Spontaneous presentation of immunogenic insulin epitopes by blood leukocytes . (A–C) Blood leukocytes were obtained from NOD mice with or without a prior glucose challenge and were directly cultured with CD4 T cell hybridomas with defined specificities to InsB:13-21 (A), InsB:12-20 (B), and Ins1C:51-61 (C). IL-2 production from the culture supernatants was measured to indicate the T cell responses. The dependence of MHC-II was determined by adding the anti–I-Ag7 blocking antibody to the culture. (D) Responses of T cell hybridomas with indicated antigen specificities to blood leukocytes obtained from B6, B6g7, or NOD mice. (E) Responses of the InsB:13-21– and InsB:12-20–specific CD4 T cells to blood leukocytes obtained from NOD mice at the indicated time points after the glucose challenge. (F) Responses of the InsB:13-21– (top) and InsB:12-20– (bottom) specific CD4 T cells to blood leukocytes obtained from NOD mice with or without injection of the insulin receptor blocker S961, followed by glucose challenge. Data (mean ± SEM) summarize results pooled from 2 (D–F), 4 (C), 12 (B), and 28 (A) independent experiments; each point (A–D and F) represents one biological replicate including one to six mice. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001; Mann–Whitney test. CPM, counts per minute; Ins-ChgA, insulin–chromogranin A.