression is sufficiently robust to block established β‐cell autoimmunity, and support the use of IL‐35 to treat T1D and other T‐cell‐mediated autoimmune diseases.     

Partial and transient modulation of the CD3–T‐cell receptor complex,elicited by low‐dose regimens of monoclonal anti‐CD3, is sufficient to induce disease remission in non‐obese diabetic mice

It has been established that a total of 250 μg of monoclonal anti‐mouse CD3 F(ab′)₂ fragments, administered daily (50 μg per dose), induces remission of diabetes in the non‐obese diabetic (NOD) mouse model of autoimmune diabetes by preventing β cells from undergoing further autoimmune attack. We evaluated lower‐dose regimens of monoclonal anti‐CD3 F(ab′)₂ in diabetic NOD mice for their efficacy and associated pharmacodynamic (PD) effects, including CD3–T‐cell receptor (TCR) complex modulation, complete blood counts and proportions of circulating CD4⁺, CD8⁺ and CD4⁺ FoxP3⁺ T cells. Four doses of 2 μg (total dose 8 μg) induced 53% remission of diabetes, similarly to the 250 μg dose regimen, whereas four doses of 1 μg induced only 16% remission. While the 250 μg dose regimen produced nearly complete and sustained modulation of the CD3 –TCR complex, lower doses, spaced 3 days apart, which induced similar remission rates, elicited patterns of transient and partial modulation. In treated mice, the proportions of circulating CD4⁺ and CD8⁺ T cells decreased, whereas the proportions of CD4⁺ FoxP3⁺ T cells increased; these effects were transient. Mice with greater residual β‐cell function, estimated using blood glucose and C‐peptide levels at the initiation of treatment, were more likely to enter remission than mice with more advanced disease. Thus, lower doses of monoclonal anti‐CD3 that produced only partial and transient modulation of the CD3–TCR complex induced remission rates comparable to higher doses of monoclonal anti‐CD3. Accordingly, in a clinical setting, lower‐dose regimens may be efficacious and may also improve the safety profile of therapy with monoclonal anti‐CD3, potentially including reductions in cytokine release‐related syndromes and maintenance of pathogen‐specific immunosurveillance during treatment.     

Antigens expressed by myelinating glia cells induce peripheral cross‐tolerance of endogenous CD8+ T cells

Auto‐reactivity of T cells is largely prevented by central and peripheral tolerance. Nevertheless, immunization with certain self‐antigens emulsified in CFA induces autoimmunity in rodents, suggesting that tolerance to some self‐antigens is not robust. To investigate the fate of nervous system‐specific CD8⁺ T cells, which only recently came up as being important contributors for MS pathogenesis, we developed a mouse model that allows inducible expression of lymphocytic choriomeningitis virus‐derived CD8⁺ T‐cell epitopes specifically in oligodendrocytes and Schwann cells, the myelinating glia of the nervous system. These transgenic CD8⁺ T‐cell epitopes induced robust tolerance of endogenous auto‐reactive T cells, which proved thymus‐independent and was mediated by cross‐presenting bone‐marrow‐derived cells. Immunohistological staining of secondary lymphoid organs demonstrated the presence of glia‐derived antigens in DC, suggesting that peripheral tolerance of CD8⁺ T cells results from uptake and presentation by steady state DC.     

Thymic emigration patterns in patients with type 2 diabetes treated with metformin

