Impact of exosomes derived from adipose stem cells on lymphocyte proliferation and phenotype in mouse skin grafts.

Aim: Exosomes derived from adipose-derived stem cells (ASCs) in mice have been reported to influence immune regulation. Yet, the potential immunological effects of ASCs-derived exosomes and their interaction with lymphocytes during transplant immunity remain understudied. Methods: ASCs from BALB/c mice, along with their conditioned culture medium, were collected for the extraction, isolation, and comprehensive characterization of exosomes. Splenic cell suspensions were isolated from BALB/c mice and subsequently processed for downstream analyses. Lymphocytes were isolated via gradient centrifugation and stimulated in vitro with the purified exosomes to assess their functional responses. Lymphocyte proliferation was quantified using the CCK8 assay, and the relative frequencies of CD4+ T cells, CD8+ T cells, Treg cells, NK (natural killer) cells, macrophages, B cells, dendritic cells (DCs), and Th17 cells were determined through flow cytometric analysis. Before establishing the skin transplantation model, the mice were administered PBS, 0.5 × 10(8) exosomes, 1 × 10(8) exosomes, 1.5 × 10(8) exosomes, or ASCs via intravenous injection through the tail vein. Seven days after transplantation, the spleens, drainage lymph nodes, and blood samples were harvested for lymphocyte isolation and further downstream analyses. Results: Exosomes derived from ASCs significantly increased the CD4+/CD8+ ratio and Treg cell levels, without inducing any notable changes in Th17 cell content or CTLA-4 protein expression in CD4+ T cells. Compared to the PBS-treated group, both ASC and exosome treatment groups demonstrated an enhanced CD4+/CD8+ ratio, increased Treg cell content, and elevated CTLA-4 protein expression in spleen tissue following skin transplantation, while Th17 cell levels remained unaffected. Compared to the ASC treatment group, the exosome group exhibited a higher CD4+/CD8+ ratio and Treg cell levels, alongside a reduced proportion of PD-1+ Treg cells and lower CTLA-4 protein expression in CD3+CD4+ T cells. No significant differences were observed in the proportions of NK cells, macrophages, B cells, and DCs in the spleens across all treatment groups. In peripheral blood, an increased proportion of CD3+ T cells, macrophages, and DCs was detected, accompanied by a reduced proportion of NK cells and B cells. In the draining lymph nodes, no significant changes were observed in the proportions of CD3+ T cells and B cells, while macrophages, NK cells, and DCs showed elevated proportions. In the exosome-treated group, mouse grafts exhibited a disorganized and thinner granular layer, accompanied by focal regions of inflammatory cell infiltration. Both exosome and ASC treatments significantly extended the survival of skin grafts. Conclusion: Exosomes derived from ASCs promote lymphocyte proliferation and modulate their phenotypic profiles in mouse skin graft models, effectively extending graft survival.