Early autoimmune destruction of islet grafts is associated with a restricted repertoire of IGRP-specific CD8+ T cells in diabetic nonobese diabetic mice

beta cell replacement via islet or pancreas transplantation is currently the only approach to cure type 1 diabetic patients. Recurrent beta cell autoimmunity is a critical factor contributing to graft rejection along with alloreactivity. However, the specificity and dynamics of recurrent beta cell autoimmunity remain largely undefined. Accordingly, we compared the repertoire of CD8+ T cells infiltrating grafted and endogenous islets in diabetic nonobese diabetic mice. In endogenous islets, CD8+ T cells specific for an islet-specific glucose-6-phosphatase catalytic subunit-related protein derived peptide (IGRP206-214) were the most prevalent T cells. Similar CD8+ T cells dominated the early graft infiltrate but were expanded 6-fold relative to endogenous islets. Single-cell analysis of the TCR alpha and beta chains showed restricted variable gene usage by IGRP206-214-specific CD8+ T cells that was shared between the graft and endogenous islets of individual mice. However, as islet graft infiltration progressed, the number of IGRP206-214-specific CD8+ T cells decreased despite stable numbers of CD8+ T cells. These results demonstrate that recurrent beta cell autoimmunity is characterized by recruitment to the grafts and expansion of already prevalent autoimmune T cell clonotypes residing in the endogenous islets. Furthermore, depletion of IGRP206-214-specific CD8+ T cells by peptide administration delayed islet graft survival, suggesting IGRP206-214-specific CD8+ T cells play a role early in islet graft rejection but are displaced with time by other specificities, perhaps by epitope spread.     

Autoantigen Recognition Is Required for Recruitment of IGRP206-214-Autoreactive CD8+ T Cells but Is Dispensable for Tolerance

The progression of autoimmune responses is associated with an avidity maturation process driven by preferential expansion of high avidity clonotypes at the expense of their low avidity counterparts. Central and peripheral tolerance hinder the contribution of high-avidity clonotypes targeting residues 206-214 of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP(206-214)) during the earliest stages of autoimmune diabetes. In this study, we probe the molecular determinants and biochemical consequences of IGRP(206-214)/K(d) recognition by high-, intermediate-, and low-avidity autoreactive CD8(+) T cells, and we investigate the effects of genetic IGRP(206-214) silencing on their developmental biology. We find that differences in avidity for IGRP(206-214)/K(d) map to CDR1α and are associated with quantitative differences in CD3ε proline-rich sequence exposure and Nck recruitment. Unexpectedly, we find that tolerance of high-avidity CD8(+) T cells, unlike their activation and recruitment into the pancreas, is dissociated from recognition of IGRP(206-214), particularly in adult mice. This finding challenges the view that tolerance of pathogenic autoreactive T cells is invariably triggered by recognition of the peptide-MHC complex that drives their activation in the periphery, indicating the existence of mechanisms of tolerance that are capable of sensing the avidity, hence pathogenicity of autoreactive T cells without the need to rely on local autoantigen availability.     

Autoimmunity to both proinsulin and IGRP is required for diabetes in nonobese diabetic 8.3 TCR transgenic mice

T cells specific for proinsulin and islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) induce diabetes in nonobese diabetic (NOD) mice. TCR transgenic mice with CD8(+) T cells specific for IGRP(206-214) (NOD8.3 mice) develop accelerated diabetes that requires CD4(+) T cell help. We previously showed that immune responses against proinsulin are necessary for IGRP(206-214)-specific CD8(+) T cells to expand. In this study, we show that diabetes development is dramatically reduced in NOD8.3 mice crossed to NOD mice tolerant to proinsulin (NOD-PI mice). This indicates that immunity to proinsulin is even required in the great majority of NOD8.3 mice that have a pre-existing repertoire of IGRP(206-214)-specific cells. However, protection from diabetes could be overcome by inducing islet inflammation either by a single dose of streptozotocin or anti-CD40 agonist Ab treatment. This suggests that islet inflammation can substitute for proinsulin-specific CD4(+) T cell help to activate IGRP(206-214)-specific T cells.     

