Large-scale in vitro expansion of human regulatory T cells with potent xenoantigen-specific suppression.

Xenotransplantation is a potential solution to the organ donor shortage. Immunosuppression is required for successful application of xenotransplantation but may lead to infection and cancer. Thus, strategies for immune tolerance induction need to be developed. Polyclonal regulatory T cells (Treg) play a central role in the induction and maintenance of immune tolerance and have been shown to protect against islet xenograft rejection in vivo. However, global immune suppression may be mediated by polyclonal Treg immunotherapy and a simple method for in vitro expansion of xenoantigen-specific Treg for efficient Treg application becomes necessary. Human Treg isolated from peripheral blood mononuclear cells (PBMCs) were initially cultured with anti-CD3/CD28 beads, rapamycin and IL-2 for 7 days as polyclonal expansion. Expanded Treg were then cocultured with irradiated porcine PBMC as xenoantigen stimulation for three subsequent cycles with 7 days for each cycle in the presence of IL-2 and anti-CD3/CD28 beads. Treg phenotype and suppressive capacity were assessed after each cycle of xenoantigen stimulation. Treg expanded with one cycle of xenoantigen stimulation retained Treg suppressive phenotype but acquired no xenoantigen specificity along with poor expansion efficiency, whereas expansion with two-cycle xenoantigen stimulation resulted in not only more than 800-fold Treg expansion but highly suppressive xenoantigen-specific Treg with effector Treg phenotype. However further increase of stimulation cycles resulted in reduced Treg suppressive potency. This study provides a simple approach to obtain high numbers of xenoantigen-specific Treg for immune tolerance induction in xenotransplantation.