The expression mechanism of programmed cell death 1 ligand 1 and its role in immunomodulatory ability of mesenchymal stem cells.

Programmed cell death 1 ligand 1 (PD-L1) is an important immunosuppressive molecule, which inhibits the function of T cells and other immune cells by binding to the receptor programmed cell death-1. The PD-L1 expression disorder plays an important role in the occurrence, development, and treatment of sepsis or other inflammatory diseases, and has become an important target for the treatment of these diseases. Mesenchymal stem cells (MSCs) are a kind of pluripotent stem cells with multiple differentiation potential. In recent years, MSCs have been found to have a strong immunosuppressive ability and are used to treat various inflammatory insults caused by hyperimmune diseases. Moreover, PD-L1 is deeply involved in the immunosuppressive events of MSCs and plays an important role in the treatment of various diseases. In this review, we will summarize the main regulatory mechanism of PD-L1 expression, and discuss various biological functions of PD-L1 in the immune regulation of MSCs.

Interferon-gamma and tumor necrosis factor-alpha synergistically enhance the immunosuppressive capacity of human umbilical-cord-derived mesenchymal stem cells by increasing PD-L1 expression.

BACKGROUND: The immunosuppressive capacity of mesenchymal stem cells (MSCs) is dependent on the "license" of several proinflammatory factors to express immunosuppressive factors such as programmed cell death 1 ligand 1 (PD-L1), which determines the clinical therapeutic efficacy of MSCs for inflammatory or immune diseases. In MSCs, interferon-gamma (IFN-γ) is a key inducer of PD-L1 expression, which is synergistically enhanced by tumor necrosis factor-alpha (TNF-α); however, the underlying mechanism is unclear. AIM: To reveal the mechanism of pretreated MSCs express high PD-L1 and explore the application of pretreated MSCs in ulcerative colitis. METHODS: We assessed PD-L1 expression in human umbilical-cord-derived MSCs (hUC-MSCs) induced by IFN-γ and TNF-α, alone or in combination. Additionally, we performed signal pathway inhibitor experiments as well as RNA interference experiments to elucidate the molecular mechanism by which IFN-γ alone or in combination with TNF-α induces PD-L1 expression. Moreover, we used luciferase reporter gene experiments to verify the binding sites of the transcription factors of each signal transduction pathway to the targeted gene promoters. Finally, we evaluated the immunosuppressive capacity of hUC-MSCs treated with IFN-γ and TNF-α in both an in vitro mixed lymphocyte culture assay, and in vivo in mice with dextran sulfate sodium-induced acute colitis. RESULTS: Our results suggest that IFN-γ induction alone upregulates PD-L1 expression in hUC-MSCs while TNF-α alone does not, and that the co-induction of IFN-γ and TNF-α promotes higher expression of PD-L1. IFN-γ induces hUC-MSCs to express PD-L1, in which IFN-γ activates the JAK/STAT1 signaling pathway, up-regulates the expression of the interferon regulatory factor 1 (IRF1) transcription factor, promotes the binding of IRF1 and the PD-L1 gene promoter, and finally promotes PD-L1 mRNA. Although TNF-α alone did not induce PD-L1 expression in hUC-MSCs, the addition of TNF-α significantly enhanced IFN-γ-induced JAK/STAT1/IRF1 activation. TNF-α up-regulated IFN-γ receptor expression through activation of the nuclear factor kappa-B signaling pathway, which significantly enhanced IFN-γ signaling. Finally, co-induced hUC-MSCs have a stronger inhibitory effect on lymphocyte proliferation, and significantly ameliorate weight loss, mucosal damage, inflammatory cell infiltration, and up-regulation of inflammatory factors in colitis mice. CONCLUSION: Overall, our results suggest that IFN-γ and TNF-α enhance both the immunosuppressive ability of hUC-MSCs and their efficacy in ulcerative colitis by synergistically inducing high expression of PD-L1.

Human iPSC-MSC-Derived Xenografts Modulate Immune Responses by Inhibiting the Cleavage of Caspases.

Mesenchymal stem cells (MSCs) negatively modulate immune properties. Induced pluripotent stem cells (iPSCs)-derived MSCs are alternative source of MSCs. However, the effects of iPSC-MSCs on T cells phenotypes in vivo remain unclear. We established an iPSC-MSC-transplanted host versus graft reaction mouse model using subcapsular kidney injection. Th1, Th2, regulatory T cells (Treg), and Th17 phenotypes and their cytokines were investigated in vivo and in vitro. The role of caspases and the soluble factors involved in the effects of MSCs were examined. We found that iPSC-MSC grafts led to more cell survival and less infiltration of inflammatory cells in mice. iPSC-MSC transplantation inhibited T cell proliferation, decreased Th1 and Th2 phenotypes and cytokines, upregulated Th17 and Treg subsets. Moreover, iPSC-MSCs inhibited the cleavage of caspases 3 and 8 and inhibition of caspases downregulated Th1, Th2 responses and upregulated Th17, Treg responses. Soluble factors were determined using protein array and TGF-ß1/2/3, IL-10, and MCP-1 were found to be highly expressed in iPSC-MSCs. The administration of the soluble factors decreased Th1/2 response, upregulated Treg response and inhibited the cleavage of caspases. Our results demonstrate that iPSC-MSCs regulate T cell responses as a result of a combined action of the above soluble factors secreted by iPSC-MSCs. These factors suppress T cell responses by inhibiting the cleavage of caspases. These data provide a novel immunomodulatory mechanism for the underlying iPSC-MSC-based immunomodulatory effects on T cell responses. Stem Cells 2017;35:1719-1732.

[Research and Application of iPS Cells in Blood System].

