Figure 1. BMMSCs induce T cell apoptosis via Fas ligand (FasL).
(A) Schema of BMMSC transplantation procedure. 1×106 BMMSCs (n=5), FasL−/− gldBMMSCs (n=4) or FasL-transfected gldBMMSCs (FasL+gldBMMSCs, n=4) were infused into C57BL6 mice through the tail vein. All groups were sacrificed at indicated time points for sample collection. Zero hour represented that mice were immediately sacrificed after BMMSC injection. (B–E) BMMSC transplantation (BMMSC) induced transient reduction in the number of CD3+ T cells and increased AnnexinV+7AAD+ double positive apoptotic CD3+ T cells in peripheral blood mononuclear cells (PBMNCs; B, C) and bone marrow mononuclear cells (BMMNCs; D, E) at indicated time points, while FasL−/− BMMSCs from gld mice (gldBMMSCs) failed to reduce CD3+ T cells or elevate CD3+ T cell apoptosis in peripheral blood (B, C) and bone marrow (D, E). FasL-transfected gldBMMSC transplantation (FasL+gldBMMSC) partially rescued the capacity to reduce the number of CD3+ T cells and induce CD3+ T cell apoptosis in peripheral blood (B, C) and bone marrow (D, E). *P<0.05; **P<0.01; ***P<0.001 vs. gldBMMSC, #P<0.05; ###P<0.001 vs. FasL+gldBMMSC, $P<0.05; $$$P<0.001 vs. gldBMMSC. (F) When BMMSCs were infused into mice, TUNEL and immunohistochemistry staining showed that TUNEL-positive apoptotic cell (brown, white arrow) number in CD3-positive T cells (purple, yellow arrowhead) was higher in the BMMSC-injected group compared to the control group in bone marrow. (G) When BMMSCs were co-cultured with T cells, BMMSC-induced AnnexinV+7AAD+ double positive apoptotic T cells were significantly blocked by anti-FasL neutralizing antibody (1μg/mL) compared to IgG antibody control group. (H) TUNEL and immunohistochemistry staining showed that TUNEL-positive apoptotic T cells (brown, white arrow) were observed in CD3+ T cells (purple, yellow arrowhead) when co-cultured with BMMSCs in vitro. In the presence of anti-FasL neutralizing antibody (FasL Ab), TUNEL-positive cell percentage was significantly less than the untreated control group. (I) In addition, the number of BMMSC-induced AnnexinV+7AAD+ double positive apoptotic T cells was significantly blocked by caspase 3, 8, and 9 inhibitor treatments. The results were representative of three independent experiments. (J) Schematic diagram indicating that BMMSCs induce T cell apoptosis. (*P<0.05; **P<0.01; ***P<0.001. The bar graph represents mean±SD). See also Figures S1 and 2.