Type 1 diabetes (T1D) is a T cell-mediated
autoimmune disease in which the
insulin-producing β cells within the pancreas are destroyed. Identification of target Ags and
epitopes of the β cell-reactive T cells is important both for understanding T1D pathogenesis and for the rational development of Ag-specific
immunotherapies for the disease. Several studies suggest that
proinsulin is an early and integral target
autoantigen in T1D. However,
proinsulin epitopes recognized by human CD4+ T cells have not been comprehensively characterized. Using a
dye dilution-based T cell cloning method, we generated and characterized 24 unique
proinsulin-specific CD4+ T cell clones from the peripheral blood of 17 individuals who carry the high-risk DR3-DQ2 and/or DR4-DQ8 HLA class II haplotypes. Some of the clones recognized previously reported DR4-restricted
epitopes within the
C-peptide (C25-35) or A-chain (A1-15) of
proinsulin. However, we also characterized DR3-restricted
epitopes within both the B-chain (B16-27 and B22-C3) and
C-peptide (C25-35). Moreover, we identified DQ2-restricted
epitopes within the B-chain and several DQ2- or DQ8-restricted
epitopes within the C-terminal region of
C-peptide that partially overlap with previously reported DQ-restricted
epitopes. Two of the DQ2-restricted
epitopes, B18-26 and C22-33, were shown to be naturally processed from whole human
proinsulin. Finally, we observed a higher frequency of CDR3 sequences matching the TCR sequences of the
proinsulin-specific T cell clones in pancreatic lymph node samples compared with spleen samples. In conclusion, we confirmed several previously reported
epitopes but also identified novel (to our knowledge)
epitopes within
proinsulin, which are presented by HLA class II molecules associated with T1D risk.