CD4⁺CD25⁺ Treg cells inhibited ESAT-6–specific IFN-γ production by γδ T cells. (A) Isolation of CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells from PBMCs. The numbers in each quadrant represent the percentages of positive cells in each cell population. (B) Purification of γδ T cells from PBMCs. (C) Suppressive effects of CD4⁺CD25⁺ T cells on IFN-γ production by γδ T cells. PBMCs or purified γδ T cells were cultured alone, with CD4⁺CD25⁻ T cells, or with CD4⁺CD25⁺ T cells (at ratio of 3:1) in the presence or absence of ESAT-6 plus anti-CD28. The supernatants were collected for IFN-γ production. (D) Addition of neutralizing antibody to IL-10 promoted IFN-γ production. The results represent the average of triplicate wells (mean ± SD). *P > .05; **P < .05; ***P < .001.