Figure 7.

Transforming growth factor (TGF)-β2-induced regulatory T cells (iTregs) are equally as potent as TGF-β1-iTregs in ameliorating allograft rejection. CD4⁺ cells were purified from naïve BL6 FoxP3^GFP mice and cocultured with allogeneic splenic APCs along with anti-CD3ε (10 µg/mL), anti-CD28 (4 µg/mL), and IL-2 (10 ng/mL). Tβ1- and Tβ2-iTregs were also administered TGF-β1 (5 ng/mL) or TGF-β2 (5 ng/mL), respectively. Coculture proceeded for 3 days at which point the cells were either analyzed for FoxP3^GFP expression, or sorted into CD4⁺ FoxP3^GFP+ cells and injected intravenously into graft-recipient mice 1 day before transplantation. (B) Histograms of FoxP3-GFP expression gated on dot plots of CD4⁺CD25⁺ cells in (A). Female C57BL/6 mice were given either syn (BL6) or allo (C₃H) tail skin grafts. Mice receiving allografts were administered 1 × 10⁶ iTregs intravenously 1 day before transplant. Grafts were scored starting 7 days after transplantation and continued until mice were sacrificed on day 12. Graft-infiltrating cells (GICs), draining lymph nodes (dLNs), and blood were collected for flow cytometric analysis. (C) Survival curve of indicated groups. (D,F) Pseudocolor plots of GFP⁺, CD4⁺ co-expressing iTregs in the (D) dLN and (F) among GICs. (H) Pseudocolor plots displaying graft-infiltrating CD4⁺ and CD8⁺ naïve, memory, and effector phenotypes. (K) Dot plots displaying IFNγ, CD8⁺ CTLs. Flow cytometry results quantified in (B,E,G,I,J,L). n = 5 (iTreg), 10 (Tβ1-iTreg), or 9 (Tβ2-iTreg) mice per group. Data are presented as mean ± SEM. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001 by ANOVA with Tukey’s multiple comparisons test, or a log-rank (Mantel–Cox) test for the survival curve.