Figure 1
Figure 1. FcμR is not involved in Fas-mediated apoptosis in mouse T and B cells. (A) Expression of Fas, FasL, and FcμR by WT and FcμR−/− thymocytes and T and B cells before and after activation. To analyze Fas, FasL, and FcμR expression, cells were first incubated with a rat IgG2b anti-mouse CD16/CD32 antibody (clone 2.4G2; BD Biosciences) to block FcγR. For Fas expression, cells were stained with PE/Cy7 anti-CD95 (clone Jo2; BD Biosciences) or an isotype control (PE/Cy7 hamster IgG2, clone Ha4/8; BD Biosciences). For FasL expression, cells were stained with PE anti-CD178 (clone MFL3; Biolegend) or an isotype control (PE hamster IgG, clone HTK888; Biolegend). For FcμR expression, cells were first stained with the rat anti-mouse FcμR mAb (clone 4B5) or an isotype control (rat IgG2a; BD Biosciences), and then stained with PE anti-rat IgG2a (clone RG7/1.30; BD Biosciences). Purified spleen T and B cells were stimulated with plate-bound anti-CD3 (clone 2C11, 10 μg/mL) and CD40L for 44 hours, respectively. CD40L, CD40 ligand; Ig, immunoglobulin; mAb, monoclonal antibody; PE, phycoerythrin. (B) Normal Fas-mediated cell death in FcμR−/− thymocytes. Thymocytes were cultured with increasing concentrations of the Jo2 anti-Fas (left panel) or FasL in the presence of 0.5 μg/mL cycloheximide (right panel). (C) Normal Fas-mediated cell death in FcμR−/− spleen T cells before and after activation. Left panel, Spleen T cells were treated with increasing concentrations of FasL for 4 hours. Middle panel, Spleen T cells were cultured for 48 hours in medium alone or in the presence of plate-bound anti-CD3 (clone 2C11, 10 μg/mL) to induce AICD. Right panel, Cells were cultured in the presence of plate-bound anti-CD3 for 48 hours, and increasing concentrations of FasL were added for the last 4 hours. (D) Normal Fas-mediated cell death in FcμR−/− B cells. Purified spleen B cells (left 2 panels) or spleen B cells stimulated with CD40L for 44 hours (right 2 panels) were cultured for 4 hours in the presence of increasing concentrations of the Jo-2 anti-Fas or FasL. Cells were stained with 7-AAD, and the cell death was calculated by the following formula: (experimental cell death − spontaneous cell death)/(100 − spontaneous cell death).9,10 7-AAD, 7-Aminoactinomycin D.

FcμR is not involved in Fas-mediated apoptosis in mouse T and B cells. (A) Expression of Fas, FasL, and FcμR by WT and FcμR−/− thymocytes and T and B cells before and after activation. To analyze Fas, FasL, and FcμR expression, cells were first incubated with a rat IgG2b anti-mouse CD16/CD32 antibody (clone 2.4G2; BD Biosciences) to block FcγR. For Fas expression, cells were stained with PE/Cy7 anti-CD95 (clone Jo2; BD Biosciences) or an isotype control (PE/Cy7 hamster IgG2, clone Ha4/8; BD Biosciences). For FasL expression, cells were stained with PE anti-CD178 (clone MFL3; Biolegend) or an isotype control (PE hamster IgG, clone HTK888; Biolegend). For FcμR expression, cells were first stained with the rat anti-mouse FcμR mAb (clone 4B5) or an isotype control (rat IgG2a; BD Biosciences), and then stained with PE anti-rat IgG2a (clone RG7/1.30; BD Biosciences). Purified spleen T and B cells were stimulated with plate-bound anti-CD3 (clone 2C11, 10 μg/mL) and CD40L for 44 hours, respectively. CD40L, CD40 ligand; Ig, immunoglobulin; mAb, monoclonal antibody; PE, phycoerythrin. (B) Normal Fas-mediated cell death in FcμR−/− thymocytes. Thymocytes were cultured with increasing concentrations of the Jo2 anti-Fas (left panel) or FasL in the presence of 0.5 μg/mL cycloheximide (right panel). (C) Normal Fas-mediated cell death in FcμR−/− spleen T cells before and after activation. Left panel, Spleen T cells were treated with increasing concentrations of FasL for 4 hours. Middle panel, Spleen T cells were cultured for 48 hours in medium alone or in the presence of plate-bound anti-CD3 (clone 2C11, 10 μg/mL) to induce AICD. Right panel, Cells were cultured in the presence of plate-bound anti-CD3 for 48 hours, and increasing concentrations of FasL were added for the last 4 hours. (D) Normal Fas-mediated cell death in FcμR−/− B cells. Purified spleen B cells (left 2 panels) or spleen B cells stimulated with CD40L for 44 hours (right 2 panels) were cultured for 4 hours in the presence of increasing concentrations of the Jo-2 anti-Fas or FasL. Cells were stained with 7-AAD, and the cell death was calculated by the following formula: (experimental cell death − spontaneous cell death)/(100 − spontaneous cell death).9,10  7-AAD, 7-Aminoactinomycin D.

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