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Background: Although islet xenotransplantation is a promising therapy for type 1 diabetes, its clinical application has been hampered by cellular rejection and the requirement for high levels of immunosuppression. The aim of this study was to determine the role of Foxp3 regulatory T (Treg) cells in costimulation blockade-induced dominant tolerance to porcine neonatal islet cell cluster (NICC) xenografts in mice., Methods: Porcine-NICC were transplanted under the renal capsule of BALB/c or C57BL/6 recipients and given a single dose of CTLA4-Fc at the time of transplant and 4doses of anti-CD154 mAb to day 6. Depletion of Foxp3Treg cell was performed in DEpletion of REGulatory T cells mice at day 80 posttransplantation. Foxp3Treg cell from spleens of treated BALB/c mice (tolerant Treg cell), and splenocytes were cotransferred into islet transplanted nonobese diabetic background with severe combined immunodeficiency mice to assess suppressive function., Results: In treated mice, increased numbers of Foxp3Treg cell were identified in the porcine-NICC xenografts, draining lymph node, and spleen. Porcine-NICC xenografts from treated mice expressed elevated levels of TGF-β, IL-10 and IFN-γ. Porcine-NICC xenograft tolerance was abrogated after depletion of Foxp3Treg cell. Tolerant Treg cell produced high levels of IL-10 and had diverse T cell receptor Vβ repertoires with an oligoclonal expansion in CDR3 of T cell receptor Vβ14. These tolerant Treg cells had the capacity to transfer dominant tolerance and specifically exhibited more potent regulatory function to porcine-NICC xenografts that naive Treg cell., Conclusions: This study demonstrated that short-term costimulation blockade-induced dominant tolerance and that Foxp3Treg cell played an essential role in its maintenance. Foxp3Treg cells were activated and had more potent regulatory function in vivo than naive Treg cells.

Purpose of Review: Clinical transplant tolerance has been most successfully achieved combining hematopoietic chimerism with kidney transplantation. This review outlines this strategy in animal models and human transplantation, and possible clinical challenges., Recent Findings: Kidney transplant tolerance has been achieved through chimerism in several centers beginning with Massachusetts General Hospital's success with mixed chimerism in human leukocyte antigen (HLA)-mismatched patients and the Stanford group with HLA-matched patients, and the more recent success of the Northwestern protocol achieving full chimerism. This has challenged the original view that stable mixed chimerism is necessary for organ graft tolerance. However, among the HLA-mismatched kidney transplant-tolerant patients, loss of mixed chimerism does not lead to renal-graft rejection, and the development of host Foxp3+ regulatory T cells has been observed. Recent animal models suggest that graft tolerance through bone marrow chimerism occurs through both clonal deletion and regulatory immune cells. Further, Tregs have been shown to improve chimerism in animal models., Summary: Animal studies continue to suggest ways to improve our current clinical strategies. Advances in chimerism protocols suggest that tolerance may be clinically achievable with relative safety for HLA-mismatched kidney transplants.

Background: Cellular rejection of xenografts is predominantly mediated by CD4 T cells. Little is known of the effectiveness of CD4CD25 T regulatory (Treg) cells at suppressing this strong T-cell mediated immune response. In this study, we evaluated the activity of fresh Treg cells and expanded Treg cells to suppress the xeno immune response in vitro., Methods: Human Treg cells were preferentially expanded by CD3/CD28 expand beads, interleukin (IL)-2, and rapamycin. Human CD4CD25 T cells were stimulated with irradiated porcine peripheral blood mononuclear cells in the presence or absence of fresh or expanded human Treg cells for 5 days before proliferation assay. In a separate experiment, the porcine xenoantigen-stimulated CD4CD25 T cells were separated from Treg cells by transwells and assessed for cytotoxicity of porcine peripheral blood mononuclear cells target cells. Cytokine-producing cells and cytokine release in the cocultures were examined by enzyme-linked immunosorbent spot and enzyme-linked immonosorbent assay, respectively., Results: Human Treg were expanded up to 3,500-fold after 14 days in culture. The addition of fresh Treg suppressed the T-cell mediated xenoimmune response. Compared with fresh Treg cells, expanded Treg cells were more potent at suppressing CD4CD25 T-cell-mediated antiporcine xenogeneic responses. This suppression required cell contact. However, the enhanced suppression by expanded Treg cells was associated with increased secretion of IL-4 and IL-10 when compared with their nonexpanded Treg counterparts., Conclusion: This study shows that expanded human Treg cells were capable of suppressing antiporcine xenogeneic responses in vitro and involve both contact dependent and cytokine mediated mechanisms.

Background: Porcine antigen primed and CD4+ T-cell-activated macrophages are capable of both recognition and rejection of porcine xenografts. However, the specific signaling mechanisms involved remains to be addressed. The aim of this study was to examine the role of chemokine receptor and CD40 signaling in macrophage recruitment and graft destruction., Methods: Macrophages were isolated from rejecting CCR2, CCR5, CD40 and control C57BL/6 mice that were recipients of neonatal porcine pancreatic cell cluster (NPCC) xenografts and were transferred to NPCC recipient NOD-SCID mice., Results: Macrophages isolated from rejecting NPCC xenografts in CD40 and wildtype C57BL/6 mice demonstrated upregulated expression of macrophage activation markers as well as CCR5 and CCR2 genes, and caused pig islet xenograft destruction 8 days after transfer to NOD-SCID recipients. Graft infiltrating macrophages from rejecting CCR2 mice showed a similar activation phenotype and destroyed NPCC xenografts 10 days after transfer to NOD-SCID mice. Blockade of MCP-1 by anti-MCP-1 mAb did not prolong graft survival in CD4+ T cell reconstituted NPCC recipient NOD-SCID mice. By contrast, the graft infiltrating macrophages from rejecting CCR5 recipients showed impaired macrophage activation when compared to control C57BL/6 recipients, and transfer of these macrophages did not result in xenograft destruction in NOD-SCID recipients until day 16 after transfer. Analysis of graft infiltrating macrophages from these rejecting NOD-SCID mice showed an impaired activation phenotype., Conclusion: These results demonstrate that CCR5 is involved in both the activation and recruitment of macrophages to rejecting islet xenografts but other pathways are involved.

Background: In this study, the mechanisms by which CD4+ T cells interact with the innate immune system in xenograft rejection were investigated., Methods: Fetal pig pancreas (FPP) grafts were transplanted into female SCID mice. The FPP recipient SCID mice were reconstituted with exogenous leukocytes obtained from male BALB/c mice., Results: Although nonreconstituted SCID recipients or recipients reconstituted with CD4+ T cell-depleted leukocytes showed indefinite FPP graft survival with very few macrophages infiltrating their grafts, reconstitution of SCID recipients with as few as 2x10(5) CD4+ T cells was sufficient to induce rapid xenograft rejection. CD4+ T cells secreted interferon-gamma but not interleukin-4 and initiated the activation and accumulation of macrophages and natural killer cells, that were responsible for the rapid graft destruction. Suppression of interferon-gamma prolonged graft survival and suppressed the macrophages and natural killer cell accumulation and activation., Conclusions: These results demonstrate that CD4+ T cell-dependent cellular xenograft rejection was a result of macrophage and natural killer cell accumulation and activation, but was not mediated by eosinophils. Consistent with this was the finding that interferon-gamma but not interleukin-4 was in part responsible for mediating this effect.