T cells recognizing a peptide contaminant undetectable by mass spectrometry

Synthetic peptides are widely used in immunological research as epitopes to stimulate their cognate T cells. These preparations are never completely pure, but trace contaminants are commonly revealed by mass spectrometry quality controls. In an effort to characterize novel major histocompatibility complex (MHC) Class I-restricted β-cell epitopes in non-obese diabetic (NOD) mice, we identified islet-infiltrating CD8+ T cells recognizing a contaminating peptide. The amount of this contaminant was so small to be undetectable by direct mass spectrometry. Only after concentration by liquid chromatography, we observed a mass peak corresponding to an immunodominant islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)(206-214) epitope described in the literature. Generation of CD8+ T-cell clones recognizing IGRP(206-214) using a novel method confirmed the identity of the contaminant, further underlining the immunodominance of IGRP(206-214). If left undetected, minute impurities in synthetic peptide preparations may thus give spurious results.     

On how monospecific memory-like autoregulatory CD8+ T cells can blunt diabetogenic autoimmunity: a computational approach

We have recently shown that during progression to autoimmune diabetes in NOD mice, memory autoreactive regulatory CD8(+) T cells arising from low-avidity precursors can be expanded to therapeutic levels using nanoparticles coated with disease-relevant peptide-major histocompatibility complexes (pMHCs). Here we examine the dynamics of memory autoregulatory CD8(+) T cells specific for islet-specific glucose-6-phosphatase catalytic subunit-related protein(206-214), a prevalent β cell autoantigen; their high-avidity counterparts (dominant effectors); and all other autoreactive non-islet-specific glucose-6-phosphatase catalytic subunit-related protein(206-214)-specific CD8(+) T cell specificities (subdominant effectors) in response to pMHC-coated nanoparticle (pMHC-nanoparticle) therapy. We combine experimental data with mathematical modeling to investigate the clonal competition dynamics of these T cell pools. To mimic the response diversity observed in NOD mice, we simulated many individual mice, using a wide range of parameters, and averaged the results as done experimentally. We find that under certain circumstances, pMHC-nanoparticle-induced expansion of autoregulatory CD8(+) T cells can effectively suppress the expansion of dominant and subdominant effectors simultaneously but, in some few cases, can lead to the substitution (or switching) of one effector population by another. The model supports the idea that disease suppression is based on the elimination of autoantigen-loaded APCs by the expanded autoregulatory CD8(+) T cells. The model also predicts that treatment strategies that operate by selectively inhibiting autoantigen-loaded APCs, such as the pMHC-nanoparticle approach, have the highest promise to blunt polyclonal, multiantigen-specific autoimmune responses in vivo without impairing systemic immunity.     

Low-avidity self-specific T cells display a pronounced expansion defect that can be overcome by altered peptide ligands

Thymic expression of self-Ags results in the deletion of high-avidity self-specific T cells, but, at least for certain Ags, a residual population of self-specific T cells with low-affinity TCRs remains after negative selection. Such self-specific T cells are thought to play a role in the induction of T cell-mediated autoimmunity, but may also be used for the induction of antitumor immunity against self-Ags. In this study, we examine the functional competence of a polyclonal population of self-specific CD8+ T cells. We show that low-affinity interactions between TCR and peptide are associated with selective loss of critical T cell functions. Triggering of low levels of IFN-gamma production and cytolytic activity through low-affinity TCRs readily occurs provided high Ag doses are used, but IL-2 production and clonal expansion are severely reduced at all Ag doses. Remarkably, a single peptide variant can form an improved ligand for the highly diverse population of low-avidity self-specific T cells and can improve their proliferative capacity. These data provide insight into the inherent limitations of self-specific T cell responses through low-avidity TCR signals and the effect of modified peptide ligands on self-specific T cell immunity.     

