The RIP-B7.1 diabetes model.

<p>(<b>A</b>) Map of ppins antigens. The expression vectors encoding the ppins and the mutant ppinsΔA_12–21 are shown. The signal peptide (SP), the insulin B- and A- chains, the C-peptide and the position and sequences of the K^b/A_12–21 epitope (•), its K^b/A_12-N21A variant and of the newly identified K^b/B_22–29 epitope (○) are indicated. (<b>B,C</b>) RIP-B7.1 tg mice were immunized with pCI (groups 1, n = 6), pCI/ppins (groups 2, n = 6) or pCI/ppinsΔA_12–21 (groups 3, n = 6). At indicated times after immunization, blood glucose levels (<b>B</b>) and cumulative diabetes incidences (<b>C</b>) were determined. The statistical significance of diabetes induction in immunized mice was determined using the log-rank test. Values of P<0.05 were considered significant. (<b>D</b>) CD8 T-cells were prepared from pancreata of pCI/ppinsΔA_12–21-immune and diabetic RIP-B7.1 tg mice. Pancreatic cell preparations from ten mice were pooled and restimulated <i>ex vivo</i> for 16 hours with a ppins-specific peptide library (i.e., 10 mers with two amino acids offset) and frequencies of IFNγ⁺ CD8 T-cells were determined by by flow cytometry (FCM). The mean % of IFNγ⁺ CD8 T-cells in the pancreatic CD8 T-cell population (obtained from two independent experiments) are shown. CD8 T-cell frequencies <0.05% are defined negative. (<b>E</b>) RIP-B7.1 tg mice were immunized with pCI (group 1), pCI/ppins (group 2), pCI/ppinsΔA_12–21 (group 3) or pCI/ppins and pCI/ppinsΔA_12–21 (group 4). In group 4, the indicated plasmids were injected into the right and the left tibialis anterior muscles, respectively. CD8 T-cells were prepared from pancreata of diabetic (groups 2–4) or non-diabetic (group 1) mice and restimulated <i>ex vivo</i> with A_12-N21A or B_22–29 peptides. Specific IFNγ⁺ CD8 T-cell frequencies were determined by FCM. The mean % of IFNγ⁺ CD8 T-cells in the pancreatic CD8 T-cell population (±SD) of a representative experiment (n = 3 mice per group) is shown. The statistical significance of differences between A_12-N21A- (groups 2 and 4) and K^b/B_22–29-specific CD8 T-cell frequencies (groups 3 and 4) was determined by the unpaired Student’s t-test (ns, not significant).