NKG2D is induced de novo on naive CD4⁺ T cells under Th1- and Th17-polarizing conditions. (A) Representative FC analysis of NKG2D expression on Th0, Th1, or Th2 cells on day 10 of culture (n = 6). (B) Frequency of NKG2D⁺ Th1 cells, differentiated using plate-bound α-CD3 stimulation, at indicated time points of the polarizing culture. Data shown are mean ± SEM (n = 5–15), Kruskal–Wallis test with multiple comparison correction. ns, not significant. (C) Representative FC analysis of NKG2D expression on Th17 cells, polarized as indicated, on day 10. Asterisk (*) indicates that IL-12 was added in the second week of culture. (D) Frequency of NKG2D⁺ Th17 cells, polarized as indicated, as measured on day 8 or 10 of culture; black inverted triangle (n = 7), black triangle (n = 18), and black circle (n = 7). Each symbol represents an individual culture replicate; lines represent mean ± SEM. Kruskal–Wallis test with multiple comparison correction. (E and F) Representative FC analysis (E) and frequency of Il17a^Kata expression in Th17 cells (F), from Il17a^fm(YFP) × Il17a^Kata double reporter mice–derived naive CD4⁺ T cells, polarized as indicated, at day 10 of culture (n = 3). Each symbol represents an individual culture replicate; lines represent mean ± SEM. (G) Representative FC analysis of T-bet and Il17a^(fm)YFP expression in Th17 cells, polarized as in E. (H) Frequency of Il17a^fm-T-bet⁺ and Il17a^fm+T-bet⁺ cells among Th17 cells polarized as indicated (n = 5). Data shown as mean ± SEM; Mann–Whitney U test. (I) Representative FC analysis of NKG2D expression in Th17 cell subsets, polarized with IL-1β, IL-6, IL-23, and IL-12*, indicated in G; blue square, T-bet⁺fm⁻; red square, T-bet⁺fm⁺; green square, T-bet⁻fm⁺ cells. (J) Frequency of NKG2D⁺ Th17 cells, quantification of I. Each symbol represents an individual culture replicate; data shown as mean ± SEM (n = 11). Kruskal–Wallis test with multiple comparison correction. In A–J, data are pooled from at least three independent experiments.