Previous studies in type 1 diabetes (T1D) in the nonobese diabetic mouse demonstrated that a crucial insulin epitope (B:9-23) is presented to diabetogenic CD4 T cells by IA g7 in a weakly bound register. The importance of antigenic peptides with low-affinity HLA binding in human autoimmune disease remains less clear. The objective of this study was to investigate T-cell responses to a lowaffinity self-epitope in subjects with T1D. HLA-DQ8 tetramers loaded with a modified insulin peptide designed to improve binding the lowaffinity register were used to visualize T-cell responses following in vitro stimulation. Positive responses were only detectable in T1D patients. Because the immunogenic register of B:9-23 presented by DQ8 has not been conclusively demonstrated, T-cell assays using substituted peptides and DQ8 constructs engineered to express and present B:9-23 in fixed binding registers were used to determine the immunogenic register of this peptide. Tetramer-positive T-cell clones isolated from T1D subjects that responded to stimulation by B:11-23 peptide and denatured insulin protein were conclusively shown to recognize B:11-23 bound to HLA-DQ8 in the low-affinity register 3. These T cells also responded to homologous peptides derived from microbial antigens, suggesting that their initial priming could occur via molecular mimicry. These results are in accord with prior observations from the nonobese diabetic mouse model, suggesting a mechanism shared by mouse and man through which T cells that recognize a weakly bound peptide can circumvent tolerance mechanisms and play a role in the initiation of autoimmune diseases, such as T1D.antigen presentation | self-antigen | MHCII tetramers T ype 1 diabetes (T1D) is a polygenic T-cell-mediated autoimmune disease with strong genetic linkages to the MHC class II and insulin promoter regions (1). Class II molecules are fundamental for CD4 + T-cell activation, whereas the insulin promoter polymorphism can modulate insulin levels in the thymus thereby influencing the threshold of selection for insulinspecific autoreactive T cells (2, 3). In the nonobese diabetic (NOD) mouse model of autoimmune mediated diabetes, insulinspecific IA g7 -restricted CD4 + T cells have been strongly implicated in β-cell destruction. In prediabetic NOD mice, 50% of the T-cell clones established from islet-infiltrating lymphocytes were insulin-specific and the majority of these clones recognized the insulin B:9-23 (B:9-23 SHLVEALYLVCGERG) epitope (4, 5). Moreover, substitution of a single residue in the B:9-23 region abrogated development of diabetes in a transgenic mouse model (6, 7). Thus, in the murine NOD model, the IA g7 -restricted B:9-23 epitope is considered to be pivotal in the development of diabetes.The position or "register" that B:9-23 occupies within the IA g7 binding groove has been a controversial but important question. At least three binding registers (R) have been considered, defined here by which B:9-23 amino acid occupies the first binding pocket (p1) position in the IA g7 ...

The frequency of epitope specific naïve CD4+ T cells in humans has not been extensively examined. In this study, a systematic approach was used to examine the frequency of CD4+ T cells that recognize the Protective Antigen of Bacillus anthracis in both Anthrax Vaccine Adsorbed vaccinees and non-vaccinees with HLA-DRB1*01:01 haplotypes. Three epitopes were identified that had distinct degrees of immunodominance in subjects that had received the vaccine. Average naïve precursor frequencies of T cells specific for these different epitopes in the human repertoire ranged from 0.2 to 10 per million naïve CD4+ T cells, which is comparable to precursor frequencies observed in the murine repertoire. Frequencies of protective Antigen-specific T cells were two orders of magnitude higher in immunized subjects than in nonvaccinees. The frequencies of epitope specific memory CD4+ T cells in vaccinees were directly correlated with the frequencies of precursors in the naïve repertoire. At the level of TCR usage, at least one preferred Vβ in the naïve repertoire was present in the memory repertoire. These findings implicate naïve frequencies as a crucial factor in shaping the epitope specificity of memory CD4+ T cell responses.

It is commonly perceived that the human immune system is naive to the newly emerged H5N1 virus. In contrast, most adults have been exposed to influenza A H1N1 and H3N2 viruses through vaccination or infection. Adults born before 1968 have likely been exposed to H2N2 viruses. We hypothesized that CD4+ T cells generated in response to H1N1, H3N2, and H2N2 influenza A viruses also recognize H5N1 epitopes. Tetramer-guided epitope mapping and Ag-specific class II tetramers were used to identify H5N1-specific T cell epitopes and detect H5N1-specific T cell responses. Fifteen of 15 healthy subjects tested had robust CD4+ T cell responses against matrix protein, nucleoprotein, and neuraminidase of the influenza A/Viet Nam/1203/2004 (H5N1) virus. These results are not surprising, because the matrix protein and nucleoprotein of influenza A viruses are conserved while the neuraminidase of the H5N1 virus is of the same subtype as that of the circulating H1N1 influenza strain. However, H5N1 hemagglutinin-reactive CD4+ T cells were also detected in 14 of 14 subjects examined despite the fact that hemagglutinin is less conserved. Most were cross-reactive to H1, H2, or H3 hemagglutinin epitopes. H5N1-reactive T cells were also detected ex vivo, exhibited a memory phenotype, and were capable of secreting IFN-γ, TNF-α, IL-5, and IL-13. These data demonstrate the presence of H5N1 cross-reactive T cells in healthy Caucasian subjects, implying that exposure to influenza A H1N1, H3N2, or H2N2 viruses through either vaccination or infection may provide partial immunity to the H5N1 virus.

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. IGRP23–35 and IGRP247–259 were identified as DRA1*0101/DRB1*0401-restricted epitopes. IGRP13–25 and IGRP226–238 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 γ-IFN and IL-10. DRA1*0101/DRB1*0401 IGRP247–259-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.

The significance of islet antigen-reactive T cells found in peripheral blood of type 1 diabetes (T1D) subjects is unclear, partly because similar cells are also found in healthy control (HC) subjects. We hypothesized that key disease-associated cells would show evidence of prior antigen exposure, inferred from expanded T cell receptor (TCR) clonotypes, and essential phenotypic properties in their transcriptomes. To test this, we developed single-cell RNA sequencing (RNA-seq) procedures for identifying TCR clonotypes and transcript phenotypes in individual T cells. We applied these procedures to analysis of islet- antigen reactive CD4+ memory T cells from the blood of T1D and HC individuals following activation with pooled immunodominant islet peptides. We found extensive TCR clonotype sharing in antigen-activated cells, especially from individual T1D subjects, consistent with in vivo T cell expansion during disease progression. The expanded clonotype from one T1D subject was detected at repeat visits spanning more than 15 months, demonstrating clonotype stability. Notably, we found no clonotype sharing between subjects, indicating a predominance of “private” TCR specificities. Expanded clones from two T1D subjects recognized distinct IGRP peptides, implicating this molecule as a trigger for CD4+ T cell expansion. While overall transcript profiles of cells from HC and T1D subjects were similar, profiles from the most expanded clones were distinctive. Our findings demonstrate that islet- antigen reactive CD4+ memory T cells with unique antigen specificities and phenotypes are expanded during disease progression and can be detected by single-cell analysis of peripheral blood.