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. Author manuscript; available in PMC: 2013 May 4.
Published in final edited form as: Cell Stem Cell. 2012 Apr 26;10(5):544–555. doi: 10.1016/j.stem.2012.03.007

Figure 5. Fas plays an essential role in BMMSC-mediated CD3+ T cell apoptosis and up-regulation of Tregs via regulating monocyte chemotactic protein 1 (MCP-1) secretion.

Figure 5

(AD) BMMSC transplantation (BMMSC) induced transient reduction in the number of CD3+ T cells and increase in the number of AnnexinV+7AAD+ double positive apoptotic CD3+ T cells in peripheral blood mononuclear cells (PBMNCs; A, B) and bone marrow mononuclear cells (BMMNCs, n=5; C, D) at indicated time points, while Fas−/− BMMSC from lpr mice (lprBMMSC, n=5) failed to reduce the number of CD3+ T cells or increase the number of CD3+ apoptotic T cells in peripheral blood (A, B) and bone marrow (C, D). (E, F) lprBMMSC transplantation failed to elevate Treg levels (E) and TGF-β (F) in C57BL6 mice compared to the BMMSC transplantation group at indicated time points. (G) lprBMMSC induced activated T cell apoptosis in a BMMSC/T cell in vitro co-cultured system, which was blocked by anti-FasL neutralizing antibody (1μg/mL). (H–K) Activated T cells (green) migrate to BMMSCs (red) in a transwell co-culture system (H). lprBMMSCs showed a significantly reduced capacity to induce activated T cell migration (I), which could be partially rescued by overexpression of MCP-1 (J) and totally rescued by overexpression of Fas (K) in lprBMMSCs. The results were representative of three independent experiments. (L) Quantitative RT-PCR analysis showed no significant difference between BMMSC and lprBMMSC in terms of MCP-1 expression level. However, overexpression of MCP-1 and Fas in lprBMMSC significantly elevated gene expression level of MCP-1. (M) Western blot showed that lprBMMSCs express a higher cytoplasm level of MCP-1 than BMMSC. Overexpression of Fas in lprBMMSC reduced the expression level of MCP-1 in cytoplasm. (N) ELISA analysis showed that MCP-1 secretion in culture supernatant was significantly reduced in lprBMMSCs compared to BMMSC. Overexpression of MCP-1 and Fas in lprBMMSCs significantly elevated MCP-1 secretion in culture supernatant. (O) ELISA data showed that knockdown Fas expression using siRNA resulted in reduction of MCP-1 level in culture medium compared to control siRNA group. (P–Q) Fas siRNA-treated BMMSCs (Q) showed reduced T cell migration in transwell co-culture system compared to control siRNA group (P). (*P<0.05, **P<0.01, ***P<0.001. The bar graph represents mean±SD). See also Figure S6.