NKG2D-expressing antigen-specific CD4⁺ T cells accumulate in vivo during OIA. (A) Schematic representation of the experimental workflow during OIA. Mice were s.c. immunized with catOVA/CFA. Disease was induced with i.a. injection of catOVA (OIA). Control animals received only i.a. injection of catOVA (ctrl). Cells from draining LN were analyzed on day 10 after disease induction and after in vitro restimulation with catOVA. (B and C) Representative FC analysis (B) and quantification of the frequency of Il17a^(fm)+ cells among CD44⁺ CD4⁺ T cells (C) in control and OIA animals 10 d after disease induction. (D and E) Representative FC analysis (D) and quantification of the frequency of Il17a^(fm)+ cells (E) among CD44⁺ CD40L⁻ CD4⁺ T cells in control and CD44⁺ CD40L⁻ and CD44⁺ CD40L⁺ CD4⁺ T cells in OIA animals 10 d after disease induction. In C and E, each symbol represents an individual mouse; data shown as mean ± SEM; n = 2 for control and n = 4 for OIA animals. Data are pooled from two independent experiments. (F) Frequency and absolute numbers of NKG2D⁺CD44⁺CD4⁺ T cells in the dLN of mice with OIA at indicated time points after disease induction. Data show mean ± SEM from two independent experiments (n = 6); Kruskal–Wallis test with multiple comparison correction. (G) Representative FC analysis of NKG2D expression on CD44⁺ CD40L⁺ CD4⁺ T cells from Klrk1^flox mice 10 d after disease induction. (H) Frequency of NKG2D-expressing CD44⁺ CD4⁺ T cells among indicated subsets in mice with OIA (n = 10); Wilcoxon test. Line shows mean ± SEM pooled from three independent experiments. (I and J) Representative FC analysis (I) and quantification of intracellular cytokine expression in NKG2D⁺ and NKG2D⁻ CD40L⁺ CD4⁺ T cells of Klrk1^flox mice (n = 9) and CD40L⁺ CD4⁺ T cells from Klrk1^floxCd4^Cre mice (n = 7; J); Mann–Whitney U test. Each symbol represents an individual mouse; data show mean ± SEM from three independent experiments. (K) Frequency of CD40L⁺ CD4⁺ T cells in Klrk1^flox (n = 14) and Klrk1^floxCd4^Cre (n = 11) mice; Mann–Whitney U test. ns, not significant. Each symbol represents an individual mouse; data show mean ± SEM from three independent experiments.