Enhanced Suppression of the Xenogeneic T-Cell Response In Vitro by Xenoantigen Stimulated and Expanded Regulatory T Cells

Background Strategies to prevent xenograft rejection while minimizing long-term immunosuppression need to be developed for successful xenotransplantaion. Polyclonally expanded human regulatory T cells (Treg) are known to suppress xenogeneic responses in vivo and in vitro. However, the risk of opportunistic infection and malignancy and the requirement for large numbers of Treg for effective suppression remain drawbacks to their clinical application. This study aimed to expand human Treg with xenoantigen stimulation and to assess their effectiveness at suppressing the xenoimmune response. Methods Human CD4(+)CD25(+)CD127(lo) Treg were stimulated with anti-CD3/CD28 beads, interleukin (IL)-2, and rapamycin for polyclonal expansion. After 7 days, Treg were further expanded with two subsequent cycles of either polyclonal stimulation or xenoantigen stimulation with irradiated porcine peripheral blood mononuclear cells with or without anti-pig SLA CII monoclonal antibody. Treg phenotype and suppressive capacity were assessed after xenoantigen stimulation. Results Porcine xenoantigen-stimulated Treg retained Treg phenotype but had an increased expression of human leukocyte antigen-DR, inducible costimulator, and CD45RO when compared with their polyclonally stimulated counterparts. In a pig-human mixed lymphocyte reaction (MLR), xenoantigen-stimulated Treg demonstrated an enhanced suppressive capacity at higher ratios of responder cells:Treg and secreted higher concentrations of IL-10 and IL-35, although they were equally suppressive as polyclonally stimulated Treg in an allogeneic or polyclonal MLR. When Treg expanded in the presence of anti-pig SLA monoclonal antibody were used in the same pig-human MLR, their suppressive capacity was reduced substantially. Conclusions Xenoantigen-stimulated Treg show enhanced suppressive capacity in the pig-human MLR most likely via an IL-10-mediated pathway.