NKG2D signaling promotes IFN-γ and GM-CSF expression. (A) Representative FC analysis of NKG2D on WT or Klrk1^−/− Th1 cells after gating on CD94⁺ cells on day 10 of culture. (B) Representative FC analysis of ERK1/2 phosphorylation after antibody cross-linking of CD3, CD3 and NKG2D (clone A10), or CD3 and CD28 in WT and Klrk1^−/− Th1 cells. (C) Frequency of pERK1/2⁺ Th1 cells (left) and per-cell content (right) of pERK1/2, quantification of B. Data shown are pooled from individual cocultures; line shows mean ± SEM (n = 7); Friedman test with Dunn´s multiple comparison test. GeoMFI, geometric mean fluorescence intensity. (D and E) Th1 and Th17 (IL-1β, IL-6, IL-23, or IL-12*) cells were sorted as NKG2D⁺ using A10 (activating; black circles) or CX5 (blocking; open circles) α-NKG2D antibody, cross-linked by plate-bound secondary antibody in the presence of α-CD3 for 6 h, and analyzed by FC for intracellular cytokine expression. Frequency of IFN-γ⁺ (n = 10) and GM-CSF⁺ Th1 (n = 6; D) and IFN-γ⁺ (n = 11), GM-CSF⁺ (n = 10), and IL-17A⁺ (n = 9) Th17 (E). Each symbol represents the value of the individual coculture; lines connect matching samples; Wilcoxon test. ns, not significant. Data are pooled from at least three independent experiments.