Position β57 of I-Ag7 controls early anti-insulin responses in NOD mice, linking an MHC susceptibility allele to type 1 diabetes onset

The class II region of the major histocompatibility complex (MHC) locus is the main contributor to the genetic susceptibility to type 1 diabetes (T1D). The loss of an aspartic acid at position 57 of diabetogenic HLA-DQ beta chains supports this association; this single amino acid change influences how TCRs recognize peptides in the context of HLA-DQ8 and I-A(g7) using a mechanism termed the P9 switch. Here, we built register-specific insulin peptide MHC tetramers to examine CD4(+) T cell responses to Ins(12-20) and Ins(13-21) peptides during the early prediabetic phase of disease in nonobese diabetic (NOD) mice. A single-cell analysis of anti-insulin CD4(+) T cells performed in 6- and 12-week-old NOD mice revealed tissue-specific gene expression signatures. TCR signaling and clonal expansion were found only in the islets of Langerhans and produced either classical T(H)1 differentiation or an unusual T-reg phenotype, independent of TCR usage. The early phase of the anti-insulin response was dominated by T cells specific for Ins(12-20), the register that supports a P9 switch mode of recognition. The presence of the P9 switch was demonstrated by TCR sequencing, reexpression, mutagenesis, and functional testing of TCR alpha beta pairs in vitro. Genetic correction of the I-A beta 57 mutation in NOD mice resulted in the disappearance of D/E residues in the CDR3 beta of anti-Ins(12-20) T cells. These results provide a mechanistic molecular explanation that links the characteristic MHC class II polymorphism of T1D with the recognition of islet autoantigens and disease onset.