Protective Effect And Underlying Mechanisms Of TGF-β1 Gene-modified Mesenchymal Stem Cells On Alleviation Of Acute Graft Versus Host Disease Post Allogeneic Hematopoietic Stem Cell Transplant In Murine Models

BackgroundGraft versus host disease(GVHD)is a common complication post allogeneic hematopoietic stem cell transplantation(allo-HSCT),which can be life-threatening.Mesenchymal stem cells(MSCs)have demonstrated efficacy in steroid resistant aGVHD.However,the outcome of aGVHD treated with MSCs in clinical trials varied and its underlying mechanism is still unclear.TGF-β1 is a potent cytokine,which plays a key role in immunoregulation.In this scenario,the combination of MSC and TGF-β1 may result in a robust function to treat aGVHD.Methods1.Normal bone marrow derived MSC was extracted and identified by morphology,immunophenotype and ability of mesoderm differentiation.2.TGF-β1 was transduced in MSC cells(MSC-TGF-β1)via lentivirus.The expression of TGF-β1 was detected by RT-PCR and Western blot.The features of MSC-TGF-β1 were tested by fluorescence microscope and flow cytometry.3.Immunosuppressive effect of MSC-TGF-β1 on lymphocytes proliferation in vitro was investigated using CFSE cell division kits.4.Prophylactic and therapeutic effects of MSC-TGF-β1 on murine aGVHD model were assessed by survival analysis,clinical and pathologic scoring.5.Finally,the underlining mechanism of the MSC-TGF-β1’s immunosuppressive effect was explored by detecting the profiles of macrophages and Treg cells.Results1、Murine MSC were successfully isolated as plastic adherent long or polygonal cells with similar morphology as fibroblasts.The isolated cells presented typical MSC immunophenotype,with positive expression of CD29(98.7%),Sca-1(99.8%)and lack of CD11c(0.019%)marker,also showed the ability of differentiating into osteogenic,chondrogenic and adipogenic lineages.TGF-β1 was efficiently transduced into mouse MSCs and high TGF-β1 expression was detected in mRNA and protein levels by RT-PCR and Western Blot assays respectively.MSC-TGF-β1 shared same morphology and immunotypic features of normal MSC.2、In vitro,MSC-TGF-β1 exerted the most marked suppression on lymphocytes proliferation.Also,the concentrate of IFN-y is lowest in MSC-TGF-β1 co-cultured group(p<0.01).3、In vivo,MSC-TGF-β1 showed enhanced amelioration on the severity of aGVHD both in prophylactic and therapeutic murine models.During the prophylactic experiments,the median survival of mice injected with BMC+SC,BMC+SC+MSC,BMC+SC+MSC-GFP-puro,BMC+SC+MSC-TGF-β1 was 26,29,28,37 days respectively;The mice treated with MSC-TGF-β1 developed the mildest aGVHD clinical manifestation,and correspondingly had the longest median survival;Histopathological examination shows that MSC-TGF-β1 reduced tissue damage mostly.During the therapeutic experiments,the mice treated with weekly injection of MSC-TGF-β1(3×105)after allo-HSCT for consecutive 5 doses resulted sixty percent of mice achieved more than 50 days of survival post transplantation.4、Finally,the macrophages derived from MSC-TGF-β1 prophylactic treated mice showed a remarkably shift from pro-inflammatory M1-like macrophages to anti-inflammatory M2-like phenotype.Furthermore,the differentiation of CD4+CD25+Foxp3+ Treg cells was also significantly increased in MSC-TGF-β1 prophylactic treated group.ConclusionsTaken together,we proved that MSC-TGF-β1 showed enhanced alleviation of aGVHD severity in mice by skewing macrophages into a M2 like phenotype or increasing the proportion of Treg cells,which opens a new frontier in the treatment of aGVHD.