| ObjectiveThe aim of this study was to investigate the effects of human umbilical cord mesenchymal stem cells derived exosomes（MSC-Exo）on the activity and function of umbilical cord blood-derived regulatory T cells（UCB-Treg）,and to further explore the related molecular mechanisms.Methods（1）CD25+Treg cells were isolated and expanded from umbilical cord blood,which were co-cultured with MSC by direct or indirect method.FCM,WB and PCR were used to detect the phenotype and functional markers of Treg.The suppressive ability of Treg in different treatment groups was evaluated by mixed lymphocyte assay（MLR）and ELISA for IL-10.Cell apoptosis induced by low serum and IL2 was assessed by Annexin-V/PI,and the expression of apoptosis-related proteins were determined by WB.（2）MSC conditioned medium（MSC-CM）was collected,and then exosomes（Exo）and exosomes-free conditioned medium（MSC-CM w/o Exo）were obtained.Exo was identified and quantified.The amplified UCB-Treg was treated in MSC-CM with or without Exo and in the medium contained Exo with different concentrations.Phenotypic and functional markers of Tregs were detected by FCM.Oxidative phosphorylation was detected by Seahorse.Viability was assessed by Annexin-V/PI and MTS.Suppressive ability was tested by MLR.After inflammatory induction,the concentrations of inflammatory factors such as IL17a and INF-γwere analyzed by FCM and CBA.What’s more,the apoptosis-regulated and functional proteins and related signaling pathways were detected by WB.（3）The Xenograft-versus-host disease（X-GVHD）model of NCG mice were established by caudal vein infusion with human PBMC.Mice were treated with PBS,Treg,Exo and Treg+Exo,respectively.The body weight,hair,survival status and clinical features of the mice were observed.The pathological changes of target organs,the proportion of human T cells and Tregs in the blood of mice,the concentration of IL17a and IL10 in serum was used to determine the incidence of X-GVHD in each group.Results（1）The expression level of CD25 in Tregs which co-cultured with MSC by the way of direct contact or indirectly trans-well were significantly increased compared to control Tregs which treated with conventional medium,along with the m RNA expression of FOXP3,CTLA4,TIGIT and TIM3 gene raised markedly.Tregs in MSC co-culture group could dramatically inhibit the proliferation of PBMC stimulated by CD3CD28 antibody,and the concentration of IL10 in supernatant was higher than control group.The assay of apoptosis induced by serum starvation showed that the survival rate of Treg in MSC co-culture group was increased,the expressions of Cleaved Caspase3 and 9 and pro-apoptotic protein Bax were down-regulated,and the anti-apoptotic protein p-Bcl2 was up-regulated.The results showed that there was no significant difference between Tregs co-cultured with MSC by the direct and indirect way.（2）Compared with the control group,the number of Treg in MSC-CM group was significantly increased,the basic and max oxygen consumption of Treg was raised,and the viability of Treg was enhanced under the induction of apoptosis.The proportion and MFI of CD4+CD25highTregs in MSC-CM group was increased.The expressions of FOXP3,CTLA4,Helios,PD1 and CD39 of Tregs treated with MSC-CM were up-regulated in m RNA and protein levels.At the same time,MSC-CM cultured Tregs were more suppressive in both polyclonal and allogeneic responses and were resistant to inflammatory stimulation in vitro compared to the controls.The secretion of inflammatory factors such as IFN-γand IL17a were decreased for Treg treated with MSC-CM under the inflammatory environment,which also promoted the secretion of IL10.However,The enhanced suppressive activity and stability of Treg cultured in MSC-CM was reduced when exosomes were depleted from MSC-CM.（3）The number of Treg treated with Exo were significantly increased compared to the control Tregs.To a certain extent,exosomes enhanced oxidative phosphorylation of Treg in a dose-dependent manner and reduced serum starvation-induced apoptosis.At the same time,the levels of CD25,CD39,PD1,FOXP3,Helios and CTLA4 of Tregs treated with Exo were dramatically up-regulated,which could more signally inhibit the proliferation of PBMC and maintain the stability under inflammatory stimulation.In addition,we detected that Exo can up-regulate the expression of autophagy marker LC3II/I by activating PI3K and Akt,as well as enhance phosphorylated JAK3、Stat5,which promote Treg survival,and regulate FOXP3 expression in Tregs.（4）In the X-GVHD model of NCG mice,the Exo group vs.the PBS group,Treg+Exo group vs.Treg group,the survival time of mice treated with Exo was prolonged,and the evaluation of clinical score showed that the symptoms of X-GVHD were reduced.Pathological lesions in target organs were improved and inflammatory cell infiltration was reduced in the mice treated with Exo,to further enhance the efficacy of Treg treatment.At the same time,Exo treatment increased the proportion of Treg and CD4+T cells in peripheral blood,decreased the content of CD8+T cells,with reducing the level of inflammatory factors in plasma to a certain extent.Conclusion（1）Direct or indirect co-culture of Treg and MSC can enhance the inhibition ability and activity of Treg,and the effects of the two co-culture methods on Treg are similar,confirming that MSC can prolong the survival of Treg and maintain its functional stability through paracrine effect.（2）MSC-CM can not only enhance the activity and inhibition ability of Treg,but also maintain the stable function of Treg under inflammatory conditions.Exo plays an important role in this process and is dose-dependent in a certain concentration range.（3）MSC-Exo could activate PI3K/Akt to promote the autophagy,as well as enhance phosphorylated JAK3 and Stat5 to up-regulate FOXP3,which is to maintain activity and functional stability of Tregs.（4）Exo enhanced the effect of Treg on inhibiting X-GVHD by reducing inflammatory cell infiltration in target organs,increasing Treg cell recruitment and regulating cytokine secretion. |
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