IL-35 Stabilizes Treg Phenotype to Protect Cardiac Allografts in Mice

Abstract

Background. Interleukin-35 (IL-35), secreted by regulatory T cells (Treg) and B cells, is immunosuppressive under both physiological and pathological conditions. However, the role of IL-35 in all responses has yet to be investigated. Here, we demonstrate that IL-35 protects allografts by stabilizing the Treg phenotype and suppressing CD8+ T-cell activation in a mouse heart transplantation model. Methods. The effect of IL-35 on immune cell infiltration in grafts and secondary lymphoid organs was examined using mass cytometry, flow cytometry, and immunofluorescence. Moreover, using quantitative real-time polymerase chain reaction, flow cytometry, and phospho-flow assays, we demonstrated that IL-35 maintains Treg phenotypes to restrain CD8+ T cells via the gp130/signal transducer and activator of transcription 1 pathway. Results. Mass cytometry analysis of intragraft immune cells showed that IL-35 decreased CD8+ T-cell infiltration and increased Foxp3 and IL-35 expressions in Treg. In vitro, we demonstrated that IL-35 directly promoted Treg phenotypic and functional stability and its IL-35 secretion, generating a positive feedback loop. However, Treg are required for IL-35 to exert its suppressive effect on CD8+ T cells in vitro. After depleting Treg in the recipient, IL-35 did not prolong graft survival or decrease CD8+ T-cell infiltration. Mechanistically, we found that IL-35 sustained Treg stability via the gp130/signal transducer and activator of transcription 1 signaling pathway. Conclusions. Our findings highlight that IL-35 stabilizes the Treg phenotype to ameliorate CD8+ T-cell infiltration in the allograft, which has never been described in the transplanted immunological milieu.