Effect And Mechanisms Of MTOR Signaling Pathway On Regulation The Immunomdulatory Ability Of Mescnchymal Stem Cells

Background:Mesenchymal stem cells (MSCs) is a kind of adult stem cells which exist in the bone marrow and various other tissues. Due to the extensive immunoregulatory function, MSCs have been employed to treat graft-versus-host disease (GVHD) and autoimmune disease. Chronic GVHD (cGVHD) is a major complication which affects the outcome of allogeneic hematopoietic stem cell transplantation (HSCT). cGVHD is known to have characteristic clinical manifestations that resemble those observed in autoimmune diseases. It has been reported that there is increased senescence and apoptosis, impaired proliferation potential of MSCs in some autoimmune diseases, which may be associated with the pathogenesis of these diseases. However, it is not clear whether MSCs in cGVHD patients have similar impairment, and whether autologous MSCs is suitable for treatment in this disease.MSCs need the activation by inflammatory microenvironment to exert immunoregulatory function. However, the impairment and increased immunogenicity of MSCs under the influence of inflammatory cytokines limits their suppressive activity in treatment for GVHD and other diseases. mTOR signaling pathway regulates various immune responses, and plays an important role in activation and differentiation of T cells, B cells. Furthermore, mTOR signaling pathway regulates the MHC molecules expression on the surface of various cells such as B16melanoma cells. However, whether mTOR signaling pathway regulates the immunosuppressive ability and immunogenicity of MSCs has not been investigated.In our study, we intend to clarify the role of MSCs in pathogenesis of cGVHD, and explore the feasibility of autologous cell therapy in patients with cGVHD; explore the effects and mechanisms of mTOR signaling pathway on regulation the immunosuppressive ability and immunogenicity of MSCs. Our research will provide the experimental evidences for improving MSCs therapy in GVHD and other diseases.Part1Biology characteristics of bone marrow mesenchymal stem cells in patients with chronic graft-versus-host diseaseAim:Comparative analysis the phenotypical and functional characterization of bone marrow MSCs from patients with cGVHD and without cGVHD following HSCT, also from healthy donors. To clarify the role of MSCs in pathogenesis of cGVHD, and explore the feasibility of autologous cell therapy in patients with this desease.Methods:Bone marrow MSCs were isolated from25patients with cGVHD,16patients without cGVHD following HSCT, and19healthy donors. Passage3-5MSCs were used for comparative analysis. Chimerism was analyzed by short tandem repeat (STR)-PCR. MSC frequency was evaluated by counting colony forming unit fibroblasts (CFU-F) in106bone marrow mononuclear cells. Surface markers, cellular apoptosis and immunosuppressive ability were assessed by flow cytometry. Osteogenic and adipogenic differentiation were assessed by Alizarin Red and Oil Red O stains respectively. Senescence-associated β-galactosidase (SA-β-gal) assay was used for evaluating senescence of MSCs. Supernatant cytokines from MSCs normal culture or co-culture with peripheral blood mononuclear cells (PBMCs) was detected by ELISA. Real time PCR was used for evaluating the expression of pluripotency-associated genes, senescence-associated genes and differentiation-associated genes. The migration potential was evaluated using transwell chambers.Results:Both bone marrow MSCs from patients with or without cGVHD following HSCT were of host origin, and highly expressed CD73, CD90, CD105, moderately expressed HLA-ABC, while they were negative for CD34, CD45, CDllb, CD19and HLA-DR. The frequency of MSCs did not differ significantly between two groups. They showed similar morphology, proliferation potential, self-renewal capacity, differentiation and migration potential. No significant differences were observed in the cellular senescence and apoptosis between two groups. The immunomodulatory potential of two groups was also identical, they were both capable of inhibiting phytohemagglutinin (PHA)-activated PBMCs proliferation and inducing CD4+CD25hiCD127" Tregs after co-culturing with CD4+T cells, the immunosuppressive factors including TGF-βl, IL-10, HGF and PGE2were secreted