ALDH1A1 expression and efflux capacity both appear to contribute to protection from mafosfamide-induced cytotoxicity. Human MLC was performed as per Figure 1, except that some groups received CsA (multidrug resistance [MDR] transporter inhibitor), PK11195 (broader MDR transporter inhibitor), or DEAB (ALDH inhibitor) from days 0 to 3. All drugs were washed off after the 1-hour treatment with vehicle or mafosfamide on day 3; CsA, PK11195, and DEAB were not added back to the cultures. (A-B) Blocking ALDH and/or MDR transporter activity sensitized CD8⁺ T cells to mafosfamide with the combination of PK11195 and DEAB having a maximal effect, suggesting that both pathways may contribute to CD8⁺ T-cell resistance to mafosfamide. (C-D) T cells were isolated from healthy donor PBMCs and plated either alone (C) or in MLC (D) as in Figure 1; some groups received exogenous IL-2 (1 IU/mL) or IL-7 (10 ng/mL) on day 0, and the IL-2 or IL-7 was not washed out until after mafosfamide (or vehicle) treatment on day 3. The addition of IL-2 or IL-7 before mafosfamide increased the percentage of viable CD8⁺ T cells on day 7. For IL-7 but not IL-2, this was in part because of significantly increased proliferation and apparent expansion of surviving T cells after IL-7 treatment. Combined results from 3 (A) or 2 (B-D) independent experiments are shown. *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. Comparisons for (B) were performed between all groups. For (C-D), mafosfamide-treated groups were only compared with each other, as were non-mafosfamide-treated groups, to assess the relative impact of IL-2 or IL-7 administration, the scientific question of interest.