CD8⁺Foxp3⁺ and CD4⁺Foxp3⁺ cells share phenotypic markers and exhibit potent suppressive function. Irradiated CD45.2⁺ B6D2F1 mice (H-2^bxd) were injected with splenocytes and T cell–depleted BM from CD45.1⁺ FoxP3.GFP mice (H-2^b). (A) On day 8 after transplantation, CD45.1⁺ CD4⁺ T cells (top panels) and CD8⁺ T cells (bottom panels) were analyzed for expression of CD25, CD122, OX40, GITR, and CTLA4. The shaded and open histograms represent FoxP3⁺ and FoxP3⁻ cells, respectively. (B) CD4⁺ and CD8⁺ Tregs (GFP⁺) and CD8⁺ Tconvs (GFP⁻) were FACS sorted from spleens on day 8 after transplantation and cocultured with CFSE-labeled CD4⁺ Tconvs, irradiated splenocytes, and anti-CD3 Ab at the indicated T cell:Tconv ratios. A representative CFSE dilution plot by Tconvs is shown in cultures without Tregs or at a 1:1 T cell:Tconv ratio. (C) A corresponding percent divided value was calculated with FlowJo Version 8.8.7 software and plotted against all T cell:Tconv ratios tested. One representative of at least 2 independent experiments is shown.