Figure 6

SHP-1, but not SHP-2 or SHIP, is required for CD300a mediated inhibition of BCR stimulated activation. (A) DT40 cells, DT40 cells lacking SHP-1, DT40 cells lacking SHP-2, or DT40 cells lacking SHIP, all expressing CD300a WT were loaded with Fluo-4 and Fura-Red. Then, cells were acquired in a flow cytometer and stimulated with anti-chicken BCR plus isotype control antibody (black line) or anti-chicken BCR plus anti-CD300a (grey line) mAb for 30 seconds and then co-crosslinked with a secondary antibody (GAM). Ca²⁺ mobilization is expressed as the ratio of Fluo-4/Fura-Red as a function of time. These results are representative of two independent experiments. (B) DT40 cell lines expressing CD300a WT were transiently transfected with a NFAT luciferase reporter plasmid and stimulated with anti-chicken BCR plus isotype control or anti-chicken BCR plus anti-CD300a mAb. Cells were lysed and supernatants assayed for luciferase activity. Results were normalized to the activity obtained when cells were treated with PMA plus ionomycin. Data are presented as percentage of inhibition of CD300a vs. isotype control and they are the average ± SEM for three separate experiments. (C) The indicated DT40 cells lines stably expressing CD300a WT and human SHP-1 WT, SHP-1 CS, SHP-2 WT or SHP-2 CS were loaded with Fluo-4 and Fura-Red. Ca²⁺ mobilization was assessed as in A. These results are representative of three independent experiments. (D) KIR-CD300a WT Jurkat T cells transfected with non target (NT), SHP-1 and SHP-2 siRNA 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. In the left panel, the percentage of inhibition of CD69 expression, calculated as shown in material and methods, is presented. The scatter plot represents the average ± SEM. In the center panel, the expression of SHP-1, SHP-2 and actin (loading control) was assessed by western blot analysis in lysates from siRNA transfected KIR-CD300a WT Jurkat T cells. In the right panel, the relative amount of SHP-1 mRNA and SHP-2 mRNA from siRNA transfected KIR-CD300a WT Jurkat T cells is shown. The bar graph represents the average ± SEM. Results are from three independent experiments.