Figure 5.
Anti-CD44 blocks the FcγR IgG binding site. The effect of anti-CD44 on macrophage FcγR expression and blockade of the FcγR IgG binding site was evaluated using blocking and nonblocking monoclonal antibodies to FcγRs. Macrophages were treated with anti-CD44 or controls for 30 minutes at 37°C, followed by washing, formaldehyde fixation, and addition of fluorescent anti-FcγR antibody. (A-B) Blocking and nonblocking anti-FcγRIII antibodies were used to distinguish FcγR blockade of the IgG binding site vs loss of FcγR expression. (A) Binding of a blocking FcγRIII-specific antibody (clone 275003) to macrophages that were treated or not (Nil) with IgG1 anti-CD44 (KM114) or IgG2b anti-CD44 (IM7). A representative line graph (left panel) and quantitation of 3 experiments (right panel) are shown. The gray shaded area (right panel) indicates the background fluorescence. Counts (%max) are counts expressed as a percentage relative to the mode (100%). (B) Binding of a nonblocking anti-FcγRIIb/III antibody (clone 015) to macrophages that were treated or not (Nil) with IgG1 anti-CD44 (KM114) or IgG2b anti-CD44 (IM7). A representative line graph (left panel) and quantitation of 3 experiments (right panel) are shown. The dotted line (right panel) indicates the background fluorescence. (C) The requirement of the CD44 antibody Fc region to block binding of the anti-FcγRIII antibody (275003 binding) was evaluated with unmodified anti-CD44 (Intact), anti-CD44 F(ab′)2 variants, or respective isotype controls (n = 3-8 experiments). The dotted line indicates the level of 275003 binding on untreated macrophages (100%). (D) Binding of an FcγRIV-blocking antibody (clone 9E9) after macrophage incubation with unmodified anti-CD44, anti-CD44 F(ab′)2 variants, or respective isotype controls (n = 3-5 experiments). The dotted line indicates the level of 9E9 binding on untreated macrophages (100%). The percentage of anti-FcγR antibody binding in (C) and (D) was calculated relative to fluorescent antibody MFI on untreated macrophages. Error: mean ± standard error of the mean. ****P < .0001, ***P < .001, *P < .05, 1-way analysis of variance with Tukey’s post hoc test. α-CD44, anti-CD44; MFI, mean fluorescence intensity.

Anti-CD44 blocks the FcγR IgG binding site. The effect of anti-CD44 on macrophage FcγR expression and blockade of the FcγR IgG binding site was evaluated using blocking and nonblocking monoclonal antibodies to FcγRs. Macrophages were treated with anti-CD44 or controls for 30 minutes at 37°C, followed by washing, formaldehyde fixation, and addition of fluorescent anti-FcγR antibody. (A-B) Blocking and nonblocking anti-FcγRIII antibodies were used to distinguish FcγR blockade of the IgG binding site vs loss of FcγR expression. (A) Binding of a blocking FcγRIII-specific antibody (clone 275003) to macrophages that were treated or not (Nil) with IgG1 anti-CD44 (KM114) or IgG2b anti-CD44 (IM7). A representative line graph (left panel) and quantitation of 3 experiments (right panel) are shown. The gray shaded area (right panel) indicates the background fluorescence. Counts (%max) are counts expressed as a percentage relative to the mode (100%). (B) Binding of a nonblocking anti-FcγRIIb/III antibody (clone 015) to macrophages that were treated or not (Nil) with IgG1 anti-CD44 (KM114) or IgG2b anti-CD44 (IM7). A representative line graph (left panel) and quantitation of 3 experiments (right panel) are shown. The dotted line (right panel) indicates the background fluorescence. (C) The requirement of the CD44 antibody Fc region to block binding of the anti-FcγRIII antibody (275003 binding) was evaluated with unmodified anti-CD44 (Intact), anti-CD44 F(ab′)2 variants, or respective isotype controls (n = 3-8 experiments). The dotted line indicates the level of 275003 binding on untreated macrophages (100%). (D) Binding of an FcγRIV-blocking antibody (clone 9E9) after macrophage incubation with unmodified anti-CD44, anti-CD44 F(ab′)2 variants, or respective isotype controls (n = 3-5 experiments). The dotted line indicates the level of 9E9 binding on untreated macrophages (100%). The percentage of anti-FcγR antibody binding in (C) and (D) was calculated relative to fluorescent antibody MFI on untreated macrophages. Error: mean ± standard error of the mean. ****P < .0001, ***P < .001, *P < .05, 1-way analysis of variance with Tukey’s post hoc test. α-CD44, anti-CD44; MFI, mean fluorescence intensity.

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