Figure 6.

In vitro–generated Foxp3⁺ iTreg cells prevented graft rejection by naive CD4 T cells and allowed some naive T cells to become Foxp3⁺ themselves. Female B6.RAG^−/− received a male CBA.RAG^−/− skin graft in conjunction with either 5 × 10⁵ in vitro–cultured female hCD2⁺ RAG^−/− Marilyn.Foxp3^hCD2 DBYT cells (squares, n = 6), 1 × 10⁵ naive CD4⁺ cells from RAG^−/− Marilyn^WT mice (circles, n = 6), or a mixture of both (diamonds, n = 8). (A) All of the mice that received 1 × 10⁵ naive CD4⁺ cells rejected the male skin compared with none of those that received 5 × 10⁵ hCD2⁺ cells (P = 0.0066) and half that received a mixture (P = 0.0247). After 100 d, the mice were killed and Foxp3⁺ cell composition was analyzed. (B) Total white cell spleen counts were similar between the three groups, but there were more CD4⁺ cells in mice that received a mixture of hCD2⁺ and naive CD4⁺ cells. (C) FACS analysis demonstrated conversion of naive CD4⁺ cells into Foxp3⁺ T cells. Over 98% of Foxp3⁺ cells in mice that received 5 × 10⁵ isolated hCD2⁺ RAG^−/− Marilyn.Foxp3^hCD2 cells alone were hCD2⁺, whereas 18% of Foxp3⁺ cells in the draining lymph and 24% in the spleen of mice that received a 5:1 mixture (and had accepted the male graft) were hCD2 negative (P = 0.0009). Error bars represent SEM.