The CD318/CD6 axis limits type 1 diabetes islet autoantigen-specific human T cell activation.

CD6 is a glycoprotein expressed on CD4 and CD8 T cells involved in immunoregulation. CD318 has been identified as a CD6 ligand. The role of CD318 in T cell immunity is restricted as it has only been investigated in a few mice autoimmune models but not in human diseases. CD318 expression was thought to be limited to mesenchymal-epithelial cells and, therefore, contribute to CD6-mediated T cell activation in the CD318-expressing tissue rather than through interaction with antigen-presenting cells. Here, we report CD318 expression in a subpopulation of CD318+ myeloid dendritic (mDC), whereas the other peripheral blood populations were CD318 negative. However, CD318 can be induced by activation: a subset of monocytes treated with LPS and IFNγ and in vitro monocyte derived DCs were CD318+. We also showed that recombinant CD318 inhibited T cell function. Strikingly, CD318+ DCs suppressed the proliferation of autoreactive T cells specific for GAD65, a well-known targeted self-antigen in Type 1 Diabetes (T1D). Our study provides new insight into the role of the CD318/CD6 axis in the immunopathogenesis of inflammation, suggesting a novel immunoregulatory role of CD318 in T cell-mediated autoimmune diseases and identifying a potential novel immune checkpoint inhibitor as a target for intervention in T1D which is an unmet therapeutic need.

Alteration of select gene expression patterns in individuals infected with HIV-1.

Multiple human proteins have been shown to both support and restrict viral replication, and confirmation of virus-associated changes in the expression of these genes is relevant for future therapeutic efforts. In this study a well-characterized panel of 49 individuals either infected with HIV-1 or uninfected was compiled and analyzed for the effect of HIV infection status, viral load, and antiretroviral treatment on specific gene expression. mRNA was extracted and reverse transcribed from purified CD4+ cells, and quantitative real-time PCR was utilized to scrutinize differences in the expression of four host genes that have been demonstrated to either stimulate (HSP90 and LEDGF/p75) or restrict (p21/WAF1 and APOBEC3G) proviral integration. HIV infection status was associated with slight to moderate alterations in the expression of all four genes. After adjusting for age, mRNA expression levels of HSP90, LEDGF/p75 and APOBEC3G were found to all be decreased in infected patients compared to healthy controls by 1.43-, 1.26-, and 4.71-fold, respectively, while p21/WAF1 expression was increased 2.35-fold. Furthermore, individuals receiving raltegravir exhibited a 1.28-fold reduction in LEDGF/p75 compared to those on non-raltegravir antiretroviral treatment. Identification of these and similar HIV-induced changes in gene expression may be valuable for delineating the extent of host cell molecular mechanisms stimulating viral replication.

Recognition of mRNA Splice Variant and Secretory Granule Epitopes by CD4+ T Cells in Type 1 Diabetes.

A recent discovery effort resulted in identification of novel splice variant and secretory granule antigens within the HLA class I peptidome of human islets and documentation of their recognition by CD8+ T cells from peripheral blood and human islets. In the current study, we applied a systematic discovery process to identify novel CD4+ T cell epitopes derived from these candidate antigens. We predicted 145 potential epitopes spanning unique splice junctions and within conventional secretory granule antigens and measured their in vitro binding to DRB1*04:01. We generated HLA class II tetramers for the 35 peptides with detectable binding and used these to assess immunogenicity and isolate T cell clones. Tetramers corresponding to peptides with verified immunogenicity were then used to label T cells specific for these putative epitopes in peripheral blood. T cells that recognize distinct epitopes derived from a cyclin I splice variant, neuroendocrine convertase 2, and urocortin-3 were detected at frequencies that were similar to those of an immunodominant proinsulin epitope. Cells specific for these novel epitopes predominantly exhibited a Th1-like surface phenotype. Among the three epitopes, responses to the cyclin I peptide exhibited a distinct memory profile. Responses to neuroendocrine convertase 2 were detected among pancreatic infiltrating T cells. These results further establish the contribution of unconventional antigens to the loss of tolerance in autoimmune diabetes.

Technical Validation and Utility of an HLA Class II Tetramer Assay for Type 1 Diabetes: A Multicenter Study.

CONTEXT: Validated assays to measure autoantigen-specific T-cell frequency and phenotypes are needed for assessing the risk of developing diabetes, monitoring disease progression, evaluating responses to treatment, and personalizing antigen-based therapies. OBJECTIVE: Toward this end, we performed a technical validation of a tetramer assay for HLA-DRA-DRB1*04:01, a class II allele that is strongly associated with susceptibility to type 1 diabetes (T1D). METHODS: HLA-DRA-DRB1*04:01-restricted T cells specific for immunodominant epitopes from islet cell antigens GAD65, IGRP, preproinsulin, and ZnT8, and a reference influenza epitope, were enumerated and phenotyped in a single staining tube with a tetramer assay. Single and multicenter testing was performed, using a clone-spiked specimen and replicate samples from T1D patients, with a target coefficient of variation (CV) less than 30%. The same assay was applied to an exploratory cross-sectional sample set with 24 T1D patients to evaluate the utility of the assay. RESULTS: Influenza-specific T-cell measurements had mean CVs of 6% for the clone-spiked specimen and 11% for T1D samples in single-center testing, and 20% and 31%, respectively, for multicenter testing. Islet-specific T-cell measurements in these same samples had mean CVs of 14% and 23% for single-center and 23% and 41% for multicenter testing. The cross-sectional study identified relationships between T-cell frequencies and phenotype and disease duration, sex, and autoantibodies. A large fraction of the islet-specific T cells exhibited a naive phenotype. CONCLUSION: Our results demonstrate that the assay is reproducible and useful to characterize islet-specific T cells and identify correlations between T-cell measures and clinical traits.

