Dnmt1^−/− Tregs lack suppressive function in vitro and in vivo. (A) In vitro Treg assays assessing their ability to inhibit the TCR-induced proliferation of CFSE+ CD4+ CD25– Tcon cells. Upper panels compare Dnmt1^−/− Tregs isolated from CD4-Cre/dnmt1^−/− mice versus WT Tregs; lower panels compare Dnmt1^−/− Tregs isolated from Foxp3-Cre/dnmt1^−/− mice versus WT Tregs; the proportions of proliferating Tcon cells is shown at each ratio of Treg:Tcon cells. (B) Cumulative data (mean ± SD) from 4 such assays as shown in previous panel. (C) Lack of Dnmt1^−/− Treg suppressive function in vivo in homeostatic proliferation assays involving transfer of WT Tcon cells plus WT or Dnmt1^−/− Tregs to B6 RAG^−/− mice; data (mean ± SD) from 4 mice per group, and comparable results were observed in 2 separate experiments. (D) Cardiac allograft survival in B6 Rag^−/− mice engrafted with BALB/c hearts and reconstituted with B6 CD4+CD25– T con cells and either WT or Dnmt1^−/− Treg cells; Kaplan-Meier plot with 4 animals per group, and comparable data were observed in 2 separate experiments.