HOD-specific CD4 T-cell activation is not dependent on MZ B cells. (A) Gating strategy and line graph showing proliferation, as measured by CFSE dilution, of CFSE-labeled CD4⁺Thy1.1⁺ OTII T cells obtained from recipient splenocytes 5 days following HOD RBC transfusion. (B) MZ-KO mice (blue) and MZ B-cell littermate control (MZ-KO Ctrl) recipients (red) received transfusion with HOD RBCs, followed by examination of splenocytes for OTII T-cell proliferation on days 1 (D1), 3 (D3), and 5 (D5); representative line graphs are shown. (C-D) OTII T cells were stained for activation markers, including CD44 (C) and CD62L (D). (E-G) B6 control recipients received HOD RBC transfusion alone (B6), MZ B-cell–depleting antibodies followed by HOD RBC transfusion (MZ B dep), or IC antibodies followed by HOD RBC transfusion (IC). Splenocytes were examined on D1, D3, and D5 for OTII T-cell proliferation; representative line graphs are shown (E). OTII T cells from these groups were stained for activation markers, including CD44 (F) and CD62L (G). Shaded graphs represent CFSE proliferation in a control mouse that received CFSE-labeled OTII T cells but did not receive HOD RBC transfusion. The red line indicates MZ B-cell–depleted mice, and the black line represents B6 control group following HOD RBC transfusion. Error bars represent standard deviation; results are a combination of 2 independent experiments with ≥3 mice per time point per group. Statistical significance was assessed using a nonparametric unpaired Student t test or 1-way ANOVA with Tukey’s post hoc test. ns, not significant.