In Vitro Expansion of Highly Suppressive Xenoantigen-Specific Human Regulatory T Cells for Immunotherapy in Xenotransplantation

Abstract

Introduction: Lifelong immunosuression is required to prevent xenograft rejection. Strategies to modulate the xenoimmune response for clinical benefit while avoiding long-term immunosuppression need be developed. Poyclonally expanded human regulatory T cells (Treg) have shown to suppress xenogeneic response in vivo. However the risk for opportunistic infection and tumor-growth and also the requirement of large numbers of polyclonally expanded Treg for effective suppression remain drawbacks for their clinical application. These difficulties could be overcome by using antigen-specific Treg. This study aimed to expand human Treg with xenoantigen-specificity in vitro for immunomodulation in xenotransplantation. Methods: Human CD4+CD25+CD127- Treg were isolated from peripheral blood mononuclear cells (PBMC) and stimulated with anti-CD3/CD28 beads, IL-2 and rapamycin for polyclonal expansion. 7 days after polyclonal expansion, Treg were further expanded with two subsequent cycles (7 days for each cycle) of either polyclonal stimulation or xenoantigen stimulation with irradiated porcine PBMC at a 4:1 ratio of porcine PBMC:Treg. Treg phenotype and their suppressive capacity were assessed after xenoantigen stimulation. Results: Porcine xenoantigen stimulation did not affect expression of Treg markers CD25, Foxp3, CTLA-4, CD62L and GITR but slightly increased average fold expansion of Treg (807-fold with xenoantugen stimulation vs. 720-fold with polyclonal stimulation) when compared to polyclonal stimulation. In addition, Treg expanded with xenoantigen but not polyclonal stimulation revealed acquired an effector Treg phenotype with upregulated expression of HLA-DR, CD45RO, ICOS, LAG-3, IL-10 and IL-35. The acquisition of Treg effector phenotype led to enhanced Treg suppressive capacity via IL-10 in xeno-stimulated MLR, as indicated by strong suppression of xenogeneic responses (>50% suppression by xenoantigen stimulated Treg vs. 15% suppression by polyclonally stimulated Treg with Tresp:Treg ratios of 16:1). However, regardless of expansion with polyclonal or xenoantigen stimulation, Treg demonstrated a similar potency in inhibition of allo- or polyclonal-stimulated MLR. Conclusion: This study shows a feasible strategy to obtain human functional xenoantigen-specific Treg in large numbers for immunomodulation. This provides useful information for the clinical application of Treg in xenotransplantation aiming at inducing tolerance to porcine xenoantigen by minimizing general immunesuppression.