CD8⁺CD25⁺ thymocytes do not proliferate in MLCs and suppress the proliferation in MLCs of CD4⁺CD25^- thymocytes via a contact-dependent mechanism. (A) Proliferative response of purified CD4⁺CD25^-, CD4⁺CD25⁺, CD8⁺CD25^-, or CD8⁺CD25⁺ human thymocytes to allogeneic stimulation. On day 5, cells were harvested and their proliferation assessed by measuring ³H-TdR uptake. Mean values (± SD) obtained in 9 separate experiments are reported. (B) Suppression by CD4⁺CD25⁺ or CD8⁺CD25⁺ thymocytes, but not by CD8⁺CD25^- thymocytes, of the proliferative response of autologous CD4⁺CD25^- thymocytes to allogeneic T-cell-depleted PBMNCs. On day 5, cells were harvested and proliferation assessed by measuring ³H-TdR uptake. Mean values (± SD) obtained in 9 separate experiments are reported. (C) Cell contact dependency of suppressive activity exerted by CD8⁺CD25⁺ thymocytes. CD4⁺CD25^- thymocytes were stimulated with irradiated T-cell-depleted allogeneic PBMNCs in the lower chamber of a transwell plate in the absence or presence of CD8⁺CD25⁺ thymocytes, which were placed in the same chamber or in the top chamber. CD8⁺CD25⁺ thymocytes placed in the upper chamber were cultured in medium alone or stimulated with 10⁶ irradiated T-cell-depleted allogeneic PBMNCs in the presence or absence of 5 × 10⁵ CD4⁺CD25^- cells. On day 5, cells present in the bottom chamber were harvested and their proliferation assessed by measuring ³H-TdR uptake. The mean values (± SD) obtained in 4 separate experiments are reported. ^*P < .01.