Preactivated WKO nTreg cells can suppress T-cell but not B-cell proliferation. nTreg, Tconv, and CD8⁺ T cells were isolated from WT and WKO mice and preactivated with anti-CD3 and IL-2. (A) Proliferation of freshly isolated Tconv (5 × 10⁴) from WT mice cocultured with the indicated number of WT and WKO preactivated nTreg or Tconv cells in the presence of anti-CD3 (2 μg/mL) and irradiated, T-depleted APCs. (B) B cells (5 × 10⁴) from WT mice were stimulated with LPS (3 μg/mL) and cocultured with the indicated number of preactivated nTreg, Tconv, or CD8⁺ cells from both WT or WKO mice in the presence of soluble anti-CD3 (2 μg/mL) and irradiated T-depleted APCs. (C) B cells isolated from WKO mice were cultured as in panels A and B. Proliferation was then assessed as incorporation of [³H]-thymidine after the cells were pulsed for the last 16 hours during a total of 72 hours of cultures. Note that the proliferation of effector T cells (nTreg, Tconv, and CD8 T cells) cultured alone in the presence of soluble anti-CD3 and LPS stimulation is also shown. Statistical significance of differences between samples was calculated using the 2-tailed Mann-Whitney test with 95% confidence intervals. Results are mean of triplicate cultures and are representative of at least 3 experiments. P < .05.