Influence of CD4+CD25+ regulatory T cells on low/high-avidity CD4+ T cells following peptide vaccination

We have recently reported that NY-ESO-1-specific naive CD4+ T cell precursors exist in most individuals but are suppressed by CD4+CD25+ regulatory T cells (Tregs), while memory CD4+ T cell effectors against NY-ESO-1 are found only in cancer patients with spontaneous Ab responses to NY-ESO-1. In this study, we have analyzed mechanisms of CD4+ T cell induction following peptide vaccination in relation to susceptibility to Tregs. Specific HLA-DP4-restricted CD4+ T cell responses were elicited after vaccination with NY-ESO-1(157-170) peptide (emulsified in IFA) in patients with NY-ESO-1-expressing epithelial ovarian cancer. These vaccine-induced CD4+ T cells were detectable from effector/memory populations without requirement for in vitro CD4+CD25+ T cell depletion. However, they were only able to recognize NY-ESO-1(157-170) peptide but not naturally processed NY-ESO-1 protein and had much lower avidity compared with NY-ESO-1-specific pre-existing naive CD4+CD25- T cell precursors or spontaneously induced CD4+ T cell effectors of cancer patients with NY-ESO-1 Ab. We propose that vaccination with NY-ESO-1(157-170) peptide recruits low-avidity T cells with low sensitivity to Tregs and fails to modulate the suppressive effect of Tregs on high-avidity NY-ESO-1-specific T cell precursors.     

Very low-dose tolerance with nucleosomal peptides controls lupus and induces potent regulatory T cell subsets

We induced very low-dose tolerance by injecting lupus prone (SWR x NZB)F1 (SNF1) mice with 1 mug nucleosomal histone peptide autoepitopes s.c. every 2 wk. The subnanomolar peptide therapy diminished autoantibody levels and prolonged life span by delaying nephritis, especially by reducing inflammatory cell reaction and infiltration in kidneys. H4(71-94) was the most effective autoepitope. Low-dose tolerance therapy induced CD8+, as well as CD4+ CD25+ regulatory T (Treg) cell subsets containing autoantigen-specific cells. These adaptive Treg cells suppressed IFN-gamma responses of pathogenic lupus T cells to nucleosomal epitopes at up to a 1:100 ratio and reduced autoantibody production up to 90-100% by inhibiting nucleosome-stimulated T cell help to nuclear autoantigen-specific B cells. Both CD4+ CD25+ and CD8+ Treg cells produced and required TGF-beta1 for immunosuppression, and were effective in suppressing lupus autoimmunity upon adoptive transfer in vivo. The CD4+ CD25+ T cells were partially cell contact dependent, but CD8+ T cells were contact independent. Thus, low-dose tolerance with highly conserved histone autoepitopes repairs a regulatory defect in systemic lupus erythematosus by generating long-lasting, TGF-beta-producing Treg cells, without causing allergic/anaphylactic reactions or generalized immunosuppression.     

Quantification of repertoire diversity of influenza-specific epitopes with predominant public or private TCR usage

The H-2Db-restricted CD8 T cell immune response to influenza A is directed at two well-described epitopes, nucleoprotein 366 (NP366) and acid polymerase 224 (PA224). The responses to the two epitopes are very different. The epitope NP366-specific response is dominated by TCR clonotypes that are public (shared by most mice), whereas the epitope PA224-specific response is private (unique within each infected animal). In addition to being public, the NP366-specific response is dominated by a few clonotypes, when T cell clonotypes expressing the Vbeta8.3 element are analyzed. Herein, we show that this response is similarly public when the NP366+Vbeta4+ CD8 T cell response is analyzed. Furthermore, to determine whether these features resulted in differences in total TCR diversity in the NP366+ and PA224+ responses, we quantified the number of different CD8 T clonotypes responding to each epitope. We calculated that 50-550 clonotypes recognized each epitope in individual mice. Thus, although the character of the response to the two epitopes appeared to be different (private and diverse vs public and dominated by a few clonotypes), similar numbers of precursor cells responded to both epitopes and this number was of similar magnitude to that previously reported for other viral CD8 T cell epitopes. Therefore, even in CD8 T cell responses that appear to be oligoclonotypic, the total response is highly diverse.     

A novel approach to the analysis of specificity, clonality, and frequency of HIV-specific T cell responses reveals a potential mechanism for control of viral escape

Escape from the CD8(+) T cell response through epitope mutations can lead to loss of immune control of HIV replication. Theoretically, escape from CD8(+) T cell recognition is less likely when multiple TCRs target individual MHC/peptide complexes, thereby increasing the chance that amino acid changes in the epitope could be tolerated. We studied the CD8(+) T cell response to six immunodominant epitopes in five HIV-infected subjects using a novel approach combining peptide stimulation, cell surface cytokine capture, flow cytometric sorting, anchored RT-PCR, and real-time quantitative clonotypic TCR tracking. We found marked variability in the number of clonotypes targeting individual epitopes. One subject recognized a single epitope with six clonotypes, most of which were able to recognize and lyse cells expressing a major epitope variant that arose. Additionally, multiple clonotypes remained expanded during the course of infection, irrespective of epitope variant frequency. Thus, CD8(+) T cells comprising multiple TCR clonotypes may expand in vivo in response to individual epitopes, and may increase the ability of the response to recognize virus escape mutants.     

