Introduction: Porcine NICC xenotransplantation has been proposed as a novel treatment of type 1 diabetes. However T-cell-mediated rejection remains a major impediment to its clinical application. The ability to manufacture xenoantigen-specific Treg may be a strategy to achieve effective suppression. Previously, we identified a HLA-DR+CD27+ subset of Treg that was separated from xenoantigen-stimulated human Treg (XnTreg) and showed potent and xenoantigen-specific suppression in vitro. The aim of this study was to confirm their suppressive function and antigen specificity in vivo.
Methods: Human Treg isolated from healthy donor peripheral blood mononuclear cells  were expanded for 3 or 4 cycles with anti-CD3/CD28 beads and irradiated porcine PBMC as xenoantigen to produce antigen-stimulated Treg (XnTreg). Human XnTreg were separated by cell sorting using the Treg cell surface markers CD27 and HLADR into non-selected XnTreg, HLADR+CD27+ and HLADR+CD27+ depleted-Treg (Dep-Treg) subsets prior to cotransfer into NOD-SCID IL2rg-/- mice recipients of porcine NICC with autologous PBMC and Treg at a 1:25 ratio of Treg (4x10^5): PBMC (1x10^7). Serum, spleen and NICC xenografts were harvested from recipient mice at day 60 for analysis of xenograft survival and Treg function in vivo.
Results: Human PBMC engraftment was confirmed by flow cytometry by day 60 after adoptive transfer. Mice reconstituted with human PBMC without Treg rejected their xenografts completely within 35 days. However, in mouse recipients of NICC xenografts that were reconstituted with human PBMC and HLADR+CD27+ Treg, immunohistochemical analysis revealed more intact NICC grafts and insulin positive-staining compared to those reconstituted with non-selected Xn Treg group at day 60. In the Dep-Treg group, very few insulin positive-staining cells were seen and a large number of human CD4 and CD8 Tcells infiltrated the islet graft. However in  HLADR+CD27+Treg group there were few CD4 and CD8 cells surrounding but not infiltrating the islet graft. The highest serum c-peptide levels were found in the HLADR+CD27+ Treg (458.83±78.1 pmol/l) transferred group compared to Xn-Treg (196.3±76.8 pmol/l), Dep-Treg (123.4±46.5 pmol/l) or Pc-Treg (144.1±19.0 pmol/l) transferred groups.  Immunofluorescent showed that HLADR+CD27+ Treg mediated islet xenograft protection was associated with a larger proportion of intragraft CD4+Foxp3+ Treg. Furthermore, the TaqMan Low Density Array analysis of human immune gene expression demonstrated upregulated expression of the Treg functional molecues (CTLA-4 and IL10) along with downregulated proinflammatory gene expression in mice co-transferred with HLADR+CD27+ Treg and PBMC.
Conclusion: CD27+HLADR+ Treg were more potent than naïve Treg at preventing islet xenograft rejection and could be achieved at much smaller Treg:Tcell ratios suggesting they may be more specific for the xenograft and may have potential as an effective immunomodulatory cell therapy for transplantation.