Characterizing T cell responses to enzymatically modified beta cell neo-epitopes.

Introduction: Previous studies verify the formation of enzymatically post-translationally modified (PTM) self-peptides and their preferred recognition by T cells in subjects with type 1 diabetes (T1D). However, questions remain about the relative prevalence of T cells that recognize PTM self-peptides derived from different antigens, their functional phenotypes, and whether their presence correlates with a specific disease endotype. Methods: To address this question, we identified a cohort of subjects with T1D who had diverse levels of residual beta cell function. Using previously developed HLA class II tetramer reagents, we enumerated T cells that recognize PTM GAD epitopes in the context of DRB1*04:01 or PTM IA2 epitopes in the context of DQB1*03:02 (DQ8). Results: Consistent with prior studies, we observed higher overall frequencies and a greater proportion of memory T cells in subjects with T1D than in HLA matched controls. There were significantly higher numbers of GAD specific T cells than IA2 specific T cells in subjects with T1D. T cells specific for both groups of epitopes could be expanded from the peripheral blood of subjects with established T1D and at-risk subjects. Expanded neo-epitope specific T cells primarily produced interferon gamma in both groups, but a greater proportion of T cells were interferon gamma positive in subjects with T1D, including some poly-functional cells that also produced IL-4. Based on direct surface phenotyping, neo-epitope specific T cells exhibited diverse combinations of chemokine receptors. However, the largest proportion had markers associated with a Th1-like phenotype. Notably, DQ8 restricted responses to PTM IA2 were over-represented in subjects with lower residual beta cell function. Neo-epitope specific T cells were present in at-risk subjects, and those with multiple autoantibodies have higher interferon gamma to IL-4 ratios than those with single autoantibodies, suggesting a shift in polarization during progression. Discussion: These results reinforce the relevance of PTM neo-epitopes in human disease and suggest that distinct responses to neo-antigens promote a more rapid decline in beta cell function.

Shared recognition of citrullinated tenascin-C peptides by T and B cells in rheumatoid arthritis.

Tenascin-C (TNC), an extracellular matrix protein that has proinflammatory properties, is a recently described antibody target in rheumatoid arthritis (RA). In this study, we utilized a systematic discovery process and identified 5 potentially novel citrullinated TNC (cit-TNC) T cell epitopes. CD4+ T cells specific for these epitopes were elevated in the peripheral blood of subjects with RA and showed signs of activation. Cit-TNC-specific T cells were also present among synovial fluid T cells and secreted IFN-γ. Two of these cit-TNC T cell epitopes were also recognized by antibodies within the serum and synovial fluid of individuals with RA. Detectable serum levels of cit-TNC-reactive antibodies were prevalent among subjects with RA and positively associated with cyclic citrullinated peptide (CCP) reactivity and the HLA shared epitope. Furthermore, cit-TNC-reactive antibodies were correlated with rheumatoid factor and elevated in subjects with a history of smoking. This work confirms cit-TNC as an autoantigen that is targeted by autoreactive CD4+ T cells and autoantibodies in patients with RA. Furthermore, our findings raise the possibility that coinciding epitopes recognized by both CD4+ T cells and B cells have the potential to amplify autoimmunity and promote the development and progression of RA.

Hybrid Insulin Peptides Are Recognized by Human T Cells in the Context of DRB1*04:01.

T cells isolated from the pancreatic infiltrates of nonobese diabetic mice have been shown to recognize epitopes formed by the covalent cross-linking of proinsulin and secretory granule peptides. Formation of such hybrid insulin peptides (HIPs) was confirmed through mass spectrometry, and responses to HIPs were observed among the islet-infiltrating T cells of pancreatic organ donors and in the peripheral blood of individuals with type 1 diabetes (T1D). However, questions remain about the prevalence of HIP-specific T cells in humans, the sequences they recognize, and their role in disease. We identified six novel HIPs that are recognized in the context of DRB1*04:01, discovered by using a library of theoretical HIP sequences derived from insulin fragments covalently linked to one another or to fragments of secretory granule proteins or other islet-derived proteins. We demonstrate that T cells that recognize these HIPs are detectable in the peripheral blood of subjects with T1D and exhibit an effector memory phenotype. HIP-reactive T-cell clones produced Th1-associated cytokines and proliferated in response to human islet preparations. These results support the relevance of HIPs in human disease, further establishing a novel posttranslational modification that may contribute to the loss of peripheral tolerance in T1D.

