Figure 3

KIR-CD300a mediates inhibition of SED stimulated Jurkat T cell activation. (A) Gating strategy for assessing CD69 expression on superantigen activated cells. Jurkat T cells were electronically gated by size and forward scatter and by the absence of expression of CD19, a marker of 721.221 cells, and then the expression of CD69 was assessed. The gating strategy for assessing CD25 expression was the same. (B) KIR CD300a WT and KIR-CD300a 4F Jurkat T cells were co-cultured with 721.221 or 721.221-Cw3 cells, loaded or not with SED. Cultures were harvested and Jurkat T cells were assessed for CD69 expression by flow cytometry. Results are representative of three independent experiments. (C) Cells were cultured as in B, loaded (white bar) or not (black bars) with SED, and the expression of CD25 was assessed. The bar graph represents average ± SEM of the percentage of CD25+ Jurkat T cells. Results are from three independent experiments. (D) Untransfected E6.1, KIR-CD300a WT and KIR-CD300a 4F Jurkat T cells were transiently transfected with a NFAT luciferase reporter plasmid. Following coculture with 721.221, 721.221-Cw3 and 721.221-Cw6 loaded (withe bars) or not (black bars) with SED, cells were lysed and supernatants were assayed for luciferase activity. Data were normalized by the activity obtained with cells treated with PMA plus ionomycin. Results are representative of three independent experiments.