HOD alloantibody formation is boosted in KEL-primed recipients subsequently transfused with RBCs expressing HOD and KEL antigens. (A) Experimental schematic, wherein B6 recipients were either not primed or primed against KEL in the presence of 100 μg PIC prior to transfusion of HOD RBCs, KEL RBCs, (HOD × KEL) RBCs, or HOD RBCs mixed with KEL RBCs (HOD + KEL) 14 days later. Anti-HOD IgM (B) and IgG (C) alloantibodies were examined at days 5 (D5) and 14 (D14), respectively, after transfusion using an indirect immunofluorescence staining using HOD and B6 RBCs. The MFI in panels B-C were calculated by normalizing the MFI of HOD RBCs to the MFI of background control B6 RBCs. Error bars represent mean ± SEM. The mean of each group is depicted as a horizontal line. Statistics were generated using a 1-way ANOVA with a Tukey’s post-test. All panels show combinatorial data from 2 experiments, with each repeat consisting of 5 mice per group. **P < .01; ****P < .0001.