similarly in both MSCs whether in normal culture or co-culture with PBMCs. In addition, MSCs from cGVHD patients displayed normal phenotype and function compared to their counterparts in healthy donors, although reduced frequency in bone marrow mononuclear cell fraction was observed in these patients.Conclusion:MSCs do not seem to contribute to the pathogenesis of cGVHD. MSCs isolated from patients with cGVHD preserve similar phenotypical and functional characterization as those from healthy donors and could therefore be considered in an autologous setting in patients with this disease.Part2Effects and mechanisms of mTOR signaling pathway on regulation the immunosuppressive ability of bone marrow mesenchymal stem cellsAim:To clarify the effects and mechanisms of mTOR signaling pathway on regulation the immunosuppressive ability of MSCs.Methods:After pretreatment with different concentrations of rapamycin, MSCs were co-cultured with activated PBMCs, mouse splenocytes and CD4+T cells for5days. The proliferation of PBMCs, mouse splenocytes and proportion of CD4+CD25hiCD127-cells were detected by flow cytometry. The mRNA expression of IFN-y, TNF-a receptors and immunosuppressive factors were detected by real time PCR. Western blotting was used for evaluating the expression of total protein and nuclear protein. Lentivirus-mediated shRNA interference was employed to verify the role of COX-2in immunosuppressive function of MSCs. The expression of PGE2of supernatant from MSCs which pretreated with rapamycin was assessed by ELIS A.Results:mTOR inhibiton by pretreatment with rapamycin significantly enhanced the immunosuppressive function of MSCs by inhibiting the proliferation of PBMCs. The ability of induction Tregs, however, was not influenced. Pretreatment with rapamycin also enhanced the ability of MSCs to inhibit the proliferation of splenocytes activated by anti-CD3/CD28antibodies. The expression of IFN-y, TNF-a receptors were not altered by mTOR inhibition. The mRNA and protein levels of COX-2and the production of PGE2were significantly upregulated by mTOR inhibition. The expression of COX-2and PGE2were also upregulated after treating with IFN-y and TNF-a, and pretreatment with rapamycin could further increase the expression level. After adding with NS-398, a specific COX-2inhibitor, or COX-2-targeting shRNA, the enhancement of immunosuppressive function by rapamycin was reduced. Rapamycin also increased the nuclear translocation of NF-kB, and the expression of COX-2upregulated by rapamycin was attenuated when added with NF-kB inhibitor PDTC.Conclusion:mTOR inhibition by pretreatment with rapamycin significantly enhanced the immunosuppressive function of MSCs partly through COX-2and PGE2.Part3Effects of mTOR signaling pathway on regulation the immunogenicity of bone marrow mesenchymal stem cellsAim:To clarify the effects of mTOR signaling pathway on regulation the expression of HLA-ABC and HLA-DR on MSCs.Methods:After pretreatment with rapamycin, HLA-ABC+HLA-DR+cell proportion and mean fluorescence intensity in normal culture or treated with IFN-y were evaluated by flow cytometry. The mRNA expression of IRF1, CIITA, proteasome subunits and MHC-I assembly related molecular chaperones were detected by real time PCR. Western blotting was used for evaluating the phosphorylation of Statl.Results:Rapamycin has no effect on the expression of HLA-ABC. The mean fluorescence intensity of HLA-ABC was elevated by treating with IFN-y for3days. Proteasome subunits LMP2, LMP7, LMP10and molecular chaperones, including Tapasin, were also upregulated. Rapamycin has no effect on the expression of HLA-ABC in culture treated with IFN-y as well, however, it increased the expression of Tapasin, LMP7and LMP10. MSCs were negative for HLA-DR in normal culture, however, its expression was significantly elevated by IFN-y. The phosphorylation of Statl and mRNA expression of IRF1, CIITA were also upregulated. Rapamycin has no influence on the Statl phosphorylation and IRF1, CIITA expression, however, pretreatment with high dose rapamycin significantly reduced the HLA-DR+cell proportion and mean fluorescence intensity in culture treated with IFN-y.Conclusion:mTOR inhibition by rapamycin has no effect on the expression of HLA-ABC, however, it reduced the expression of HLA-DR which upregulated by IFN-y.