Figure 6.

IL-27 pre-stimulated third party human iTregs partially reduced mortality of xenogeneic GVHD. To generate human iTregs, sorted human naïve CD4 T cells (CD3⁺CD4⁺CD25⁻CD127⁺) were cultured with immobilized anti-CD3 (2 μg/ml), anti-CD28 (2 μg/ml), IL-2 (100 U/ml) and TGFβ1 (5 ng/ml) for 5 days. iTregs were restimulated with immobilized anti-CD3 (2 μg/ml), anti-CD28 (2 μg/ml), IL-2 (100 U/ml) in the presence or absence of rIL-27 (10 ng/ml) for 24 h. (A) Foxp3 expression. (B) IL-2 production. (C) Surface Lag3 expression. Isotype control (filled), Control iTregs (dashed line), IL-27 iTregs (solid line). (D) In vitro suppression activity. Histogram shown represents one of three independent experiments. Data are mean ± SEM. (E) To induce xenogeneic GvHD (xGvHD), NSG mice were received 200 cGy irradiation and transplanted with 10 × 10⁶ PBMCs with/without 1 × 10⁶ iTregs. Survival and GVHD score were examined. Data shown are from three independent experiments (11–13 mice per group). (F) Serum cytokines were analyzed at day 14 post-xGVHD induction. Data are mean ± SEM, *p < 0.05, **p < 0.01.