TCR recognition of D^d activates CD3^-CD5⁺ D^d-ζ killer cells. (A) D^d-ζ killer cells are activated by a D^d-restricted T-cell hybridoma. A total of 3 million homozygous 130 strain D^d-ζ splenocytes were incubated for 2 days with 1 million paraformaldehyde-fixed B4.2.3 cells in the presence and absence of the cognate p18 peptide. The D^d-ζ cells were then tested for cytotoxicity against EL4 and P815 target cells in a ⁵¹Cr release assay. No killing of P815 target cells was observed. Error bars show the standard deviation (SD) for triplicate samples. ▪ indicates unstimulated cells; •, B4 stimulators; and ▴, B4 + p18 stimulators. (B) Spleen cells from 130 D^d-ζ transgenic mice were stimulated by incubation with irradiated D^d-primed splenocytes for 4 days and were left either unsorted or were sorted by FACS into CD3⁺CD5⁺ and CD3^–CD5⁺ populations. The large dot plot on the left is before sorting and shows the sort gates. The 2 smaller plots on the right show the sort purity. Percentages of viable cells are indicated for each quadrant. (C) Unsorted (▪) and sorted (•, CD3⁺CD5⁺; ▴, CD3^–CD5⁺) cells were tested in a ⁵¹Cr release assay for cytotoxic activity against EL4 (H-2^b, Ly-49A⁺). Anti–Ly-49A blocked killing of EL4 target cells by unsorted D^d-ζ cells (unpublished observations, January 25, 2001). Specific lysis was not observed against syngeneic (H-2^d) P815 target cells (unpublished observations, January 25, 2001). Error bars show the SD for triplicate samples.