Background: Our previous proteome study revealed that haptoglobin was involved in adipose-derived stem cell (ASC) modulation of allotransplant survival and T-cell regulation to induce immune tolerance. This study investigated whether ASCs could modulate T-cell regulation through haptoglobin and the downstream heme oxgenase-1 (HO-1) pathway in vitro. Methods: Splenocytes were isolated from LEW-rat spleens and then CD3+T cells were purified using anti-CD3+ beads. ASCs were harvested from LEW rats and co-cultured with the T-cells. After transwell co-culture at different time periods, we analyzed cell proliferation with a BrdU assay. Cell extractions and culture supernatants were collected for further analysis. HO-1 and related protein expression levels from the ASCs and T-cells were detected using Western blotting. The related cytokine expression levels were analyzed with ELISA kits. Flow cytometry was used to detect the regulatory T-cell proportion. Results: The results revealed that ASCs significantly suppressed T-cell proliferation. The regulatory T-cell percentages were significantly increased in the ASCs that were co-cultured with T-cells compared with T-cells alone without ASC co-culture. HO-1 expression in concanavalin A (ConA) stimulated T cells that were co-cultured with ASCs revealed a significant increase compared with ConA stimulated T-cells alone. Cytokine assays of the culture supernatants revealed that TGF-[beta] and IL-10 were significantly increased and IFN-[gamma] was statistically decreased in the ASC-co-cultured T-cell group compared with other groups; however, blockade with a HO-1 inhibitor (ZnPP IX) protected against these changes. Conclusions: ASCs modulate T-cell proliferation and enhance regulatory T-cell expression, and this correlated with HO-1 expression and related cytokine pathway changes. (C)2016American Society of Plastic Surgeons
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