Figure 4: For flow cytometric analysis, cells were gated on lymphocytes and then, on the CD4⁺ population to evaluate CD25⁺Foxp3⁺ Treg frequency in the spleen.

(A) Phase 1 treatment of NOD mice with tobacco expressing 500 μg GAD (n = 7) or 250 μg CTB-hpINS (n = 14) without ATRA did not affect Treg frequency in the spleen compared to treatment with WT (n = 14), P = 0.73. (B) Treatment with ATRA + WT tobacco (n = 14) did not affect Treg frequency compared to WT tobacco-treated mice (n = 14), P = 0.62 (ANOVA).At (C) 6, (D) 10, and (E) 14 weeks of age as well as (F,G) at T1D onset or 32 weeks of age, fresh splenocytes were stained for CD4, CD8, CD25, LAP, and Foxp3 for flow cytometric analysis. Live lymphocytes were analyzed for the percent of CD4⁺CD25⁺Foxp3⁺ Tregs. (C–E) There was no difference in Treg frequency at cross-sectional time points, P = ns (all). (F) At study endpoint (T1D onset or 32 weeks of age), mice treated with CTB-hpINS tobacco, alone or in combination with GAD tobacco, demonstrated significantly greater Treg frequencies compared to untreated NOD mice, P < 0.05. (G) Among animals that received combination therapy, Treg frequency was significantly higher in those that were euglycemic at 32 weeks of age compared to diabetic animals, P < 0.001 (ANOVA). No Tx indicates untreated control NOD mice.