Figure 2
Figure 2. SLP-76 is necessary for optimal NK-cell function and proliferation downstream of Ly49D stimulation. Ly49D+ splenic NK cells from WT or SLP-76 KO mice were enriched and expanded in IL-2 for 6 days. (A) Expanded NK cells (CD3–NK1.1+ lymphocytes) were analyzed for the expression of Ly49D and represented as mean MFI ± standard error of the mean of 5 independent experiments. (B) Expanded NK cells were left unstimulated or stimulated with soluble isotype Ct Ab or anti-Ly49D Ab for 1 or 10 minutes and analyzed for total PLC-γ2 (loading control), pAKT, and pERK by western blot. One representative of 3 independent experiments is shown. (C) Expanded NK cells from WT or SLP-76 KO mice were stimulated with plate-immobilized Ct Ab, anti-Ly49D Ab, or soluble PMA/ionomycin for 4 hours in the presence of Monensin followed by detection of IFN-γ and CD107a expression by flow cytometry. Ly49D expression of NK cells at the time of stimulation is shown. All plots are gated on CD4–CD8–NK1.1+ lymphocytes. (D) Expanded NK cells from WT or SLP-76 KO mice were stimulated with plate-immobilized anti-Ly49H Ab or anti-NK1.1 Ab for 4 hours in the presence of Monensin followed by detection of IFN-γ and CD107a expression by flow cytometry. Ly49H and NK1.1 expression of NK cells at the time of stimulation is shown. All plots are gated on CD4–CD8–NK1.1+ lymphocytes. Dot plots are representative of 4 (Ly49H) or 2 (NK1.1) independent experiments. (E) Data compiled from 5 (IFN-γ) or 4 (CD107a) Ly49D stimulation independent experiments were normalized to the WT NK-cell response and expressed as mean ± standard error of the mean (SEM). (F) CFSE-labeled T-cell–depleted WT or SLP-76 KO splenocytes were stimulated with plate-immobilized control Ab or anti-Ly49D Ab in the presence of IL-2 (40 to 100 U/mL) for 3 days followed by flow cytometric analysis. Plots are gated on CD4–CD8–NK1.1+Ly49D+ NK cells. Cells to the left of gate have diluted CFSE and represent cells that have proliferated. One representative of 4 independent experiments is shown. (G) The percent of maximal proliferation induced by Ly49D stimulation is expressed as mean ± SEM of 4 independent experiments. * indicates statistical significance of P < .05 by paired t test; n.s., not significant.

SLP-76 is necessary for optimal NK-cell function and proliferation downstream of Ly49D stimulation. Ly49D+ splenic NK cells from WT or SLP-76 KO mice were enriched and expanded in IL-2 for 6 days. (A) Expanded NK cells (CD3–NK1.1+ lymphocytes) were analyzed for the expression of Ly49D and represented as mean MFI ± standard error of the mean of 5 independent experiments. (B) Expanded NK cells were left unstimulated or stimulated with soluble isotype Ct Ab or anti-Ly49D Ab for 1 or 10 minutes and analyzed for total PLC-γ2 (loading control), pAKT, and pERK by western blot. One representative of 3 independent experiments is shown. (C) Expanded NK cells from WT or SLP-76 KO mice were stimulated with plate-immobilized Ct Ab, anti-Ly49D Ab, or soluble PMA/ionomycin for 4 hours in the presence of Monensin followed by detection of IFN-γ and CD107a expression by flow cytometry. Ly49D expression of NK cells at the time of stimulation is shown. All plots are gated on CD4–CD8–NK1.1+ lymphocytes. (D) Expanded NK cells from WT or SLP-76 KO mice were stimulated with plate-immobilized anti-Ly49H Ab or anti-NK1.1 Ab for 4 hours in the presence of Monensin followed by detection of IFN-γ and CD107a expression by flow cytometry. Ly49H and NK1.1 expression of NK cells at the time of stimulation is shown. All plots are gated on CD4–CD8–NK1.1+ lymphocytes. Dot plots are representative of 4 (Ly49H) or 2 (NK1.1) independent experiments. (E) Data compiled from 5 (IFN-γ) or 4 (CD107a) Ly49D stimulation independent experiments were normalized to the WT NK-cell response and expressed as mean ± standard error of the mean (SEM). (F) CFSE-labeled T-cell–depleted WT or SLP-76 KO splenocytes were stimulated with plate-immobilized control Ab or anti-Ly49D Ab in the presence of IL-2 (40 to 100 U/mL) for 3 days followed by flow cytometric analysis. Plots are gated on CD4–CD8–NK1.1+Ly49D+ NK cells. Cells to the left of gate have diluted CFSE and represent cells that have proliferated. One representative of 4 independent experiments is shown. (G) The percent of maximal proliferation induced by Ly49D stimulation is expressed as mean ± SEM of 4 independent experiments. * indicates statistical significance of P < .05 by paired t test; n.s., not significant.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal