CD4+ T Cells From Individuals With Type 1 Diabetes Respond to a Novel Class of Deamidated Peptides Formed in Pancreatic Islets.

The ß-cell plays a crucial role in the pathogenesis of type 1 diabetes, in part through the posttranslational modification of self-proteins by biochemical processes such as deamidation. These neoantigens are potential triggers for breaking immune tolerance. We report the detection by LC-MS/MS of 16 novel Gln and 27 novel Asn deamidations in 14 disease-related proteins within inflammatory cytokine-stressed human islets of Langerhans. T-cell clones responsive against one Gln- and three Asn-deamidated peptides could be isolated from peripheral blood of individuals with type 1 diabetes. Ex vivo HLA class II tetramer staining detected higher T-cell frequencies in individuals with the disease compared with control individuals. Furthermore, there was a positive correlation between the frequencies of T cells specific for deamidated peptides, insulin antibody levels at diagnosis, and duration of disease. These results highlight that stressed human islets are prone to enzymatic and biochemical deamidation and suggest that both Gln- and Asn-deamidated peptides can promote the activation and expansion of autoreactive CD4+ T cells. These findings add to the growing evidence that posttranslational modifications undermine tolerance and may open the road for the development of new diagnostic and therapeutic applications for individuals living with type 1 diabetes.

Interferon-α promotes neo-antigen formation and preferential HLA-B-restricted antigen presentation in pancreatic ß-cells.

Interferon (IFN)-α is the earliest cytokine signature observed in individuals at risk for type 1 diabetes (T1D), but its effect on the repertoire of HLA Class I (HLA-I)-bound peptides presented by pancreatic ß-cells is unknown. Using immunopeptidomics, we characterized the peptide/HLA-I presentation in in-vitro resting and IFN-α-exposed ß-cells. IFN-α increased HLA-I expression and peptide presentation, including neo-sequences derived from alternative mRNA splicing, post-translational modifications - notably glutathionylation - and protein cis-splicing. This antigenic landscape relied on processing by both the constitutive and immune proteasome. The resting ß-cell immunopeptidome was dominated by HLA-A-restricted ligands. However, IFN-α only marginally upregulated HLA-A and largely favored HLA-B, translating into a major increase in HLA-B-restricted peptides and into an increased activation of HLA-B-restricted vs. HLA-A-restricted CD8+ T-cells. A preferential HLA-B hyper-expression was also observed in the islets of T1D vs. non-diabetic donors, and we identified islet-infiltrating CD8+ T-cells from T1D donors reactive to HLA-B-restricted granule peptides. Thus, the inflammatory milieu of insulitis may skew the autoimmune response toward epitopes presented by HLA-B, hence recruiting a distinct T-cell repertoire that may be relevant to T1D pathogenesis.

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.