HOD alloantibody production occurs independently of FO B cells. (A) General experimental design. B6 recipients received FO B-cell–depleting, total B-cell–depleting, or IC antibodies, followed by HOD RBC transfusion 2 weeks later. Serum was collected 3, 5, 7, 14, and 21 days following transfusion and examined for anti-HOD antibodies by flow cytometric analysis. (B) Quantification of absolute number of B220⁺CD23^hiCD21^lo FO B cells in splenocytes of B6 recipients that received PBS (B6), FO B-cell–depleting antibody anti-CD20 IgG1 (FO B dep), mouse IgG1 IC antibody (FO B ctrl), B-cell–depleting antibody anti-CD20 IgG2a (Total B dep), or mouse IgG2a IC antibody (Total B ctrl). (C-D) Representative line graph (C) and quantification (D) of anti-HOD IgM alloantibody formation in FO B-cell–depleted recipients (blue) compared with B6 control (red) and total B-cell–depleted (purple) recipients following HOD RBC transfusion. (E-F) Representative line graph (E) and quantification (F) of anti-HOD IgG alloantibody formation in FO B-cell–depleted recipients (blue) compared with B6 control (red) and total B-cell–depleted (purple) recipients following HOD RBC transfusion. (G) Anti-HOD IgG antibody subclass analysis on plasma collected 14 days after HOD RBC transfusion. Error bars represent standard deviation; results are representative of 2 or 3 independent experiments with ≥5 mice per group. ****P < .0001, ***P < .0004, **P < .009, *P < .04; 1-way ANOVA with Tukey’s post hoc test. D3, day 3; D5, day 5; D7, day 7; D14, day 14; D21, day 21; MFI, mean fluorescence intensity; ns, not significant.