Figure 5.

Vitamin C-treated allo-iTregs can efficiently prolong skin allograft survival. One day before skin transplantation, Foxp3^RFP+ cells (CD45.2⁺) were sorted from allo-iTreg cultures (±vitamin C) and adoptively transferred together with freshly isolated CD4⁺ naïve T cells (CD45.1⁺) into Rag2^−/− (C57BL/6) mice. One day later, mice received an allogeneic skin transplant (BALB/c) and graft survival was monitored over a period of 100 days. Rag2^−/− mice receiving a syngeneic (C57BL/6) or allogeneic (BALB/c) skin transplant without adoptive T cell transfer were taken as graft survival controls. (A) Graph depicts percentage of graft survival for each group, and data are summarized from two independent experiments; syngeneic graft survival control (n = 2, black circle), allogeneic graft survival control (n = 3, black diamond), + allo-iTregs (−vitamin C) (n = 7, blue triangle) and + allo-iTregs (+vitamin C) (n = 9, red triangle). Data were tested for significance using the long-rank (Mantel cox) test; ***p < 0.001. (B) Upon graft rejection or on day 100, Foxp3 expression among adoptively transferred allo-iTregs within spleen, mesenteric, and axial lymph nodes (LN) was analyzed by flow cytometry. Representative histograms from indicated organs show frequency of Foxp3^RFP+ cells among CD3⁺CD4⁺CD45.1⁻ cells. (C) Graphs show frequency of Foxp3^RFP+ cells among CD3⁺CD4⁺CD45.1⁻ cells in indicated groups, and each dot represents an individual mouse. Data are summarized from two independent experiments (mean ± SD) and tested for significance using Mann–Whitney test; *p < 0.05; ***p < 0.001.