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Supplementary Material 6

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Aims To investigate whether placenta-derived mesenchymal stromal cells (hPMSCs) have immunoregulatory effects on PD-1+ T cell generation by controlling ROS production and thus alleviating GVHD. Main methods Flow cytometry was used to analyze the percentage of PD-1+ T cells, as well as the generation of ROS, GSH and GST in PD-1+ T cells. The expression of GST in the spleen and liver was analyzed by western blotting. Key findings The percentage of PD-1+ T cells was increased, but the ratio of GSH/GSSG was decreased in GVHD patients and the GVHDhigh mouse model compared with that in the normal control group. hPMSCs downregulated the level of malondialdehyde (MDA) and upregulated the ratio of GSH/GSSG and the expression of glutathione S transferase (GST) in the plasma, spleen and liver of GVHD mice compared with those of PBS-treated GVHD mice. Further studies showed that the ROS level, as well as the expression of PD-1, in both CD3+ and CD4+ T cells from the spleen and liver of hPMSC-treated GVHD mice were decreased compared with those observed in PBS-treated mice. Significance hPMSCs downregulated ROS generation by increasing GSH and GST levels and further reduced the expression of PD-1 on T cells, thereby alleviating inflammation in GVHD mice.