Islet-specific glucose-6-phosphatase catalytic subunit-related protein-reactive CD4+ T cells in human subjects

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is recognized as a major autoantigen for autoimmune type 1 diabetes (T1D) in the NOD mouse model. This study was undertaken to examine CD4+ T cell responses toward IGRP in human subjects. The tetramer-guided epitope mapping approach was used to identify IGRP-specific CD4+ T cell epitopes. IGRP(23-35) and IGRP(247-259) were identified as DRA1*0101/DRB1*0401-restricted epitopes. IGRP(13-25) and IGRP(226-238) were identified as DRA1*0101/DRB1*0301-restricted epitopes. IGRP-specific tetramers were used to evaluate the prevalence of IGRP-reactive T cells in healthy and T1D subjects. More than 80% of subjects with either DRB1*0401 or DRB1*0301 haplotype have IGRP-specific CD4+ T cell responses for at least one IGRP epitope. IGRP-specific T cells from both healthy and T1D groups produce both gamma-IFN and IL-10. DRA1*0101/DRB1*0401 IGRP(247-259)-restricted T cells also show cross-reactivity to an epitope derived from liver/kidney glucose-6-phosphatase. The detection of IGRP-reactive T cells in both type 1 diabetic subjects and healthy subjects and recent reports of other autoreactive T cells detected in healthy subjects underscore the prevalence of potentially autoreactive T cells in the peripheral immune system of the general population.     

Systemic administration of agonist peptide blocks the progression of spontaneous CD8-mediated autoimmune diabetes in transgenic mice without bystander damage

Insulin-dependent diabetes is an autoimmune disease targeting pancreatic beta-islet cells. Recent data suggest that autoreactive CD8+ T cells are involved in both the early events leading to insulitis and the late destructive phase resulting in diabetes. Although therapeutic injection of protein and synthetic peptides corresponding to CD4+ T cell epitopes has been shown to prevent or block autoimmune disease in several models, down-regulation of an ongoing CD8+ T cell-mediated autoimmune response using this approach has not yet been reported. Using CL4-TCR single transgenic mice, in which most CD8+ T cells express a TCR specific for the influenza virus hemagglutinin HA512-520 peptide:Kd complex, we first show that i.v. injection of soluble HA512-520 peptide induces transient activation followed by apoptosis of Tc1-like CD8+ T cells. We next tested a similar tolerance induction strategy in (CL4-TCR x Ins-HA)F1 double transgenic mice that also express HA in the beta-islet cells and, as a result, spontaneously develop a juvenile onset and lethal diabetes. Soluble HA512-520 peptide treatment, at a time when pathogenic CD8+ T cells have already infiltrated the pancreas, very significantly prolongs survival of the double transgenic pups. In addition, we found that Ag administration eliminates CD8+ T cell infiltrates from the pancreas without histological evidence of bystander damage. Our data indicate that agonist peptide can down-regulate an autoimmune reaction mediated by CD8+ T cells in vivo and block disease progression. Thus, in addition to autoreactive CD4+ T cells, CD8+ T cells may constitute targets for Ag-specific therapy in autoimmune diseases.     

The proline-rich sequence of CD3epsilon as an amplifier of low-avidity TCR signaling

Engagement of peptide-MHC by the TCR induces a conformational change in CD3epsilon that exposes a proline-rich sequence (PRS) and recruits the cytoskeletal adaptor Nck. This event, which precedes phosphorylation of the CD3epsilon ITAM, has been implicated in synapse formation and T cell function. However, there is compelling evidence that responsiveness to TCR ligation is CD3epsilon PRS independent. In this study, we show that the CD3epsilon PRS is necessary for peptide-MHC-induced phosphorylation of CD3epsilon and for recruitment of protein kinase Ctheta to the immune synapse in differentiated CD8+ T lymphocytes. However, whereas these two events are dispensable for functional T cell responsiveness to high-avidity ligands, they are required for responsiveness to low-avidity ones. Thus, in at least certain T cell clonotypes, the CD3epsilon PRS amplifies weak TCR signals by promoting synapse formation and CD3epsilon phosphorylation.     

