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

Abstract

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 twocycle 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.

Altered expression of CD39 on memory regulatory T cells in type 1 diabetes patients

“…Studies of human memory Tregs have mostly relied on phenotypic characterization and in vitro assays. In previous studies, Tregs were found to expressed more CD45RO and less CD45RA after antigen stimulation, indicating transition from resting Tregs to activated Tregs after antigen stimulation . Thus, human T cells are often termed memory cells if they express the CD45RO.…”

“…In previous studies, Tregs were found to expressed more CD45RO and less CD45RA after antigen stimulation, indicating transition from resting Tregs to activated Tregs after antigen stimulation. 12,17 Thus, human T cells are often termed memory cells if they express the CD45RO. In the present study, T1D patients had a higher percentage of effector/memory Tregs than healthy controls, with a higher percentage of memory Tregs in T1D patients than in healthy controls.…”

“…Isolated PBMCs were used for flow cytometric analysis of Treg phenotype, as described previously . Briefly, cells were surfaced stained with fluorescently coupled antibodies against CD3, CD4, CD25, CD127, CD45RO, CCR7, CD27 and CD39 (BioLegend, San Diego, California), with fluorescently coupled Foxp3 antibody (BioLegend) used for intracellular staining.…”

“…Isolated PBMCs were used for flow cytometric analysis of Treg phenotype, as described previously. 12,17 Briefly, cells were surfaced stained with fluorescently coupled antibodies against CD3, CD4, CD25, CD127, CD45RO, CCR7, CD27 and CD39 (BioLegend, San Diego, California), with fluorescently coupled Foxp3 antibody (BioLegend) used for intracellular staining. The fluorescently labeled cells were detected using a FACSAria SORP flow cytometer (Becton Dickinson, San Jose, California) and the data collected were analyzed using FlowJo version 10.0.2 (Treestar, Ashland, Oregon).…”

Regulatory T cells from allo‐ to xenotransplantation: Opportunities and challenges

“…In these studies, suppressive baboon or human, polyclonal 61 or pig-specific, [71][72][73] CD4 + Treg were expanded ex vivo. Autologous natural CD4 + Treg (nTreg) were isolated and cocultured with either xenogeneic pig cells, or autologous dendritic cells loaded with pig-derived antigens, 74,75 in the presence of interleukin-(IL-) 2 and rapamycin.…”