Activated and expanded CD8⁺ T cells recognize targets through a NKG2D-dependent and TCR-independent pathway. (A) Cytolysis of 721-221 cell lines by activated and expanded CD8⁺ T cells either alone or in the presence of isotype control antibodies or antibody against NKG2D (at 20 μg/mL). (B) Redirected cytolysis against murine P815 cells either alone, with isotype control antibody, anti-NKG2D, or anti-NKG2D and anti-CD3 (at 20 μg/mL). Error bars indicate standard deviation (C) FACS analysis after NKG2D-specific siRNA. Shown is the FACS analysis of activated and expanded T cells at day 14 to 21 of culture treated with siRNA complementary to human NKG2D (hNKG2D) or controls (nontransfected cells and cells transfected with mCD8 siRNA). siRNA complementary to NKG2D induces a more than 95% inhibition of NKG2D expression at 72 hours after transfection, but has no effect on either CD3 or αβ TCR relative to controls. (D) Cytotoxicity of cells treated with siRNA. Untreated cells or cells treated with control siRNA (against mCD8) induced significant cytotoxicity against the U266 cell line, whereas cells transfected with hNKG2D siRNA or nontransfected cells treated with NKG2D-blocking antibodies had no cytotoxicity. All are representative of 3 or more experiments.