Suppression mediated by CD8⁺CD25⁺ thymocytes is mediated by CTLA-4 and TGF-β1 and is due to the inhibition of IL-2Rα chain expression. (A) CD4⁺CD25^- thymocytes were stimulated with allogeneic irradiated T-cell-depleted PBMNCs in the presence of different numbers of autologous purified CD8⁺CD25⁺ thymocytes without (IgG1 + IgG2a isotype control; black bars) or with anti-CTLA-4 (dark gray bars), anti-TGF-β1 (light gray bars), or a mixture of both (anti-mix; white bars) mAbs. Cell proliferation was measured by assessing ³H-TdR incorporation. Mean values (± SD) obtained in 4 separate experiments are reported. (B) Effect of the addition of CD8⁺CD25⁺ thymocytes on the expression of CD25 and CD69 by CD4⁺CD25^- autologous thymocytes, as detected on day 5 of culture by flow cytometry. Mean values (± SD) obtained in 4 separate experiments are reported. ▪ indicates isotype mAb; and □ indicates anti-mix. (C) IL-15, but not IL-2, restores the proliferative response of CD4⁺CD25^- thymocytes suppressed by CD8⁺CD25⁺ thymocytes. Cell proliferation was measured by assessing ³H-TdR incorporation. Mean values (± SD) obtained in 4 separate experiments are reported. □ indicates CD4⁺CD25^- thymocytes; and ▦ indicates CD4⁺CD25^- plus CD8⁺CD25⁺ (ratio 1:1).