Xi Jin1, Ya Wang, Wayne J Hawthorne, Min Hu, Shounan Yi, Philip O'Connell. 1. 1 Center for Transplant and Renal Research, Westmead Millennium Institute, Westmead Hospital, Westmead, New South Wales, Australia. 2 Present address: Key Laboratory of Transplant Engineering and Immunology, Ministry of Health; Regenerative Medicine Research Center; West China Hospital, Sichuan University, Chengdu, Sichuan, China. 3 Transplantation Research Center, The 2nd Xiangya Hospital, Central South University, Changsha, Hunan, China. 4 Address for correspondence to: Shounan Yi, M.D., Ph.D., or Philip O'Connell, M.D., Ph.D., Centre for Transplant and Renal Research, Westmead Millennium Institute, Westmead Hospital, Westmead, New South Wales 2145, Australia.
Abstract
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 CD4CD25CD127 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.
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 CD4CD25CD127 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-pigSLA 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-pigSLA 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.