CD27-Expressing Xenoantigen-Expanded Human Regulatory T Cells Are Efficient in Suppressing Xenogeneic Immune Response

Clinically, xenotransplantation often leads to T-cell-mediated graft rejection. Immunosuppressive agents including polyclonal regulatory T cells (poly-Tregs) promote global immunosuppression, resulting in serious infections and malignancies in patients. Xenoantigen-expanded Tregs (xeno-Tregs) have become a promising immune therapy strategy to protect xenografts with fewer side effects. In this study, we aimed to identify an efficient and stable subset of xeno-Tregs. We enriched CD27⁺ xeno-Tregs using cell sorting and evaluated their suppressive functions and stability in vitro via mixed lymphocyte reaction (MLR), real-time polymerase chain reaction, inflammatory induction assay, and Western blotting. A STAT5 inhibitor was used to investigate the relationship between the function and stability of CD27⁺ xeno-Tregs and the JAK3-STAT5 signaling pathway. A humanized xenotransplanted mouse model was used to evaluate the function of CD27⁺ xeno-Tregs in vivo. Our results show that CD27⁺ xeno-Tregs express higher levels of Foxp3, cytotoxic T-lymphocyte antigen-4 (CTLA4), and Helios and lower levels of interleukin-17 (IL-17) than their CD27^- counterparts. In addition, CD27⁺ xeno-Tregs showed enhanced suppressive function in xeno-MLR at ratios of 1:4 and 1:16 of Tregs:responder cells. Under inflammatory conditions, a lower percentage of CD27⁺ xeno-Tregs secretes IL-17 and interferon-γ (IFN-γ). CD27⁺ xeno-Tregs demonstrated an upregulated JAK3-STAT5 pathway compared with that of CD27^- xeno-Tregs and showed decreased Foxp3, Helios, and CTLA4 expression after addition of STAT5 inhibitor. Mice that received porcine skin grafts showed a normal tissue phenotype and less leukocyte infiltration after reconstitution with CD27⁺ xeno-Tregs. Taken together, these data indicate that CD27⁺ xeno-Tregs may suppress immune responses in a xenoantigen-specific manner, which might be related to the activation of the JAK3-STAT5 signaling pathway.