Granzyme B-expressing Tr1 regulatory cells exhibit perforin-dependent cytotoxicity. (A) An example of the flow-based cytotoxicity assay (FloKA) using CFSE-labeled target cells, which can be specifically gated upon and analyzed for 7-AAD incorporation. (B) Representative example of cytotoxicity induced by Tr1 regulatory cells (ie, CD3/CD46-activated CD4⁺ T lymphocytes), demonstrating time-dependent 7-AAD incorporation in human U937 target cells. Cytotoxicity was blocked by EGTA (4 mM), which blocks calcium-dependent perforin polymerization. Percentage shown indicates the percentage of cells shown in each regional gate depicted. (C) Representative example of cytotoxicity by IL-2-activated CD4⁺ T lymphocytes versus Tr1 regulatory cells (ie, CD3/CD46 activated), against human target cells (U937 and K562) at 4 and 8 hours. Effector to target (E/T) ratios and EGTA controls are shown. (D) Inhibition of cytotoxicity by concanamycin A (CMA; 100 nM). Data are representative of at least 4 independent experiments showing similar results. Values shown are the mean ± SD; ^*P < .05; ^**P < .01; ^***P < .001.