Effect of anti-CD3 activation on thymic subsets and their Fas expression. After 24 hours of culture with immobilized anti-CD3 antibody or control IgG1, thymocytes were first labeled with anti-Fas antibody, then with biotin-coupled antimouse antibody, and lastly with Quantum Red-conjugated streptavidine and anti-CD4 and anti-CD8 antibodies; only the last two steps were performed in staining controls. Fas expression was analyzed in total cells and thymic subsets. (A) Representative analysis of CD4 and CD8 expression by thymocytes cultured with immobilized anti-CD3 antibody or IgG1. In comparison with the IgG1 control, anti-CD3 significantly reduced the proportion of CD4⁺CD8⁺ thymocytes and increased the intermediate subset CD8⁺CD4^lo. (B) Effect of immobilized anti-CD3 antibody on Fas expression by thymic subsets. Data are means ± SEM of 5 experiments. (C) A representative analysis of total thymocytes and thymic subsets is shown. Fas staining is shown as solid profiles and staining controls as open profiles. For each stain, the proportion of Fas^hithymocytes and MFI of Fas staining are indicated. Anti-CD3 significantly increased the Fas^hi thymocyte proportion among CD4⁺ and CD4⁺CD8⁺ cells but not among CD8⁺CD4^lo or CD8⁺cells.