CD161⁺⁺ CD8αα T cells can be derived from CD161⁺⁺CD8αβ cells in culture. (A) CD161⁺⁺, CD161⁺ and CD161⁻ populations of the CD8 enriched/CD8β⁺ selected PBMCs remain stable over the 7 days of culture in R10 media alone. (B) Representative FACS plots demonstrating change in CD8β expression of CD161⁺⁺, CD161⁺ and CD161⁻ populations of the CD8 enriched/CD8β⁺ selected PBMCs in media alone over 7 days of culture. CD8αα population seen only in CD161⁺⁺ population. (C) Representative histograms showing change in CD8β expression of the CD161⁺⁺CD8α⁺ population of the CD8 enriched/CD8β⁺ selected PBMCs over 7 days of culture (top panel). Cumulative data shown (bottom panel), *P < .05, **P < .001, and ***P < .0001 (1-way ANOVA). Data representative of 4 experiments. (D) Cumulative data showing no significant change in the CD8α⁺ T-cell population of cultures over course of experiment (1-way ANOVA). (E) Ki-67 proliferation data for sorted CD8β⁺ cells left in culture media alone or with CD3/CD28 beads for 48 hours. Baseline Ki-67 staining (gray), after culture Ki-67 staining (black). Data representative of 2 experiments.