Very diverse CD8 T cell clonotypic responses after virus infections

We measured CD8 T cell clonotypic diversity to three epitopes recognized in C57BL/6 mice infected with mouse hepatitis virus, strain JHM, or lymphocytic choriomeningitis virus. We isolated epitope-specific T cells with an IFN-gamma capture assay or MHC class I/peptide tetramers and identified different clonotypes by Vbeta chain sequence analysis. In agreement with our previous results, the number of different clonotypes responding to all three epitopes fit a log-series distribution. From these distributions, we estimated that >1000 different clonotypes responded to each immunodominant CD8 T cell epitope; the response to a subdominant CD8 T cell epitope was modestly less diverse. These results suggest that T cell response diversity is greater by 1-2 orders of magnitude than predicted previously.     

Identification of CD4+ T cell-specific epitopes of islet-specific glucose-6-phosphatase catalytic subunit-related protein: a novel beta cell autoantigen in type 1 diabetes

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) has been identified as a novel CD8(+) T cell-specific autoantigen in NOD mice. This study was undertaken to identify MHC class II-specific CD4(+) T cell epitopes of IGRP. Peptides named P1, P2, P3, P4, P5, P6, and P7 were synthesized by aligning the IGRP protein amino acid sequence with peptide-binding motifs of the NOD MHC class II (I-A(g7)) molecule. Peptides P1, P2, P3, and P7 were immunogenic and induced both spontaneous and primed responses. IGRP peptides P1-, P2-, P3-, and P7-induced responses were inhibited by the addition of anti-MHC class II (I-A(g7)) Ab, confirming that the response is indeed I-A(g7) restricted. Experiments using purified CD4(+) and CD8(+) T cells from IGRP peptide-primed mice also showed a predominant CD4(+) T cell response with no significant activation of CD8(+) T cells. T cells from P1-, P3-, and P7-primed mice secreted both IFN-gamma and IL-10 cytokines, whereas P2-primed cells secreted only IFN-gamma. Peptides P3 and P7 prevented the development of spontaneous diabetes and delayed adoptive transfer of diabetes. Peptides P1 and P2 delayed the onset of diabetes in both these models. In summary, we have identified two I-A(g7)-restricted CD4(+) T cell epitopes of IGRP that can modulate and prevent the development of diabetes in NOD mice. These results provide the first evidence on the role of IGRP-specific, MHC class II-restricted CD4(+) T cells in disease protection and may help in the development of novel therapies for type 1 diabetes.     

Defective CD8+ T cell peripheral tolerance in nonobese diabetic mice.

Nonobese diabetic (NOD) mice develop spontaneous autoimmune diabetes that involves participation of both CD4+ and CD8+ T cells. Previous studies have demonstrated spontaneous reactivity to self-Ags within the CD4+ T cell compartment in this strain. Whether CD8+ T cells in NOD mice achieve and maintain tolerance to self-Ags has not previously been evaluated. To investigate this issue, we have assessed the extent of tolerance to a model pancreatic Ag, the hemagglutinin (HA) molecule of influenza virus, that is transgenically expressed by pancreatic islet beta cells in InsHA mice. Previous studies have demonstrated that BALB/c and B10.D2 mice that express this transgene exhibit tolerance of HA and retain only low-avidity CD8+ T cells specific for the dominant peptide epitope of HA. In this study, we present data that demonstrate a deficiency in peripheral tolerance within the CD8+ T cell repertoire of NOD-InsHA mice. CD8+ T cells can be obtained from NOD-InsHA mice that exhibit high avidity for HA, as measured by tetramer (K(d)HA) binding and dose titration analysis. Significantly, these autoreactive CD8+ T cells can cause diabetes very rapidly upon adoptive transfer into NOD-InsHA recipient mice. The data presented demonstrate a retention in the repertoire of CD8+ T cells with high avidity for islet Ags that could contribute to autoimmune diabetes in NOD mice.     

