Figure 3.
AMIS-inducing antibodies promote RBC antigen loss, RBC membrane loss, and RBC membrane transfer to macrophages in vitro. HOD-RBCs fluorescently labeled with PKH67 (PKH67+HOD-RBC) and sensitized with anti-HEL pIgG or anti-Duffy MIMA29 IgG2a were incubated with or without RAW macrophages for 30 minutes or 3 hours at 37°C as indicated. Nonsensitized PKH67+HOD-RBCs (PBS) were used as negative controls. RBCs were recovered after incubation, and the residual external RBCs were lysed. The RBCs recovered from each condition were resensitized with additional pIgG anti–HEL or anti-Duffy MIMA29, followed by APC-labeled anti–mouse IgG and further analyzed by flow cytometry. Representative histograms of (A) HEL antigen and (B) Duffy antigen detected on the recovered RBCs after 3 hours of incubation with (dashed blue lines) or without (solid red line) macrophages. The filled red histogram represents the fluorescent RBC gate (ie, PKH67+HOD-RBC) incubated only with the secondary antibody. (C-D) Cumulative data depicting the median fluorescence intensity (MFI) of detectable (C) HEL antigen and (D) Duffy antigen on HOD-RBCs after incubation in the presence vs absence of macrophages. (E-F) MFI of the PKH67 signal of the PKH67+HOD-RBCs recovered as indicated. (G) Macrophages incubated with nonsensitized and sensitized RBCs were analyzed by flow cytometry, and the MFI of PKH67 from RBCs in the macrophage gate was determined. Gray-shaded area represents the MFI of PKH67 signal in the PKH67+ macrophages detected after incubation with RBCs sensitized with TER-119, an antibody that induces robust phagocytosis. The dotted line represents background fluorescence of macrophages without added RBCs. Statistical analyses of the data represented in graphs of panels C and E were performed by two-way ANOVA with Tukey multiple comparisons post hoc test, mixed-effects analysis with Tukey multiple comparisons post hoc test for panel D graph, and two-way ANOVA with Sidak multiple comparisons post hoc test for graphs of panels F and G (∗P = .05; ∗∗P = .01; ∗∗∗P = .001; ∗∗∗∗P = .0001).

AMIS-inducing antibodies promote RBC antigen loss, RBC membrane loss, and RBC membrane transfer to macrophages in vitro. HOD-RBCs fluorescently labeled with PKH67 (PKH67+HOD-RBC) and sensitized with anti-HEL pIgG or anti-Duffy MIMA29 IgG2a were incubated with or without RAW macrophages for 30 minutes or 3 hours at 37°C as indicated. Nonsensitized PKH67+HOD-RBCs (PBS) were used as negative controls. RBCs were recovered after incubation, and the residual external RBCs were lysed. The RBCs recovered from each condition were resensitized with additional pIgG anti–HEL or anti-Duffy MIMA29, followed by APC-labeled anti–mouse IgG and further analyzed by flow cytometry. Representative histograms of (A) HEL antigen and (B) Duffy antigen detected on the recovered RBCs after 3 hours of incubation with (dashed blue lines) or without (solid red line) macrophages. The filled red histogram represents the fluorescent RBC gate (ie, PKH67+HOD-RBC) incubated only with the secondary antibody. (C-D) Cumulative data depicting the median fluorescence intensity (MFI) of detectable (C) HEL antigen and (D) Duffy antigen on HOD-RBCs after incubation in the presence vs absence of macrophages. (E-F) MFI of the PKH67 signal of the PKH67+HOD-RBCs recovered as indicated. (G) Macrophages incubated with nonsensitized and sensitized RBCs were analyzed by flow cytometry, and the MFI of PKH67 from RBCs in the macrophage gate was determined. Gray-shaded area represents the MFI of PKH67 signal in the PKH67+ macrophages detected after incubation with RBCs sensitized with TER-119, an antibody that induces robust phagocytosis. The dotted line represents background fluorescence of macrophages without added RBCs. Statistical analyses of the data represented in graphs of panels C and E were performed by two-way ANOVA with Tukey multiple comparisons post hoc test, mixed-effects analysis with Tukey multiple comparisons post hoc test for panel D graph, and two-way ANOVA with Sidak multiple comparisons post hoc test for graphs of panels F and G (∗P = .05; ∗∗P = .01; ∗∗∗P = .001; ∗∗∗∗P = .0001).

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