Figure 5.

Regulatory T cells (Tregs) in allogeneic mixed lymphocyte reaction (MLR). Cells were harvested and phenotyped after 7, 10, or 14 days of allogeneic MLR. Each antibody was used at a concentration of 10 µg/ml. Data displayed as mean ± SD. Data were analyzed using two-way ANOVA followed by Dunnett’s multiple comparison test (*p < 0.05, ***p < 0.001, ****p < 0.0001) with no antibody controls serving as the comparison data set. Tregs were identified as CD4⁺ CD127⁻ CD25⁺ FoxP3⁺ (A) Tregs as % of CD4⁺ T cells over time (N = 12 donor pairs). Siplizumab significantly enriched Tregs among CD4⁺ T cells after 10 (p = 0.0340) and 14 (p < 0.0001) days of allogeneic MLR. Similarly, rATG increased the percentage of Tregs among CD4⁺ T cells on all time points (p < 0.0001 for all time points). (B) Percentage of Tregs among proliferated (VPD450low) CD4⁺ T cells (N = 12 donor pairs). Siplizumab (p = 0.0004; p < 0.0001; p < 0.0001) and rATG (p = 0.0007; p < 0.0001; p < 0.0001) significantly enriched Tregs among proliferated CD4⁺ T cells after 7, 10, and 14 days of allogeneic MLR. (C) FoxP3 promoter methylation status of regulatory T cells (N = 5 donor pairs) with Tregs from no antibody controls serving as the comparison data set. PBMC were used as a control. Lower percentage methylation indicates higher commitment to Treg phenotype. FoxP3 promoter methylation was significantly decreased in Tregs isolated from siplizumab-treated samples relative to Tregs isolated from no antibody controls after 7 days of allogeneic MLR (p = 0.0190). A decrease trending toward significance (p = 0.0529) was measured in siplizumab-treated samples after 14 days of allogeneic MLR. rATG failed to induce a significant change in FoxP3 promoter methylation, although decreases observed after 7 days trended toward significance (p = 0.0520). (D) Representative dot plots illustrating enrichment of Tregs among proliferated CD4 T cells in samples treated with 10 µg/ml siplizumab or 10 µg/ml rATG after 14 days of MLRs.