Comprehensive phenotypic characterization of CD4⁺CD161⁺Rho^loT cells by multiparameter flow cytometry. PBMCs were stained with a panel of surface markers including monoclonal antibodies against CD16, TCR Vα24, CD8β, and γδ TCR to exclude natural killer, natural killer T, CD8^dim, and γδ T cells, respectively. (A) Multiparameter analysis of gated CD4⁺CD161⁺ T cells revealed a mixture of effector (CD45RA⁻CD62L⁻CCR7⁻), central memory (CD45RA⁻CD62L⁺CCR7⁺), and naive-like (CD45RA^intCD62L⁺ CCR7⁺) subsets; cumulative data from 5 independent experiments are shown. ***P < .001. (B) Contour plots showing expression of memory markers, CD45RO (left) and CD95 (right), on CD4⁺CD95^neg, CD4⁺CD161⁺CD45RA^int, and CD4⁺CD161⁺ T cells. (C) Regulatory T cells lack ABCB1 activity. Representative fluorescence-activated cell sorter (FACS) plots showing the frequency of CD25⁺Foxp3⁺ T cells (upper) in CD4⁺CD161⁺Rho^lo (left), CD4⁺CD161⁺Rho^hi (middle), and CD4⁺CD161⁻ (right) T cells; cumulative data from 9 independent experiments are shown in right lower panel. **P < .01; ****P < .0001. Histogram in lower left panel compares the mean fluorescence intensity (MFI) of CD127 among CD4⁺CD161⁺Rho^lo (red), CD4⁺CD161⁺Rho^hi (blue), and CD4⁺CD161⁻ (green) T-cell subsets. (D) Expression of c-kit and CD57 on CD4⁺CD161⁺Rho^hi and CD4⁺CD161⁺Rho^lo T cells is mutually exclusive. Representative FACS plots show that c-kit expression is restricted to the rapidly effluxing CD4⁺CD161⁺ subset, whereas CD57 expression is restricted to the CD4⁺CD161⁺Rho^hi T-cell subset with no detectable expression on CD4⁺CD161⁺Rho^lo T cells; cumulative data from 4 independent experiments are shown. **P < .01; ****P < .0001.