HOD-specific antibodies can cause AMIS by inducing RBC antigen loss rather than RBC clearance. (A,G,M) Schematic representation of HOD-RBC with antibodies specific for each antigen portion. C57BL/6 mice were transfused with 108 PKH26+HOD-RBCs. Twenty-four hours later, mice were injected with antibodies specific for (A-F) Duffy, (G-L) OVA, and (M-R) HEL portions of the HOD antigen. The quantity of each antibody evaluated was 5 μg of anti-Duffy antibody CBC-512 wild-type or its deglycosylated variant (deCBC-512); 25 and 50 μg of a mouse pIgG specific to OVA; 5 μg of the HEL-specific mouse pIgG; and monoclonal antibodies 4B7, 2F4, or a blend of 4B7 and 2F4 recognizing non–crossblocking epitopes (4B7 + 2F4) (2.5 μg each). Mice injected with PBS were used as an alloimmunization controls. The HEL-specific (B,H,N) IgM and (C,I,O) IgG response on day 7 were evaluated by enzyme-linked immunosorbent essay. Data are presented as the mean ± standard error of the mean (SEM) of the optical density at 405 nm (OD 405nm). (D,J,P) The percentage of PKH26+HOD-RBCs in circulation 2 hours before (time = −2) and after antibody injection, as indicated. Transfused mice injected with 5 μg of anti-Duffy monoclonal antibody MIMA29 were used as a positive controls for RBC clearance. The MFI signal of the Duffy (E), OVA (K), and HEL (Q) epitope from the recovered cells from recipient mice injected with antibodies specific against each antigen (or PBS) was determined by ex vivo incubation of the cells with additional anti-Duffy CBC-512, pIgG anti–OVA, or pIgG anti–HEL respectively, followed by fluorescein isothiocyanate–labeled anti–mouse IgG. The remaining level of Duffy, OVA, and HEL epitopes at 2 and 24 hours after antibody injection was expressed as a percentage of the initial MFI before antibody injection. The dotted line indicates the MFI of the fluorescein isothiocyanate signal of cells incubated only with the secondary antibody, expressed as percentage of the initial MFI. RBC membrane loss induced by (F) anti-Duffy, (L) anti-OVA, and (R) anti-HEL antibodies was measured as the MFI of the PKH26 signal from cells recovered at each time point and expressed as the percentage initial MFI. Data in all panels represent the mean ± SEM of 3 independent experiments with the total number of mice per group as (B-F) PBS: (n = 9), CBC-512: (n = 7), deCBS-512: (n = 9), and nil; (n = 6); (H-L) PBS: (n = 8), pIgG anti–OVA 25 μg, (n = 10), 50 μg, (n = 10), and nil: (n = 8); and (N-R) PBS: (n = 14), pIgG anti–HEL: (n = 14), 4B7: (n = 10), 2F4: (n = 8), 4B7 + 2F4: (n = 8), and nil: (n = 10). Statistical significance compared with that of the positive control for alloimmunization (PBS) was determined using Kruskal-Wallis test with Dunn post hoc test in graphs of panels B,C,H,I,N,O. Data represented in graphs of panels E,F,K,L by two-way analysis of variance (ANOVA) and panels Q,R by mixed-effects analysis with Dunnett multiple comparisons test for all of them. ∗ and & indicate significant differences to the control group (PBS) at 2 hours and 24 hours, respectively. (∗P = .05; ∗∗P = .01; ∗∗∗P = .001; ∗∗∗∗P = .0001 and &P = .05; &&P = .01; &&&P = .001; &&&&P = .0001). Panels A,G,M were created with BioRender.com.
