Mafosfamide treatment on day 3 of human mixed lymphocyte culture kills many CD8⁺ T cells, but the surviving cellular composition on day 7 is more similar to the compositions within vehicle-treated cultures than within cultures treated with CsA or rapamycin. Human CD3⁺ T cells and CD3⁻ cells were obtained from the fresh blood of healthy donors via Ficoll density–gradient separation and immunomagnetic selection. CD3⁺ T cells first were labeled with CFSE 2.5 µM and then placed in mixed lymphocyte cultures (MLCs) with irradiated (30 Gy) allogeneic MHC-mismatched CD3⁻ PBMCs in a 1:1 ratio of 1 × 10⁵ of each cell type per well. Cells were either vehicle (PBS)-treated, treated with mafosfamide (Maf) 7.5 µg/ml as a 1-hour incubation on day 3, or treated with CsA (600 ng/mL) or rapamycin (Rap, 15 ng/mL) from days 0 to 7. Six wells per treatment group were pooled for analyses. Total numbers or percentages of different CD8⁺ T-cell subsets were determined using the gating schema shown in supplemental Figure 1. Combined results of 2 independent experiments are shown with n = 12 per group for parts (B-H) except for the MAIT and CD161⁺ non-MAIT total numbers and percentages in parts (F) and (I) (n = 6 per group). *P < .05, **P < .01, ***P < .001, ****P < .0001, and ns, not significantly different on repeated-measure one-way ANOVA followed by the Holm-Sidak post hoc test compared with the control group. CFSE, carboxyfluorescein succinimidyl ester; CM, central memory; EM, effector memory; MAIT, mucosal-associated-invariant-T cells; PBMC, peripheral blood mononuclear cells; TEMRA, terminally differentiated effector memory expressing CD45RA.