FIGURE 5.

KEL^lo RBC exposure fails to induce detectable changes in Treg numbers or function. (A) Representative flow plots of CD25⁺ Foxp3⁺ Tregs following C57BL/6 KEL⁻ recipient exposure to PBS (Naive), C57BL/6 (B6), KEL^lo, or KEL RBCs. Quantitative analysis of CD25⁺ Foxp3⁺ Tregs (B) or quantitation of TGF-β–producing CD25⁺ Foxp3⁺ Tregs (C) following exposure to C57BL/6 (B6) or KEL^lo RBCs prior to (D0, day 0) or following (D7, day 7 following transfer) a KEL RBC challenge. A PBS-treated control group, not exposed to KEL (Naive), was also evaluated at each time point. At D7, a group transfused with KEL only group (KEL) was included to control for endogenous Treg response to KEL RBC exposure. (D) Representative flow cytometric examination of CD4⁺ CD25⁺ T cells following injection of an anti-CD25 depleting or isotype-control Ab. (E) Quantitative analysis of CD4⁺ CD25⁺ T cells following depletion (Treg depl.) or isotype control (Iso. Cont.) injection, as indicated. (F) Examination of IgG anti-KEL Ab formation following KEL RBC transfusion (KEL) into untreated (Naive), CD4⁺ CD25⁺ T cell–depleted (Treg Depl.), or isotype control (Iso. Cont.)-treated recipients or recipients that were exposed to KEL^lo RBCs prior to depletion of CD4⁺ CD25⁺ T cell depletion (Treg Depl.) or isotype control (Iso. Cont.) treatment and then challenged with KEL RBCs (KEL^lo→KEL). (G) Evaluation of anti-KEL IgG Ab formation following KEL RBC challenge in recipients pretransfused with PBS (Naive) or adoptively transferred with 10⁷ CD4⁺ T cells from C57BL/6 naive (Naive T cells) or KEL^lo RBC–treated (KEL^lo T cells) recipients. Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA.