Fas/FasL and MHC class I are not required for Tr1-induced cytotoxicity. (A) FACS analysis for expression of FasL on CD4⁺ and CD8⁺ effector T lymphocytes. Methods of stimulation (over 4 days) are shown. (B) A FasL blocking antibody (clone DX2; 10 μg/200 μL) does not block the cytotoxicity of Tr1 regulatory cells against target cells (U937 and K562) at 4 and 8 hours. E/T ratios and EGTA controls for perforin-dependent cytotoxicity are shown. Data are representative of more than 3 independent experiments showing similar results. Values shown are the mean ± SD; ^*P < .05; ^**P < .01; ^***P < .001. (C) FACS analysis for Fas, MHC class II (DR), and MHC class I (A, B, C) on U937 and K562 cells. Percentage of total cells in each quadrant is shown in the upper right corner of each subpanel in panels A and C.