PIC enhances KEL alloimmunization through a CD4⁺ TD process. (A) Representative gating strategy and graphical demonstration of the percentage of splenic CD3⁺ CD4⁺ T cells in B6 recipients treated with PBS (B6), anti-mouse CD4-depleting antibody (CD4 depl.), or rat IgG2b isotype control antibody (Iso. Cont.) and CD4⁺ T-cell–deficient MHC II KO mice. (B) Anti-KEL IgG formation in recipients treated with PBS (B6), anti-mouse CD4-depleting antibody (CD4 depl.), and rat IgG2b isotype control antibody (Iso. Cont.) and transfused with KEL RBCs in the presence or absence of 100 μg PIC. (C) MHC Class II KO (MHC II KO) recipients transfused with KEL RBCs in the presence or absence of 100 μg PIC. Serum was collected on day 14 (D14) after transfusion, and serological analysis for anti-KEL IgG alloantibodies in panels B-C was done by indirect immunofluorescence staining using KEL and B6 RBCs. The MFI in panels B-C was derived from normalizing the MFI of serum samples incubated with KEL RBCs to the MFI of serum samples incubated with background control B6 RBCs. Error bars represent mean ± standard error of the 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 in panels A-B and a Student t test in panel C. Data in panel A are representative of 3 experiments with 3 to 5 mice per group. Panels B-C show a combination of data from 2 to 3 experiments with 4 to 6 mice per group. **P < .01; ***P < .001; ****P < .0001; n.s., nonsignificant. FSC, forward scatter; SSC, side scatter.