Agonist activity of insulin B chain epitopes and mimetopes. (a) One-letter amino acid codes are shown for insulin B chain epitopes and mimetopes. MHC-binding residues are shown in subscript and in blue for binding register 3. Residues 9–11, which do not contribute to MHC binding, are shown in gray. (b) Analysis of proliferation of insulin B:9-23–specific TCR transgenic T cells stimulated with epitopes and mimetopes. Results are the averages ± SEM of triplicate wells from four independent experiments. (c) Agonist and T reg cell conversion activity. In vivo T reg cell conversion using naive CD4⁺ insulin B:9-23 TCR transgenic T cells transferred into congenic NOD Thy1.1⁺ mice at 4 wk of age. Error bars show percentages of Foxp3⁺ T reg cells (± SEM from three independent experiments; n = 4 per group) at 2 wk after injection of cells and infusion of peptide. Peptide was delivered either by a 2-wk infusion period using osmotic minipumps or by a single i.p. injection of DEC205 fusion antibody. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus insulin B:9-23.

Development of diabetes. (a) Cumulative diabetes incidence in NOD mice, nonvaccinated, vaccinated with insulin B:9-23 or insulin mimetope 3 at 4–6 wk of age. n = 12 from three independent experiments. (b) Cumulative diabetes incidence of NOD mice, nonvaccinated, vaccinated with insulin B:9-23 or insulin mimetope 3 at 12–14 wk of age. n = 6 from two independent experiments. **, P < 0.01; ***, P < 0.001 versus insulin B:9-23.

Impact of the mTOR inhibitor everolimus on development of diabetes. Cumulative diabetes incidence of NOD mice treated with a combination of the natural insulin epitope or the mimetope plus everolimus (3 mg/kg i.p. for 14 d) or everolimus alone. n = 12 from three independent experiments. *, P < 0.05 versus insulin B:9-23 + everolimus.

Analysis of Foxp3 expression in 30-wk-old NOD mice. Foxp3 immunohistochemistry in pancreatic cryosections. (a) Co-stainings of CD4 (green) and Foxp3 (red) expression in the respective treatment groups (b–d). Representative co-stainings of insulin (green) and Foxp3 (red) expression in pancreatic sections of mice that were nonvaccinated (b), vaccinated with insulin B:9-23 (c), and vaccinated with insulin mimetope 3 (d). n = 8 per group from two independent experiments.Bars, 100 µm. (e) Quantification of Foxp3⁺ cells in pancreatic sections as described in b–d. Bars show Foxp3⁺ cells per high-power field (± SEM from two independent experiments; n = 8 per group). *, P < 0.05; **, P < 0.01 versus insulin B:9-23.

Analysis of dominant tolerance in vaccinated NOD mice. CFSE dilution profiles of insulin B:9-23 TCR transgenic T cells at day 4 after transfer into nonvaccinated or vaccinated 8- (a) or 30-wk-old (b) NOD mice. (c) Percentages of divided insulin B:9-23 TCR transgenic T cells after transfer into the vaccination groups as described in a and b. Bars represent means ± SEM from two independent experiments. n = 4 per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus insulin B:9-23. (d) Analysis of proliferation of T cells from pancreatic lymph nodes of NOD Foxp3-GFP reporter mice at 20 wk, vaccinated with either insulin B:9-23 or insulin mimetope 3 at 4–6 wk of age. Proliferation before or after depletion of Foxp3⁺ T cells in response to the insulin mimetope was analyzed. Results are the means ± SEM from two independent experiments. n = 4 per group.

Efficacy of Foxp3⁺ T reg cell vaccination as a function of IAA levels at 4 wk. (a) Naive CD4⁺CD25⁻CD62L^hiCD44^lowFoxp3GFP⁻ cells or activated CD4⁺CD25⁻CD62L^loCD44^hiFoxp3GFP⁻ cells were subjected to in vitro conversion assays (α-CD3 + α-CD28) in the presence or absence (= control) of TGFβ. After 3 d, Foxp3 expression was analyzed by FACS. Figure shows a representative dot plot from four independent experiments performed in triplicates per treatment group. (b and c) NOD mice were infused with insulin B:9-23 peptide (b) or insulin mimetope 3 (c) at 4–6 wk of age. IAA indices were measured at 4 wk of age, before vaccination. Vertical lines indicate the lower detection limit for IAAs. n = 12 for each group from three independent experiments. (d) IAA indices in NOD mice were measured at 4 wk of age (before vaccination) and at 6 wk of age (after vaccination). Nonvaccinated control mice were infused with PBS. Mice with IAA indices <1 were included. Bars show averages ± SEM of IAA indices. n = 8 from two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus insulin B:9-23.

Dose response of insulin epitope and mimetope. (a) Cumulative diabetes incidence in NOD mice that were nonvaccinated, vaccinated with insulin B:9-23, or vaccinated by insulin mimetope 3 at 4–6 wk of age (doses 2.5–20 µg/d). n = 6 from two independent experiments. (b) In vivo T reg cell conversion using naive CD4⁺ insulin B:9-23 TCR transgenic T cells transferred into congenic NOD Thy1.1⁺ mice. Percentages of Foxp3⁺ T reg cells (± SEM, n = 6 from two independent experiments) at 2 wk after vaccination with indicated doses of peptide. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus insulin B:9-23.

Histopathological evaluation of pancreas sections from either nonvaccinated or vaccinated NOD mice at the age of 20 or 40 wk. Representative hematoxylin and eosin–stained paraffin-embedded pancreas sections are shown in a and b. Bars, 100 µm. Grading of insulitis of the various treatment groups is shown in c and d. n = 12 from three independent experiments.

Foxp3⁺ T reg cells in pancreata and pancreatic lymph nodes of NOD mice. (a) Percentages and absolute numbers of Foxp3⁺ T reg cells in pancreatic islet–infiltrating T cells of 30-wk-old NOD mice from the respective treatment groups, analyzed by flow cytometry. Bars represent means ± SEM from three independent experiments. n = 4 per group. (b) Percentages and absolute number of CD4 T cells expressing Foxp3 GFP in pancreatic lymph nodes from 20-wk-old NOD Foxp3-GFP reporter mice, analyzed by flow cytometry. Bars represent means ±SEM from two independent experiments, n = 4 per group. Mice were vaccinated at 4–6 wk of age.

Characterization of effector T cells isolated from pancreatic lymph nodes of NOD Foxp3-GFP reporter mice at 20 wk. The mice were nonvaccinated, vaccinated with insulin B:9-23, or insulin mimetope 3 at 4–6 wk. Cytokine expression in T effector cells after depletion of Foxp3⁺ T cells in response to the insulin mimetope 3 was analyzed by intracellular staining (a) or by ELISA (b). *, P < 0.05 versus control. Representative dot plots are shown. Data are representative of two independent experiments. n = 4 per group. Expression of the lineage-specific transcription factors as assessed by intracellular staining is shown in c. Cells are gated on CD4⁺ T cells. Black solid line, nonvaccinated control; red solid line, insulin B:9-23 vaccinated; blue dotted line, insulin mimetope 3 vaccinated. Results are the means ± SEM from two independent experiments. n = 4 per group.