Figure 2.

Experimental design for CD4⁺CD25⁺Treg cell-mediated conversion of feline CD4⁺CD25^-T cells to iTreg cells. Single-cell suspensions were prepared from PLNs collected from FIV-infected or uninfected control cats. Cells were stained with anti-feline CD25 FITC and anti-feline CD4 PerCP labeled antibodies and CD4⁺CD25⁺ T cell populations purified by FACS. Converter CD4⁺CD25⁺ cells from control cats were stimulated with LPS/IL2 for 4 days, whereas CD4⁺CD25⁺ cells from FIV-infected cats are constitutively active and therefore were not stimulated prior to co-culture. PLN cells from FIV negative cats were FACs purified into CD4⁺CD25^- Th target cells or CD4^-CD8^- APCs and stimulated for 4 hours with ConA. Converter cells were labeled with Vybrant DiD membrane dye and co-cultured with the purified CD4⁺CD25^- Th cells at a converter to target cell ratio of 1:2 in medium supplemented with 100 U/mL IL2. After 5 days, DiD membrane negative cells were FACS purified from the co-culture and analyzed for phenotype and function. In some experiments, anti-TGFβ, anti-GARP, or anti- TGFβRII antibodies were added prior to the conversion assay or prior to the functional assay.