Recent data suggest that thymic output, which provides the naive T cells necessary for the normal functioning of T‐cell‐dependent immunosurveillance cellular immunity including anti‐cancer protection, can be disturbed in the course of type 2 diabetes. Metformin, an anti‐diabetic drug commonly confirmed as an agent with many potential anti‐cancer activities, might be helpful in this immune correction. The profile of thymic output was evaluated in the current study on the basis of the signal‐joint T‐cell receptor excision circle (sjTREC) concentration in peripheral blood polymorphonuclear cells and thymic emigrant content in peripheral blood evaluated from CD127 and/or CD132 antigen expression. It was revealed that recent thymic emigrants and more differentiated CD127⁺ CD132⁺ cell populations were decreased among naive T cells and CD8⁺ T cells, whereas RTE count was increased in CD4⁺ T cells, and the CD127⁺ CD132⁺ cell population was less numerous than in non‐diabetic participants. Terminally differentiated thymic emigrants, i.e. CD127^? CD132⁺ cells, were increased in naive T cells and in CD8⁺ T cells. Metformin affects mainly the early phases of thymic export, increasing CD127⁺ CD132^? and CD127⁺ CD132⁺ cell populations in naive T cells and the CD127⁺ CD132^? population in CD4⁺ T lymphocytes. It could be concluded that type 2 diabetes deteriorates thymic immunostasis. The decreased thymic output could be compensated by metformin, especially with regard to CD4⁺ naive T cells. It is the first time that therapy with metformin has been documented by us as particularly useful in the control and normalization of thymus function, regarding correction of early populations of thymic emigrants.     

Carbon monoxide‐treated dendritic cells decrease β1‐integrin induction on CD8+ T cells and protect from type 1 diabetes

Carbon monoxide (CO) treatment improves pathogenic outcome of autoimmune diseases by promoting tolerance. However, the mechanism behind this protective tolerance is not yet defined. Here, we show in a transgenic mouse model for autoimmune diabetes that ex vivo gaseous CO (gCO)‐treated DCs loaded with pancreatic β‐cell peptides protect mice from disease. This protection is peptide‐restricted, independent of IL‐10 secretion by DCs and of CD4⁺ T cells. Although no differences were observed in autoreactive CD8⁺ T‐cell function from gCO‐treated versus untreated DC‐immunized groups, gCO‐treated DCs strongly inhibited accumulation of autoreactive CD8⁺ T cells in the pancreas. Interestingly, induction of β1‐integrin was curtailed when CD8⁺ T cells were primed with gCO‐treated DCs, and the capacity of these CD8⁺ T cells to lyse isolated islet was dramatically impaired. Thus, immunotherapy using CO‐treated DCs appears to be an original strategy to control autoimmune disease.     

The threshold for autoimmune T cell killing is influenced by B7-1

The concept that naive CD4⁺ and CD8⁺ T cells require co-stimulatory signals for activation and proliferation is well documented. Less clear is the need for co-stimulation during the effector phase of the T cell response. Here we examined the influence of B7-1 (CD80) during the effector phase of an autoimmune response to pancreatic islets using transgenic mouse lines which expressed B7-1 in either all or only some of their β cells ( “confluent” or “patchy” RIP-B7-1 mice). Transgenic expression of B7-1 in normal mouse islets that co-expressed the pro-inflammatory cytokine, IL-2, resulted in early spontaneous autoimmunity. Islets with IL-2 and “confluent” B7-1 expression were destroyed whereas islets with IL-2 and “patchy” B7-1 expression showed selective killing of the B7-1⁺ β cells. Islet-reactive T cells, circulating in the RIP-B7-1/IL-2 mice, rejected syngeneic islet grafts, but only if these expressed B7-1. Introduction of the B7-1 transgene into the nonobese diabetic (NOD) genetic background likewise resulted in early spontaneous autoimmunity, but splenocytes from the diabetic animals could only transfer diabetes to NOD scid recipients that expressed B7-1 on their β cells. In both these transgenic models, therefore, islet destruction required continuous B7-1 expression by target β cells. Thus, although the normal repertoire contains T cells with potential islet reactivity, these T cells remain harmless because parenchymal cells like the β cell cannot normally express B7-1. Our results also have implications for tumor immunotherapy in that the ability of T cells to kill poorly immunogenic targets may be dependent upon B7-1 expression by the target cell itself.     

