Activated CD8⁺CD25⁺ human thymocytes do not produce cytokines, but express CTLA-4 and TGF-β1 on their surface. (A) CD8⁺CD25^- or CD8⁺CD25⁺ thymocytes were incubated in the presence of insolubilized anti-CD3 and anti-CD28 mAbs. On day 6, cells were stimulated for 6 hours with PMA plus ionomycin, and the last 2 hours of stimulation occurred in the presence of brefeldin A, and cytokine synthesis at the single-cell level was analyzed by flow cytometry. (B) Detection of CTLA-4 and TGF-β1 by flow cytometry on the surface of the same cell populations. Histograms in the bottom panels represent stainings obtained with isotype-matched mAbs. A representative experiment is shown.