Fig 2: Characterization of autoreactive CD8 T-cell responses in PD-L1-/- mice.(A) PD-L1-/- mice were immunized with pCI (group 1, n = 3), pCI/ppins (group 2, n = 10) or pCI/ppins?A12–21 (group 3, n = 20) and cumulative diabetes incidences (%) were determined (left panel). CD8 T-cells were prepared from pancreata of pCI/ppins-immune and diabetic PD-L1-/- mice. Pancreatic cell preparations from eight ppins-immune mice were pooled and restimulated ex vivo for 16 hours with a ppins-specific peptide library (i.e., 10 mers with two amino acids offset) and frequencies of IFN?+ CD8 T-cells were determined by FCM. The mean % of IFN?+ CD8 T-cells in the pancreatic CD8 T-cell population (obtained from two independent experiments) are shown (middle panel). Pancreatic cell preparations from ppins-immune and diabetic (group 2, n = 3), and from control (pCI) or ppins?A12–21-immune and healthy PD-L1-/- mice (groups 1 and 3, n = 3) were directly stained with Kb/B22–29-tetramers. The percentage of Kb/B22–29-tetramer+ CD8 T-cells (±SD) within the pancreas-infiltrating CD8 T-cell population is shown (right panel). (B) RIP-B7.1+/PD-L1-/- mice were immunized with pCI (group 1, n = 3) or pCI/ppins?A12–21 (group 2, n = 4) and cumulative diabetes incidences (%) (left panel) and Kb/B22–29-tetramer+ CD8 T-cells in the pancreata (right panel) were determined as described above. (C) PD-L1-/- mice were immunized with pCI (group 1, n = 2) or both, pCI/ppins+pCI/ppins?A12–21 vectors (group 2, n = 4) into the right and the left tibialis anterior muscles, respectively and cumulative diabetes incidences (%) (left panel) and Kb/B22–29-tetramer+ CD8 T-cells in the pancreata (right panel) were determined as described above. The statistical significance of diabetes induction in immunized mice (A–C) was determined using the log-rank test. Values of P<0.05 were considered significant.

Fig 3: Priming of Kb/B22–29-specific CD8 T-cell responses and EAD by mutant ppins antigens.(A) Map of the expression vectors pCI/ppins?A12–21, pCI/SP-B (encoding the ER-targeting signal peptide and the insulin B-chain) and pCI/SP-B-C (encoding the ER-targeting signal peptide up to the C-peptide). The position of the Kb/B22–29 epitope (?) is indicated. (B) RIP-B7.1 tg mice were immunized with pCI/ppins?A12–21 (group 1, n = 4), pCI/SP-B (group 2, n = 8) or pCI/SP-B-C DNA (group 3, n = 8) and cumulative diabetes incidences 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. (C) HEK-293 cells were transiently transfected with pCI (lane 1), pCI/ppins (lane 2) or pCI/ppins?A12–21 DNA (lane 3). Cells were labeled with 35S-methionine/cysteine, lysed and immunoprecipitated with an anti-insulin (H86) Ab and protein G sepharose. Immunoprecipitates were processed for SDS-PAGE, followed by fluorography of the gels. The position of pins is indicated (D) HEK-293 cells were transiently transfected with pCI/ppins (lane 1) or pCI/ppins?A12–21 (lanes 2–4). At 28 h after transfection, cells were either non-treated (lanes 1 and 2), or incubated for 6 h with the proteasome-inhibitors expoxymycin (ep; lane 3) or lactacystein (lac; lane 4) and subsequently lysed. Total cell extracts were subjected to high resolution tricine-urea-SDS-PAGE (16%) followed by anti-insulin (H86) specific western blotting.

Fig 4: Diabetes Induction in RIP-B7.1 tg Mice by Ppins-Derived Antigens Lacking the Dominant Kb/A12-21 Epitope(A) Schematic presentation of the Kb/A12-21 deleted or mutated antigens ppins?A12-21 and ppins102,105,107A.(B) HEK293 cells were transiently transfected with pCI/ppins (lane 1), pCI/ppins102,105,107A (lane 2), pCI/ppins?A12–21 (lane 3), or control pCI DNA (lane 4). At 48 hr post-transfection, cells were lysed and total cell extracts were subjected to SDS-PAGE (12.5%) followed by anti-insulin- and anti-beta-actin-specific western blotting.(C) Alternatively, HeLa cells were co-transfected with pCI/ppins or pCI/ppins102,105,107A DNA and an ER-targeted, red fluorescent protein (L-RFP-ER)-expressing vector. Ppins or insulin staining was performed using anti-insulin and FITC-conjugated anti-rabbit IgG.(D) RIP-B7.1 tg mice (n = 5 per group) were immunized with pCI/ppins (group 1), pCI/ppins?A12–21 (group 2), or pCI/ppins102,105,107A (group 3), and diabetes development was followed by determination of the blood glucose levels. Cumulative diabetes incidences (%) are shown.(E) Frequencies of Kb/B22-29-specific CD8+ T cells in the pancreata of diabetic, pCI/ppins (group 1, n = 7)-, pCI/ppins?A12–21 (group 2, n = 7)-, pCI/ppins102,105,107A-immune (group 3, n = 7), and sham-immunized (pCI) (group 4; n = 7) RIP-B7.1 tg mice were determined by Kb/B22-M29L-dimer staining. Bar graphs represent the mean percentage of Kb/B22-M29L-dimer+ cells among the CD8+ T cell population ± SD. Statistical analysis was performed using a repeated-measures one-way ANOVA followed by Tukey’s multiple comparisons. *p < 0.05; **p < 0.01; ns, not significant.

Fig 5: The RIP-B7.1 diabetes model.(A) Map of ppins antigens. The expression vectors encoding the ppins and the mutant ppins?A12–21 are shown. The signal peptide (SP), the insulin B- and A- chains, the C-peptide and the position and sequences of the Kb/A12–21 epitope (•), its Kb/A12-N21A variant and of the newly identified Kb/B22–29 epitope (?) are indicated. (B,C) RIP-B7.1 tg mice were immunized with pCI (groups 1, n = 6), pCI/ppins (groups 2, n = 6) or pCI/ppins?A12–21 (groups 3, n = 6). At indicated times after immunization, blood glucose levels (B) and cumulative diabetes incidences (C) 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. (D) CD8 T-cells were prepared from pancreata of pCI/ppins?A12–21-immune and diabetic RIP-B7.1 tg mice. Pancreatic cell preparations from ten mice were pooled and restimulated ex vivo for 16 hours with a ppins-specific peptide library (i.e., 10 mers with two amino acids offset) and frequencies of IFN?+ CD8 T-cells were determined by by flow cytometry (FCM). The mean % of IFN?+ 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. (E) RIP-B7.1 tg mice were immunized with pCI (group 1), pCI/ppins (group 2), pCI/ppins?A12–21 (group 3) or pCI/ppins and pCI/ppins?A12–21 (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 ex vivo with A12-N21A or B22–29 peptides. Specific IFN?+ CD8 T-cell frequencies were determined by FCM. The mean % of IFN?+ 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 A12-N21A- (groups 2 and 4) and Kb/B22–29-specific CD8 T-cell frequencies (groups 3 and 4) was determined by the unpaired Student’s t-test (ns, not significant).