Figure 3

CD4⁺CD25⁺Treg cells from FIV-infected cats are capable of converting CD4⁺CD25^-Th cells from FIV-negative cats into phenotypic iTreg cells. A. Untreated CD4⁺CD25^- T cells, soluble TGFβ and ConA treated CD4⁺CD25^- cells, or CD4⁺CD25^- cells from control cats co-cultured with Treg cells from FIV⁺ cats were analyzed by flow cytometry for the Treg surface phenotypic markers CD25, GARP, TGFβ and TGFβRII after the 5 days in culture. Representative dot plots are shown. The percent of cells expressing each marker is at the top right of each dot plot. B. CD4⁺CD25^- cells treated as described for (A), as well as CD4⁺CD25^- cells treated with IL2 alone or ConA alone were analyzed for expression of CD25, GARP, TGFβ, TGFβRII and FoxP3. Bars represent the mean ± SEM percent of cells expressing each marker from three independent experiments from separate cats. C and D. Target CD4⁺CD25^- cells from two FIV⁺ cats were cultured as described above then analyzed for FoxP3 (C) or GARP (D) mRNA expression by PCR. Lanes 1: freshly isolated CD4⁺CD25⁺ Treg cells from Cat A. Lanes 2 and 3: CD4⁺CD25^- cells from Cat A (2) or Cat B (3) cultured for 5 days with IL2 alone. Lanes 4 and 5: CD4⁺CD25^- cells from Cat A (4) and Cat B (5) cultured for 5 days with soluble TGFβ (10 ng/mL) and ConA (5 ug/mL). Lanes 6 and 7: ConA-activated CD4⁺CD25^- cells from Cat A (6) and Cat B (7) co-cultured for 5 days with autologous CD4⁺CD25⁺ then purified by FACS. The target cells cultured with sTGFβ/ConA or with activated Treg cells express FoxP3 and GARP mRNA.