Figure 5.

IL10 is required for long-term Tr1 induction in NOD mice following Salmonella-based combination therapy. Female 7–8 weeks old NOD mice were treated orally with 2 doses of combined vaccine with 2.5 μg/mouse of anti-CD3 mAb as described. Splenocytes were prepared from the indicated treated groups at 7 weeks and 15 weeks post-vaccination. (A) Representative FACS plots gated on live CD4 T cells indicate the frequency of CD4⁺CD49b⁺LAG3⁺ T cells in the spleens of mice from the indicated groups. (B) Scatter plots of the percentages of CD4⁺CD49b⁺LAG3⁺ Tr1 expression across the 3 groups at 7 weeks and 15 weeks post-vaccination. Data presented as means ± SD from 2 independent experiments. Statistical analysis using one-way ANOVA shows the significance between combined therapy and control group (*p < 0.05: **p < 0.01; ****p < 0.0001). (C) in vitro stimulation assays of vaccine induced Tr1. Pooled splenocytes CD4⁺CD49b⁺LAG3⁺ Tr1 cells were isolated from vaccinated and vehicle-treated mice 3 weeks post-vaccination by FACS sorting. Secretion of IL10 was determined by ELISA of culture supernatants obtained from CD4⁺CD49b⁺LAG3⁺ Tr1 cells after peptide stimulation for 3 days. Data presented as means ± SD and obtained from 4 replicas. The statistical significance was calculated with two-way ANOVA and the significance level indicated by asterisks (****p < 0.0001).