Fig. 4: NKG2D-DAP12 complex mediates NK cytotoxicity in CD27⁻CD28⁻CD8⁺ T cells.

a) Box-and-whisker plot of cell-surface CD107a expression of CD27⁺CD28⁺CD8⁺, CD27⁺CD28⁻CD8⁺, CD27⁻CD28⁻CD8⁺, and CD3⁻CD56⁺ NK cells isolated from healthy donors co-cultured with K562 cells at E:T 2:1 (median and range, n = 5). b) CD107a expression on CD28⁻CD8⁺ T cells transfected with NKG2D siRNA (siNKG2D) or control siRNA (siCtrl) and cultured with C1R-MICA or C1R (E:T ratio 2:1) for 6 hours (mean and s.e.m., n = 4). c) Immunoblots (representative of five independent experiments with similar results) and d) flow cytometry (means and s.e.m., n = 12) showing DAP12 expression on CD27⁺CD28⁺CD8⁺, CD27⁺CD28⁻CD8⁺, CD27⁻CD28⁻CD8⁺, and CD3⁻CD56⁺ NK cells. g) Expression of DAP12 in lysed CD28⁺CD8⁺ T cells and CD28⁻CD8⁺ T cells co-immunoprecipitated with antibody against NKG2D. Loading control, light-chain IgG (IgGL). Whole-cell lysate immunoblot shown as a control. Representative of two independent experiments. f) Phosphorylation of Zap70(Tyr319) and Syk(Tyr352) in freshly isolated CD27⁻CD28⁻CD8⁺ T cells after treated with CD3 mAb (OKT3, 10 μg/mL, 15 minutes) and/or NKG2D Ab (1D11, 5 μg/mL, 15 minutes). Numbers indicate relative expression normalized to total Zap70. Representative of 2 experiments. g) Granzyme B expression (left) and IFN-γ secretion (right) in CD27⁺CD28⁺CD8⁺, CD27⁺CD28⁻CD8⁺, and CD27⁻CD28⁻CD8⁺ T cells after stimulation with NKG2D Ab (5 μg/mL, means and s.e.m., n = 15 donors). h) CD107a expression in human CD28⁻CD8⁺ T cells transfected with DAP12 siRNA (siDAP12) or control siRNA (siCtrl) cultured with C1R-MICA*008 or C1R cells for 6 hours (E:T ratio 2:1; means and s.e.m., n = 4). Statistical significance determined with Kruskal-Wallis test in a) Friedman test with Dunńs correction in d), two-way ANOVA with Bonferroni correction in b, j) and one-way ANOVA with Tukey’s in g) (*p <0.05, **p <0.01, ***p <0.001).