Regulation of immunity by a novel population of Qa-1-restricted CD8alphaalpha+TCRalphabeta+ T cells

Regulatory mechanisms involving CD8+ T cells (CD8 regulatory T cells (Tregs)) are important in the maintenance of immune homeostasis. However, the inability to generate functional CD8 Treg clones with defined Ag specificity has precluded a direct demonstration of CD8 Treg-mediated regulation. In the present study, we describe the isolation of functional lines and clones representing a novel population of TCRalphabeta+ Tregs that control activated Vbeta8.2+ CD4 T cells mediating experimental autoimmune encephalomyelitis. They express exclusively the CD8alphaalpha homodimer and recognize a peptide from a conserved region of the TCR Vbeta8.2 chain in the context of the Qa-1a (CD8alphaalpha Tregs). They secrete type 1 cytokines but not IL-2. CD8alphaalpha Tregs kill activated Vbeta8.2+ but not Vbeta8.2- or naive T cells. The CD8alphaalpha Tregs prevent autoimmunity upon adoptive transfer or following in vivo activation. These findings reveal an important negative feedback regulatory mechanism targeting activated T cells and have implications in the development of therapeutic strategies for autoimmune diseases and transplantation.     

MHC class I-restricted determinants on the glutamic acid decarboxylase 65 molecule induce spontaneous CTL activity

CD4(+) T cell responses to glutamic acid decarboxylase (GAD65) spontaneously arise in nonobese diabetic (NOD) mice before the onset of insulin-dependent diabetes mellitus (IDDM) and may be critical to the pathogenic process. However, since both CD4(+) and CD8(+) T cells are involved in autoimmune diabetes, we sought to determine whether GAD65-specific CD8(+) T cells were also present in prediabetic NOD mice and contribute to IDDM. To refine the analysis, putative K(d)-binding determinants that were proximal to previously described dominant Th determinants (206-220 and 524-543) were examined for their ability to elicit cytolytic activity in young NOD mice. Naive NOD spleen cells stimulated with GAD65 peptides 206-214 (p206) and 546-554 (p546) produced IFN-gamma and showed Ag-specific CTL responses against targets pulsed with homologous peptide. Conversely, several GAD peptides distal to the Th determinants, and control K(d)-binding peptides did not induce similar responses. Spontaneous CTL responses to p206 and p546 were mediated by CD8(+) T cells that are capable of lysing GAD65-expressing target cells, and p546-specific T cells transferred insulitis to NOD.scid mice. Young NOD mice pretreated with p206 and p546 showed reduced CTL responses to homologous peptides and a delay in the onset of IDDM. Thus, MHC class I-restricted responses to GAD65 may provide an inflammatory focus for the generation of islet-specific pathogenesis and beta cell destruction. This report reveals a potential therapeutic role for MHC class I-restricted peptides in treating autoimmune disease and revisits the notion that the CD4- and CD8-inducing determinants on some molecules may benefit from a proximal relationship.     

HLA-A*0201-restricted T cells from humanized NOD mice recognize autoantigens of potential clinical relevance to type 1 diabetes

In both humans and NOD mice, particular MHC genes are primary contributors to development of the autoreactive CD4+ and CD8+ T cell responses against pancreatic beta cells that cause type 1 diabetes (T1D). Association studies have suggested, but not proved, that the HLA-A*0201 MHC class I variant is an important contributor to T1D in humans. In this study, we show that transgenic expression in NOD mice of HLA-A*0201, in the absence of murine class I MHC molecules, is sufficient to mediate autoreactive CD8+ T cell responses contributing to T1D development. CD8+ T cells from the transgenic mice are cytotoxic to murine and human HLA-A*0201-positive islet cells. Hence, the murine and human islets must present one or more peptides in common. Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is one of several important T1D autoantigens in standard NOD mice. Three IGRP-derived peptides were identified as targets of diabetogenic HLA-A*0201-restricted T cells in our NOD transgenic stock. Collectively, these results indicate the utility of humanized HLA-A*0201-expressing NOD mice in the identification of T cells and autoantigens of potential relevance to human T1D. In particular, the identified antigenic peptides represent promising tools to explore the potential importance of IGRP in the development of human T1D.