NKG2D modulates the cytokine production of CD4⁺ Th cells in vitro. (A) Frequency of indicated cytokine-producing cells and per-cell content of IFN-γ among Th17 polarized with IL-1β, IL-6, or IL-23. Each symbol represents the individual coculture replicate; line connects matching samples (n = 7–10); Wilcoxon test. Data are pooled from at least three individual experiments. GeoMFI, geometric mean fluorescence intensity. (B) Representative FC analysis of intracellular cytokine expression in Th1 cells on day 10 after sorting for NKG2D⁺ OT-II, NKG2D⁻ OT-II, and OT-IIxKlrk1^−/− and PMA/Iono restimulation. (C and D) Frequency of IFN-γ⁺ and per-cell content of IFN-γ in NKG2D⁺ (red) and Klrk1^−/− (black) 1-wk (C) or 2-wk (D) differentiated Th1 cells. Each symbol represents an individual culture; line represents mean ± SEM, n = 7 (C), n = 17 (%), or 6 (GeoMFI; D). Wilcoxon test. Data are pooled from at least three individual experiments. (E) Frequency of GM-CSF⁺ among NKG2D⁺ (red) and Klrk1^−/− (black) Th1 cells. Each symbol represents an individual culture from at least three independent experiments; line represents mean ± SEM; n = 10. (F and G) Frequency of cells undergoing 0, 1, 2, 3, 4, and more divisions (div.) among WT and Klrk1^−/− Th1 or Th17 cells (day 3) or among NKG2D⁺ WT, NKG2D⁻ WT and Klrk1^−/− Th1 or Th17 cells (day 8). Th17 cells were polarized as in A. Data shown are mean ± SEM; n = 3; pooled from at least two independent experiments.