NKG2D⁺ Th17 and Th1 cells are enriched in IFN-γ and GM-CSF producers. (A and B) Frequency of IFN-γ⁺ and GM-CSF⁺ CD44⁺ CD4⁺ T cells in spleen (A) and of IFN-γ⁺, GM-CSF⁺, and IL-17A⁺ CD44⁺ CD4⁺ T cells in siLP (B) of C57BL/6 mice (n = 6–7), Wilcoxon test. Each symbol represents a mouse; lines represent mean ± SEM; data are pooled from at least three independent experiments. (C) Representative FC analysis of intracellular cytokine expression in Th17 cells, polarized with IL-1β, IL-6, IL-23, or IL-12*, on day 10 after sorting for NKG2D⁺ OT-II, NKG2D⁻ OT-II, and OT-IIxKlrk1^−/− and PMA/Iono restimulation. (D) Frequency of indicated cytokine-producing cells and per-cell content of IFN-γ among Th17 cells, quantification of C. GeoMFI, geometric mean fluorescence intensity; ns, not significant. (E) Representative FC analysis of Il17a^fm(YFP), T-bet, and intracellular cytokine expression in NKG2D⁺ and NKG2D⁻ Th17 cells, polarized as in C. (F) Frequency of Th17 cell subsets, expressing indicated cytokines, quantification of E. In D and F, each symbol represents the individual culture replicate; lines connect matching samples from individual cocultures (n = 7–10); Wilcoxon test. Data are pooled from at least three independent experiments.