CD8⁺ T-cell subsets increase drug efflux capacity after stimulation in mixed lymphocyte culture. Human T cells were immunomagnetically isolated from fresh, healthy donor PBMCs and either assessed for Rh-123 effluxing immediately on day 0 or stimulated in MLC for 3 or 7 days before assessing for Rh-123 effluxing. Each sample was divided after Rh-123 loading, and half of the sample was treated with 600 ng/mL CsA, a multidrug resistance transporter inhibitor, during the effluxing to serve as a noneffluxing control for each sample. (A-B) Flow cytometry plots of Rh-123 are shown for a representative donor on day 0 (A) and day 3 of MLC (B). The vertical gray line indicates the threshold for effluxing determined based on the CsA control. (C) Percentages of different T-cell subsets that were Rh-123 effluxing are shown on day 0 (n = 3) and days 3 and 7 of MLC (n = 6). These percentages increased significantly for each subset between day 0 and day 3 except for the CD25⁺ subset (P = .15). These percentages overall somewhat declined between day 3 and day 7, but these differences were not significant except for the CM (P = .0086) and Tscm (P = .012) subsets.