Inflammation-Induced Citrullinated Glucose-Regulated Protein 78 Elicits Immune Responses in Human Type 1 Diabetes.

The ß-cell has become recognized as a central player in the pathogenesis of type 1 diabetes with the generation of neoantigens as potential triggers for breaking immune tolerance. We report that posttranslationally modified glucose-regulated protein 78 (GRP78) is a novel autoantigen in human type 1 diabetes. When human islets were exposed to inflammatory stress induced by interleukin-1ß, tumor necrosis factor-α, and interferon-γ, arginine residue R510 within GRP78 was converted into citrulline, as evidenced by liquid chromatography-tandem mass spectrometry. This conversion, known as citrullination, led to the generation of neoepitopes, which effectively could be presented by HLA-DRB1*04:01 molecules. With the use of HLA-DRB1*04:01 tetramers and ELISA techniques, we demonstrate enhanced antigenicity of citrullinated GRP78 with significantly increased CD4+ T-cell responses and autoantibody titers in patients with type 1 diabetes compared with healthy control subjects. Of note, patients with type 1 diabetes had a predominantly higher percentage of central memory cells and a lower percentage of effector memory cells directed against citrullinated GRP78 compared with the native epitope. These results strongly suggest that citrullination of ß-cell proteins, exemplified here by the citrullination of GRP78, contributes to loss of self-tolerance toward ß-cells in human type 1 diabetes, indicating that ß-cells actively participate in their own demise.

Human Leukocyte Antigen (HLA)-DRB1*15:01 and HLA-DRB5*01:01 Present Complementary Peptide Repertoires.

Human leukocyte antigen (HLA)-DR15 is a haplotype associated with multiple sclerosis. It contains the two DRB* genes DRB1*1501 (DR2b) and DRB5*0101 (DR2a). The reported anchor motif of the corresponding HLA-DR molecules was determined in 1994 based on a small number of peptide ligands and binding assays. DR2a could display a set of peptides complementary to that presented by DR2b or, alternatively, a similar peptide repertoire but recognized in a different manner by T cells. It is known that DR2a and DR2b share some peptide ligands, although the degree of similarity of their associated peptidomes remains unclear. In addition, the contribution of each molecule to the global peptide repertoire presented by the HLA-DR15 haplotype has not been evaluated. We used mass spectrometry to analyze the peptide pools bound to DR2a and DR2b, identifying 169 and 555 unique peptide ligands of DR2a and DR2b, respectively. The analysis of these sets of peptides allowed the refinement of the corresponding binding motifs revealing novel anchor residues that had been overlooked in previous analyses. Moreover, the number of shared ligands between both molecules was low, indicating that DR2a and DR2b present complementary peptide repertoires to T cells. Finally, our analysis suggests that, quantitatively, both molecules contribute to the peptide repertoire presented by cells expressing the HLA-DR15 haplotype.

Identification of Unique Antigenic Determinants in the Amino Terminus of IA-2 (ICA512) in Childhood and Adult Autoimmune Diabetes: New Biomarker Development.

OBJECTIVE: The characterization of diverse subtypes of diabetes is a dynamic field of clinical research and an active area of discussion. The objective of this study was to identify new antigenic determinants in the neuroendocrine autoantigen IA-2 (ICA512) and assess whether circulating autoantibodies directed to new IA-2 epitopes identify autoimmune diabetes in young and adult populations with diabetes. RESEARCH DESIGN AND METHODS: Clinically diagnosed patients with type 2 diabetes (n = 258; diabetes duration: 0.01-31 years) were evaluated using a new biomarker detecting autoantibodies directed to the extracellular domain of the neuroendocrine autoantigen IA-2 (IA-2ec). The proportion of IA-2ec autoantibodies was also evaluated in newly diagnosed patients with type 1 diabetes (n = 150; diabetes duration: 0.04-0.49 years). In addition, IA-2 (intracellular domain), GAD65, and zinc transporter 8 autoantibodies were assayed. RESULTS: IA-2ec autoantibodies were detected in patients with type 1 diabetes and, surprisingly, in 5% of patients with type 2 diabetes without serologic responses to other IA-2 antigenic epitopes or other islet autoantigens. We also assessed the ability of IA-2ec-derived peptides to elicit CD4+ T-cell responses by stimulating peripheral blood mononuclear cells from patients with type 1 diabetes (n = 18) and HLA-matched healthy subjects (n = 13) with peptides and staining with the peptide/DQ8-specific tetramers, observing disease-associated responses to previously unreported epitopes within IA-2ec. CONCLUSIONS: We developed a new antibody biomarker identifying novel antigenic determinants within the N terminus of IA-2. IA-2ec autoantibodies can be detected in patients with type 1 diabetes and in a subgroup of adult autoimmune patients with type 2 diabetes phenotype negative for conventional islet autoantibody testing. These observations suggest that islet autoimmunity may be more common in clinically diagnosed type 2 diabetes than previously observed.