Target cell killing by D^d-ζ cells via D^d–Ly-49A interaction. (A) D^d-ζ effector cells were generated from homozygous 130 splenocytes in a 4-day mixed lymphocyte culture (MLC) with irradiated, D^d-primed, FVB spleen cells and were tested in a ⁵¹Cr release cytotoxicity assay against EL4 target cells. The indicated amounts of anti–Ly-49A mAb were added to triplicate wells at the start of the cytotoxicity assay. (B) This panel shows the control for panel A in which anti–H-2^b effectors are generated by stimulating unirradiated FVB splenocytes with irradiated C57BL/6J (H-2^b) splenocytes. (C) Anti–H-2D^d mAb was tested for its ability to block D^d-ζ versus EL4 cytotoxicity assays using cells from the MLC described in panel A. (D) This panel shows a control cytotoxicity assay using the anti-H-2^b effectors from panel B with anti-H-2D^d mAb added as in panel C. The effectors described earlier showed minimal kill in cytotoxicity assays using P815 (H-2^d) target cells. This experiment was performed twice with similar results. Error bars show the SD for triplicate samples. (E) Following activation, CD3⁺CD5⁺ cells lose Dd expression, whereas CD3^–CD5⁺ cells retain D^d expression. Splenocytes from D^d-ζ transgenic mice (130 strain) were CFSE labeled and left unstimulated or stimulated with irradiated (2000 rad) splenocytes from D^d-primed mice. After 4 days of incubation, the cells were harvested, stained with anti-CD3 and anti-CD5, and evaluated by flow cytometry. The cells were gated into CD3⁺CD5⁺ and CD3^–CD5⁺ and examined for CFSE versus D^d expression.