Innate cytokines present during TCR signaling induce proliferation of CD161^hi CD8⁺ T cells. (A) Sort-purified CD161^hi CD62L⁺ (left) and CD62L⁻ (right) cells were cultured with αCD3 with or without IL-1β, IL-12, IL-18, IL-23, or αCD28 for 72 hours before assessment of proliferation by tritiated thymidine incorporation. The broken lines represent the stimulation indices of CD161^lo CD62L⁺ (left) and CD161^lo CD62L⁻ cells (right) obtained after stimulation with αCD3 alone. The P values (*P < .05, **P < .01) refer to the difference between the stimulation indices of cells stimulated with αCD3 in the presence of the indicated costimulation and cells stimulated with αCD3 alone (Nil). Data represent the mean ± SE of 4 separate experiments. (B) Costimulation with IL-1β, IL-18, and IL-23 has a more selective effect on CD161^hi cells than on CD161^lo cells. Sort-purified subsets were cultured with αCD3 with or without IL-1β, IL-12, IL-18, or IL-23 for 72 hours before assessment of proliferation. Data represent the mean ± SE of at least 4 separate experiments. (C) Sort-purified CD161^hi CD62L⁺ and CD62L⁻ cells were stimulated with PMA and ionomycin before intracellular staining with antibodies to IL-17. Data from 5 individuals are depicted. (D) CD161^hi CD62L⁺ (left) and CD62L⁻ cells (right) were stimulated with αCD3/αCD28 and IL-1β, IL-12, IL-18, or IL-23. After 14 days, the cells were stimulated with PMA/ionomycin and the fraction of IL-17 secreting cells was determined by intracellular cytokine flow cytometry. Data represent the mean ± SE of at least 5 separate experiments. P values were determined by 1-way ANOVA.