Efficacy And Mechanism Of Immunity-and Matrix-regulatory Cells For Graft-versus-host Disease

Background and objectives:Hematopoietic stem cell transplantation(HSCT)is the best therapy for hematological diseases that cannot be cured by traditional methods.During HSCT,immunocompetent lymphocytes from donor can recognize recipient antigens.These immune responses develop in a pro-inflammatory microenvironment resulting in clinical manifestations of GVHD,including rashes,elevated serum bilirubin,and diarrhea.In HSCT performed on HLA-identical siblings,aGVHD incidence is approximately 40%,reaching 80%for HLA-mismatched unrelated donors.A steroid regimen remains the standard therapy,with less than50%effectiveness and no standard second-line therapy is currently available.Patients with severe GVHD have dismal overall survival rates.GVHD poses a growing threat to patient survival.Therefore,new effective therapy is warranted.Mesenchymal stem cells are a kind of stem cells originated from mesoderm,with strong self-renewal and multi-directional differentiation ability.Studies have shown that MSC has immunomodulatory effects.Under the stimulation of high levels of pro-inflammatory cytokines,MSCs inhibit inflammatory response and prevents excessive tissue damage.Therefore,MSCs are widely used in the treatment of immune disorders,such as GVHD.Most clinical trials to examine the safety and efficacy of MSCs in the prevention or treatment of GVHD have achieved satisfactory results,but there are also some failed cases.For example,Prochymal(ex vivo cultured human MSCs)of Osiris did not achieve the expected efficacy in the phase ? clinical trial for the therapy of GVHD,resulting in Prochymal was not approved by the FDA.The reasons include:(1)immunogenicity of MSCs:The ability of MSCs to express MHC-? and MHC-? was up-regulated when exposed to inflammatory signals;(2)the quality of MSCs varies:there are differences in the quality of MSCs from different individuals;in vitro expansion of MSCs causes senescence and functional decline;cryopreservation and resuscitation lead to the decrease of MSC viability and function;(3)heterogeneity of MSCs:MSCs from different tissue sources have different functions,MSC from the same tissue source also have different functional subsets,these subsets have different immune regulation ability;(4)the influence of immune system on MSCs has two sides:different MSC doses and intervention timing will lead to different effects of the same disease.Therefore,researchers looked for other cell types that could replace MSCs,such as embryonic stem cells(ESCs)and induced pluripotent stem cells(i PSCs).However,ethical issues and potential tumorigenesis hinder the clinical transformation of ESCs and i PSCs.Our group established a clinical-grade ESC line in the early stage,and differentiated it to obtain MSC-like cells,which meet the international standard for MSCs,we named it immunity-and matrix-regulatory cells(IMRCs).Whole genome sequencing showed that compared with UC-MSCs,immunomodulatory genes and tissue repair genes of IMRCs were up-regulated,and pro-inflammatory genes were down-regulated.Enrichment analysis of differentially expressed genes confirmed that IMRCs showed reduced inflammatory response and enhanced proliferation ability.Therefore,the purpose of this study is to examine the ability of IMRCs to inhibit lymphocyte proliferation and the efficacy of IMRCs for GVHD,so as to provide an important basis for the subsequent clinical trials with IMRCs and provide new insights for cell therapy.Methods:Firstly,IMRCs were identified according to the standard for MSCs defined by the International Society of Cell Therapy.The morphology of IMRCs was observed,the markers of IMRCs were detected by flow cytometry,and the ability of IMRCs to differentiate into mesenchymal lineages was identified by chemical staining.Secondly,IMRCs were co-cultured with PBMC or CD4/8~+cells to detect whether IMRCs inhibited PBMC or CD4/8~+cells proliferation,whether IFN-?can enhance the effect of IMRCs,to detect whether 1-MT,an inhibitor of IDO,can block the inhibitory effect of IMRCs.To detect whether there is difference of inhibition ability between CD24~+and CD24~-?MRCs.To test whether IMRCs can inhibit T cell activation.Detection of cytokines in co-culture system by ELISA,RNA-seq of IMRCs co-cultured with PBMC.To detect whether the inhibitory effect of different batches of IMRCs is consistent.To test if IMRCs preparations have the same effect as freshly cultured IMRCs.Finally,aGVHD model was constructed and treated with IMRCs.The survival time,body weight and GVHD clinical score of mice were observed,and the effect of IMRCs on the proliferation and activation of donor T cells in the recipient was studied.Results:(1)Under standard culture conditions,IMRCs are spindle-shaped,grow adherently,express CD29,CD73,CD90,CD105,but do not express CD34,CD45,CD11b,CD19 and HLA-DR.IMRCs have the ability to differentiate into adipocytes,chondroblasts and osteoblasts,which conform to the definition of MSCs;(2)IMRCs can inhibit the proliferation of PBMC and CD4/8~+cells,inhibit the activation of T cells,and the inhibitory ability is dose-dependent.The inhibitory effect is related to the IDO secreted by IMRCs.If IMRCs are stimulated with IFN-?,the effect of IMRCs on inhibiting PBMC proliferation is enhanced;(3)There was no significant difference in the ability of different batches of IMRCs to inhibit the proliferation of PBMC and CD4/8~+cells;(4)IMRCs preparations also have the ability to inhibit the proliferation of PBMC and CD4/8~+cells;(5)After co-culture of GFP-?MRCs with PBMC,the transcriptome changed,and the DEGs were mainly involved in biological processes such as T cell activation,regulation of cytokine secretion,and regulation of adhesion between cells,and were mainly enriched in interactions between cytokine receptors,chemokines,cell adhesion molecules and other signal pathways;(6)After co-culture of IMRCs with PBMC,the expressions of IFN-?,TNF-?,IL-4,IL-5,IL-10,IL-13,MIP-1?and GM-CSF were significantly reduced.And the expression of IL-2,IL-6,IL-12 and MCP-1 were significantly increased;(7)IMRCs can improve the survival rate of GVHD mice and reduce the infiltration of lymphocytes in lung,intestine,liver and kidney;(8)The effect of GVHD by IMRCs does not depend on reducing the chimerism,inhibiting T cell activation and changing the proportion of lymphocytes and their subsets.Conclusions:IMRCs differentiated from clinical-grade ESCs conform to the standards of MSCs proposed by the International Society for Cell Therapy.IMRCs can inhibit the proliferation of PBMC and CD4/8~+cells,inhibit the activation of T cells,and the inhibitory ability is dose-dependent.The inhibitory effect of IMRCs is related to IDO secreted by itself.After IFN-?stimulation,the effect of IMRCs on PBMC proliferation was enhanced.After co-culture of IMRCs with PBMC,the transcriptome was significantly changed,which in turn changed the expression of cytokines.The quality of IMRCs of different batches is uniform;IMRCs preparations also have the ability to inhibit the proliferation of PBMC and CD4/8~+cells.IMRCs prolong the survival time of GVHD mice and reduce the infiltration of lymphocytes to target organs,but do not reduce the chimerism of PBMC,inhibit the activation of human T cells,and change the proportion of human T,B lymphocytes and their subpopulations.The specific mechanism of IMRCs for GVHD need to be further studied.