CD4+CD25+ Treg cells induce Foxp3 expression but not suppressor function in CD8+ lymphocyte targets. (A). CD8+ lymphocytes from FIV- or FIV+ cats were either untreated, ConA stimulated (5 ug/ml) or ConA stimulated for two hours then co-cultured with autologous CD4+CD25+ Treg cells for twenty-four hours. After twenty-four hours, RNA was isolated and reverse transcription RT PCR was performed on all sample groups. For the CD8+/CD4+CD25+ co-cultures, CD4+CD25+ cells were depleted by FACS prior to RNA isolation. Foxp3 induction was significantly higher in all treatment groups from FIV+ cats when compared to FIV- cats (asterisks, p < 0.05) and in ConA stimulated, CD8+ lymphocytes following CD4+CD25+ co- culture when compared to ConA stimulation alone (p < 0.05 arrows). Each bar represents the mean + SEM for six experiments. (B). CD8+ lymphocytes from FIV+ cats were ConA stimulated then co-cultured with autologous CD4+CD25+ cells to induce Foxp3 expression as described in part A. Following co-culture, CD4+CD25+ cells and CD8+ target cells were re-sorted and then co-cultured with ConA stimulated (2 hours before co-culture), autologous CD8+ lymphocytes for forty-eight hours in IFNγ ELISpot plates. Percent suppression was calculated by the following: ConA stimulated CD8+ lymphocyte SFCs ÷ ConA stimulated CD8+ lymphocytes + Foxp3+ CD8+ lymphocytes SFCs or ConA stimulated CD8+ lymphocyte SFCs ÷ ConA stimulated CD8+ lymphocytes + CD4+CD25+ lymphocytes SFCs. The box-whisker plots represent 5th and 95th percentiles (whisker), 25th and 75th percentiles (box) and median of percent suppression, dots represent individual cats. There was little suppression evident when CD8+ targets were co-cultured with Foxp3+CD8+ cells. As expected, CD4+CD25+ lymphocytes suppressed IFNγ production in CD8+ targets (p < 0.01, asterisks).