Induced pluripotent stem cells (iPS cells) were first constructed by Takahshi and et al in 2006. They converted the mouse fibroblasts into ES-like cells via viral transduction with four transcription factors (Oct4, Sox2, Klf4 and c-Myc). Since, the significant progress has been made and many researchers have succeeded in inducing iPS cells from other human somatic cells by some novel approaches, such as combining transcriptional factors and small chemicals. IPS cells have significant prospect in clinical application. IPS cells derived from patient somatic cells can be used as a model in studying the pathogenesis of genetic hematological disease and applied in therapeutic screenings. Recent studies suggested that iPS cells can differentiate into red blood cells and platelets in vitro, which may make up a big blood bank for transfusion in future. In this review, current understanding of both recombinant technology of iPS cells and the research progress in hematology are summarized.

iPSC-MSCs Combined with Low-Dose Rapamycin Induced Islet Allograft Tolerance Through Suppressing Th1 and Enhancing Regulatory T-Cell Differentiation.

Mesenchymal stem cell (MSC) differentiation is dramatically reduced after long-term in vitro culture, which limits their application. MSCs derived from induced pluripotent stem cells (iPSCs-MSCs) represent a novel source of MSCs. In this study, we investigated the therapeutic effect of iPSC-MSCs on diabetic mice. Streptozocin-induced diabetic mice transplanted with 400 islets alone or with 1×10(6) iPSC-MSCs were examined following rapamycin injection (0.1 mg/kg/day, i.p., from days 0 to 9) after transplantation. Our results showed that iPSC-MSCs combined with rapamycin significantly prolonged islet allograft survival in the diabetic mice; 50% of recipients exhibited long-term survival (>100 days). Histopathological analysis revealed that iPSC-MSCs combined with rapamycin preserved the graft effectively, inhibited inflammatory cell infiltration, and resulted in substantial release of insulin. Flow cytometry results showed that the proportion of CD4(+) and CD8(+) T cells was significantly reduced, and the number of T regulatory cells increased in the spleen and lymph nodes in the iPSC-MSCs combined with the rapamycin group compared with the rapamycin-alone group. Production of the Th1 proinflammatory cytokines interleukin-2 (IL-2) and interferon-γ was reduced, and secretion of the anti-inflammatory cytokines IL-10 and transforming growth factor-ß was enhanced compared with the rapamycin group, as determined using enzyme-linked immunosorbent assays. Transwell separation significantly weakened the immunosuppressive effects of iPSC-MSCs on the proliferation of Con A-treated splenic T cells, which indicated that the combined treatment exerted immunosuppressive effects through cell-cell contact and regulation of cytokine production. Taken together, these findings highlight the potential application of iPSC-MSCs in islet transplantation.

Clinical significance and therapeutic value of glutathione peroxidase 3 (GPx3) in hepatocellular carcinoma.

AIMS: We aimed to investigate the clinical significance of GPx3 in hepatocellular carcinoma (HCC) and to characterize its tumor suppressive role. METHODS: HCC patients (113) who underwent hepatectomy were recruited to examine the clinical relevance of GPx3. The tumor suppressive role of GPx3 was studied by administration of recombinant GPx3 (rGPx3) or over-expression of GPx3 in HCC cells in vitro and in vivo. The therapeutic value of GPx3 for HCC was further investigated using human induced pluripotent stem cell derived mesenchymal stem cells (hiPSC-MSCs) as its delivery vehicle. RESULTS: Down-regulation of GPx3 significantly correlated with advanced tumor stage (P = 0.024), venous infiltration (P = 0.043) and poor overall survival (P = 0.007) after hepatectomy. Lower plasma GPx3 in HCC patients was significantly associated with larger tumor size (P = 0.011), more tumor nodules (P = 0.032) and higher recurrence (P = 0.016). Over-expression of GPx3 or administration of rGPx3 significantly inhibited proliferation and invasiveness of HCC cells in vitro and in vivo. Tumor suppressive activity of GPx3 was mediated through Erk-NFκB-SIP1 pathway. GPx3 could be delivered by hiPSC-MSCs into the tumor and exhibited tumor suppressive activity in vivo. CONCLUSIONS: GPx3 is a tumor suppressor gene in HCC and may possess prognostic and therapeutic value for HCC patients.

Dendritic cells-mediated CTLs targeting hepatocellular carcinoma stem cells.

Immunotherapy, especially using dendritic cells (DCs)-based vaccine, appears promising in the treatment of hepatocellular carcinoma (HCC) following surgery. However, the therapeutic efficacy of current DC vaccines loaded with HCC antigen is limited in clinical practice. One important reason might be that the DC vaccines for the treatment of HCC were not aimed at targeting the hepatocellular carcinoma cancer stem cells (HCCCSCs). Therefore, establishing an immunotherapy to kill HCC stem cells could be a novel therapeutic strategy. In this study, we have developed an immunotherapy to target CD133(+) HCC cells in the treatment of HCC. This study had three main findings; (1) CD133(+)HCC cells RNA loaded DCs could induce special CD8(+) cytotoxic T lymphocytes (CD133(+)Huh7-CTLs) response against CD133(+) Huh7 cells in vitro. (2) Huh7 cells-induced tumor growth in vivo was effectively inhibited by CD133(+)Huh7-CTLs. (3) the great inhibition potential of CD133(+)Huh7-CTLs to Huh7-induced tumor growth might not be only associated with anti-tumor cytokines such as IFNγ, but also to CD133(+)Huh7-DCs induced specific CTLs. This study shows an experimental proof that CD133(+)HCC cells RNA loaded DC vaccine has potential in treating HCC and may provide a new therapy for clinical post operative adjuvant therapy in future.