Cultured CD8+ T cells suppress naïve T cell proliferation. (a) Dose-dependent suppression of CD4+CD25- responder T cells by cultured CD8+ T cells. CFSE-labeled CD4+CD25- naïve T cells (Tn) isolated from B6 spleens were stimulated with anti-CD3 (1.5 μg/ml) in the presence of irradiated splenic APCs with graded numbers of cultured CD8+ T cells (Tr). After 72 h in the culture, CFSE dilution in the responder CD4+ T cells was analyzed by flow cytometry. T cells in the M2 zone are undivided cells and T cells in the M3 zone with lower CFSE are divided cells. Data are representative of five independent experiments. (b) Naïve CD4+CD25- splenic T cells were cultured in the same condition as shown in (a). The cultures were pulsed with 1 μCi/well [3H]thymidine at 72 h and the level of proliferation was assessed by [3H]thymidine incorporation in the last 16 h of culture. (c) Cultured CD8+ T cells (NKT-like), freshly isolated CD4+CD25+ Treg cells and cultured CD4+ cells (cCD4) were compared for their ability to suppress the proliferation of CD4+CD25- responder T cells. Data are presented as percentage of suppression based on the CFSE dilution with standard deviation. ANOVA test suggests that the suppressive ability is significantly different between these cells (p < 10-6).