Fas expression is differently regulated by activation signals in human thymocytes. Fas and CD25 expression were analyzed after culture with the following activators: immobilized anti-CD3 (mouse IgG, 10 μg/mL), PHA (1 μg/mL), and the combination of PMA (5 ng/mL) and ionomycin (500 ng/mL). Control cultures were performed in the absence of agents or with immobilized mouse IgG1 (10 μg/mL). Thymocytes were collected after 4, 16, 24, and 48 hours of culture and Fas, CD4, and CD8 expression was examined using three-color flow cytometry. CD25 expresssion was also measured to check the level of cell activation. A representative experiment is shown. (A) Analysis of Fas expression after 24 hours of culture with the different activators. Fas staining is shown as solid profiles and staining controls as open profiles. Thymocytes were first labeled with anti-Fas antibody, then with biotin-coupled antimouse antibody, and lastly with Quantum Red-conjugated streptavidine and anti-CD25 or anti-CD4 and anti-CD8 antibodies; only the last two steps were performed in staining controls. Anti-CD3 antibody-activated thymocytes display the higher increase in the Fas^hi thymocyte proportion and in the MFI of Fas staining. (B) Kinetic study of Fas and CD25 expression on thymocytes cultured with activation factors. CD25 expression was maximal after 16 hours of activation and Fas expression after 24 hours. By contrast with Fas expression, CD25 expression was strongly increased by PMA + ionomycin but moderately increased by anti-CD3 activation.