Figure 5. InsB-g7 CAR Tregs suppress BDC 2.5 T cell–induced autoimmune diabetes.

(A) Experimental design indicating that 450 × 10³ (9:1) or 150 × 10³ (3:1) untransduced control Tregs or InsB-g7CAR Tregs were cotransferred with 50 × 10³ naive BDC2.5 CD4⁺ T cells into 6- to 10-week-old NOD.Rag1^–/– recipient mice. (B) Diabetes-free survival of mice described in A with pooled 9:1 and 3:1 Treg/BDC T cell treatment groups. Data are from 2–3 independent experiments. No Tregs, n = 14; 150 × 10³ control Tregs, n = 4; 450 × 10³ control Tregs, n = 9; 150 × 10³ 1B2 Tregs, n = 4; 450 × 10³ 1B2 Tregs, n = 9. Log-rank test with Bonferroni’s correction. *P < 0.05; ***P < 0.001. (C) Quantification of CD4⁺CD45.2⁺FOXP3⁺ Tregs isolated from spleen and pLNs from mice treated at 9:1 (filled circles) or 3:1 (open circles) Treg/Tconv, as indicated in pooled groups shown in B. Circles containing crosshairs depict diabetic mice, with all mice analyzed either 2 days after becoming diabetic or at day 30 after transfer. Two-way ANOVA with multiple comparisons. *P < 0.05; ***P < 0.001. (D) Representative flow cytometry plot illustrating gating strategy for identification of CD4⁺CD45.2⁺ InsBP8E tetramer⁺ and tetramer^neg FOXP3⁺ Tregs from InsB-g7 CAR Treg–treated mice shown in B. (E) The frequency of Ki67⁺ and gMFI of surface PD-1 and intracellular CTLA4 of tetramer^neg and tetramer⁺ InsBg7 CAR Tregs. Data are pooled from mice treated at 9:1 and 3:1 InsB-g7 CAR Tregs/BDC2.5 T cells and are from 2–3 independent experiments. n = 5–10 mice/group. Only mice with more than 10 CD4⁺CD45.2⁺ T cells (limit of detection) were included in the analysis. Student’s t test, 2-tailed. *P < 0.05; ***P < 0.001.