A subset of CD4⁺T cells efflux Rhodamine123 and daunorubicin. PBMCs were incubated with Rho for 30 to 60 minutes at 37°C, after which multicolor FACS analysis was performed on gated CD4⁺Rho^lo (effluxing subset) and CD4⁺Rho^hi cells (non-effluxing subset). (A) Representative FACS plot showing Rho efflux by CD4⁺ T cells after incubation at 37°C for 30-60 min (right) compared with negative control (left); cumulative data from 5 independent experiments are shown; ***P < .001. (B) Histograms comparing the phenotypes of effluxing (left) and non-effluxing (right) subsets within the total CD4⁺ T cell compartments. Note enrichment of Rho effluxing CD4⁺ T cell subsets within the CD161⁺ population; cumulative data from 4 independent experiments are shown; **P < .01, ***P < .001 and ****P < .0001. (C) Sort-purified CD4⁺CD161⁺Rho^lo, CD4⁺CD161⁺Rho^hi and CD4⁺CD161^- T cell subsets were stained for ABCB1 and examined by laser scanning confocal microscopy. Representative images from CD4⁺CD161⁺Rho^lo (left), CD4⁺CD161⁺Rho^hi (middle) and CD4⁺CD161^- (Right) T cell subsets are shown. ABCB1 (red) is only expressed on CD4⁺CD161⁺Rho^lo. DAPI (blue) was used as counterstaining. (D) FACS plots showing the abrogation of Rho efflux by CD4⁺CD161⁺ T cells after coincubation with inhibitors of ABCB1 and ABCC1 (cyclosporine A, PK11195 and vinblastine). (E) PBMCs were coincubated with fluorescent anthracycline (daunorubicin) for 30 to 60 min at 37°C. FACS plots show daunorubicin efflux by CD4⁺ T cells and complete abrogation of efflux upon addition of ABCB1 inhibitors (cyclosporin A and PK11195) and ABCC1 (vinblastine). (F) Histograms show that the gated daunorubicin-effluxing CD4⁺ T cells (red histogram) are enriched within the CD95⁺ CD161⁺ memory T-cell compartment compared with their non-effluxing counterpart (blue histogram); cumulative data from 3 independent experiments are shown; **P < .01 and ***P < .001.