CD4⁺CD161⁺T cells with rapid efflux activity have the capacity to proliferate and differentiate. (A) Representative histograms showing the proliferative response of CD161⁺ (blue) and CD161⁻ (red) CD4⁺ T cells to stimulation with anti-CD3/CD28 beads; cumulative data from 8 independent experiments are shown. ***P < .001. (B) Representative histograms showing the proliferative response of FACS-purified CD4⁺CD161⁺Rho^lo T cells that were further selected based on Tcm (CD45RA⁻CD62L⁺) (upper) or Tem (CD45RA⁻CD62L⁻) phenotype (bottom). Each FACS-sorted subset was stimulated under 3 different conditions: anti-CD3/anti-CD28 alone, anti-CD3/anti-CD28+IL-7/IL-15 and IL-7/IL-15 alone for 10 days. Proliferation was assessed by the CFSE dilution assay on day 10 of culture. (C) Expression levels of CD161 on sort-purified CD161⁺Rho^lo CD4⁺ Tcm and Tem cells after stimulation with anti-CD3/anti-CD28 alone, anti-CD3/anti-CD28+IL-7/IL-15, and IL-7/IL-15 alone on day 10. (D) Quantitative analysis compares CD161 expression on CD161⁺Rho^lo (left) and CD161⁺Rho^hi (right) by different proliferative stimulus for Tcm and Tem subsets. (E) Homeostatic cytokines, IL-7 and IL-15, instruct self-renewal and maintain retention of rapid effluxing function. Sort-purified CD161⁺Rho^lo, CD161⁺Rho^hi, and CD161⁻CD4⁺ T cells were stimulated with anti-CD3/anti-CD28, anti-CD3/anti-CD28+IL-7/IL-15, and IL-7/IL-15 alone for 10 days. Representative FACS plots showing Rho efflux by CD161⁺Rho^lo, CD161⁺Rho^hi, and CD161⁻CD4⁺ T cells after incubation at 37°C for 30 to 60 minutes compared with negative control on day 10.