Background: Currently,the wide clinical application of adoptive cord blood-derived regulatory T cell (CB-treg) therapy is facing challenges in the persistence time and functional stability of Tregs in vivo.Therefore,trategies are needed to improve Treg survival and stability as a means to improve the efficiency for adoptive CB-Treg transfer. In this study, we cultured CB-tregs with human MSC conditioned medium(MSC-CM) to explore whether the exosomes from MSC can enhance the protective effects on tregs in vitro by promoting the anti-apoptosis and improving the inhibition function and further discussed the related mechanism.
Materials: CB-Treg were harvested after 2 weeks’ expansion and divided to coculture in MSC-CM with or without exsomes for another 2 weeks. Extracellular flux analysis was performed  to assess the vitality .Apoptotic Treg cells were induced by serum starvation culture for 96h, in the meantime cell viability were determined by staining Annexin-V/PI and Western-blot.FACS, WB and Realtime PCR were performed to assess cell surface and function related markers.Mixed lymphocyte reaction was performed for their suppressive capacity. IL17a and INF-r were analysed by FACS to evalue the stability of tregs by inflammatory stimulation. 
Results: Compared to the control tregs,the number of Tregs treated with MSC-CM with or without exsomes both higher significantly. The expression of apoptotic protein cleaved caspase-3 was down-regulated, while autophagy marker LC3II/ LC3I was greatly up-regulated in the MSC-CM treated tregs, and the difference was more significant in the group containing exosomes than the group with exosomes cleared, indicating that exosomes from MSC play important roles in inhibiting tregs apoptosis regulated by enhanced autophagy activity. Meanwile the exosomes enhanced vitality of Tregs measured by the basal oxygen consumption rate (OCR).And exosomes of MSC-CM protected the survival of Tregs under apoptosis induction by serum starvation culture. In addition, higher propotion of CD4+CD25+ Tregs was detected  in MSC-CM group, especially for the group containing exsomes.Simultaneously ,the corresponding Tregs upregulated the functional markers on mRNA and/or protein level,such as FOXP3,CTLA4,CD39,IDO and so on.What’s more, Treg treated in MSC-CM more strongly suppressed the proliferation of allogeneic Tconv in a dose-dependent manner compared to control group,especially for the MSC-CM group with exosomes.Last but not least, we found the lowest percent of IL-17a+T cell in the MSC-CM group containing exosomes,indicating MSC derived exosomes strongly promote Treg to resist the inflammatory induction in vitro.
Conclusion: This study demonstrated that MSC derived exosomes not only enhanced tregs survival and suppressive capacity in vitro,but also maintained the stable function under inflammatory condition.It will provide a new, simple,and effective strategy for clinical application of adoptive CB-treg therapies.