CD4 T cells are required for anti-HOD IgG alloantibody formation. (A-B) Gating strategy (A) and quantification (B) of the percentage of CD3⁺CD4⁺CD8⁻ CD4 T cells in splenocytes obtained from B6 mice that received PBS (B6), CD4-depleting antibody (CD4 dep), or IC antibody. (C-D) Representative line graph (C) and quantification (D) of anti-HOD IgM alloantibody formation in CD4 T-cell–depleted mice (blue) compared with B6 control mice (red) and IC mice (green) following HOD RBC transfusion. (E-F) Representative line graph (E) and quantification (F) of anti-HOD IgG alloantibody formation in CD4 T-cell–depleted mice (blue) compared with B6 control mice (red) and IC mice (green) following 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 < .0002, **P < .006, *P < .02; 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.