Selective antigen‐specific CD4+ T‐cell,but not CD8+ T‐ or B‐cell,tolerance corrupts cancer immunotherapy

Self‐tolerance, presumably through lineage‐unbiased elimination of self‐antigen‐specific lymphocytes (CD4⁺ T, CD8⁺ T, and B cells), creates a formidable barrier to cancer immunotherapy. In contrast to this prevailing paradigm, we demonstrate that for some antigens, self‐tolerance reflects selective elimination of antigen‐specific CD4⁺ T cells, but preservation of CD8⁺ T‐ and B‐cell populations. In mice, antigen‐specific CD4⁺ T‐cell tolerance restricted CD8⁺ T‐ and B‐cell responses targeting the endogenous self‐antigen guanylyl cyclase c (GUCY2C) in colorectal cancer. Although selective CD4⁺ T‐cell tolerance blocked GUCY2C‐specific antitumor immunity and memory responses, it offered a unique solution to the inefficacy of GUCY2C vaccines through recruitment of self‐antigen‐independent CD4⁺ T‐cell help. Incorporating CD4⁺ T‐cell epitopes from foreign antigens into vaccines against GUCY2C reconstituted CD4⁺ T‐cell help, revealing the latent functional capacity of GUCY2C‐specific CD8⁺ T‐ and B‐cell pools, producing durable antitumor immunity without autoimmunity. Incorporating CD4⁺ T‐cell epitopes from foreign antigens into vaccines targeting self‐antigens in melanoma (Trp2) and breast cancer (Her2) produced similar results, suggesting selective CD4⁺ T‐cell tolerance underlies ineffective vaccination against many cancer antigens. Thus, identification of self‐antigens characterized by selective CD4⁺ T‐cell tolerance and abrogation of such tolerance through self‐antigen‐independent T‐cell help is essential for future immunotherapeutics.     

Bystander stimulation of activated CD4+ T cells of unrelated specificity following a booster vaccination with tetanus toxoid

Antigen‐specific CD4⁺ T cells are central to natural and vaccine‐induced immunity. An ongoing antigen‐specific T‐cell response can, however, influence surrounding T cells with unrelated antigen specificities. We previously observed this bystander effect in healthy human subjects following recall vaccination with tetanus toxoid (TT). Since this interplay could be important for maintenance of memory, we have moved to a mouse model for further analysis. We investigated whether boosting memory CD4⁺ T cells against TT in vivo would influence injected CD4⁺ TCR transgenic T cells (OT‐II) specific for an unrelated OVA peptide. If OT‐II cells were pre‐activated with OVA peptide in vitro, these cells showed a bystander proliferative response during the ongoing parallel TT‐specific response. Bystander proliferation was dependent on boosting of the TT‐specific memory response in the recipients, with no effect in naive mice. Bystander stimulation was also proportional to the strength of the TT‐specific memory T‐cell response. T cells activated in vitro displayed functional receptors for IL‐2 and IL‐7, suggesting these as potential mediators. This crosstalk between a stimulated CD4⁺ memory T‐cell response and CD4⁺ T cells activated by an unrelated antigen could be important in human subjects continually buffeted by environmental antigens.     

CD4+ T cells recognize diverse epitopes within GAD65: implications for repertoire development and diabetes monitoring

Type 1 diabetes is associated with T‐cell responses to β‐cell antigens such as GAD65. Single T‐cell epitopes have been investigated for immune monitoring with some success, but multiple epitopes may be required to fully characterize responses in all subjects. We used a systematic approach to examine the diversity of the GAD65‐specific T‐cell repertoire in subjects with DRB1*04:01 haplotypes. Using class II tetramers, we observed responses to 15 GAD65 epitopes, including five novel epitopes. The majority were confirmed to be processed and presented. Upon stimulation with peptides, GAD‐specific responses were equally broad in subjects with diabetes and healthy controls in the presence or absence of CD25⁺ T cells, suggesting that a susceptible HLA is sufficient to generate a potentially autoreactive repertoire. Without depleting CD25⁺ cells, GAD_113–132 and GAD_265–284 responses were significantly stronger in subjects with diabetes. Although nearly every individual responded to at least one GAD65 epitope, most were seen in less than half of the subjects tested, suggesting that multiple epitopes are recommended for immune monitoring.