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CD4+Foxp3+ regulatory T cells (Tregs) play an essential role in suppressing transplant rejection, but their role within the graft and heterogeneity in tolerance are poorly understood. Here, we compared phenotypic and transcriptomic characteristics of Treg populations within lymphoid organs and grafts in an islet xenotransplant model of tolerance. We showed Tregs were essential for tolerance induction and maintenance. Tregs demonstrated heterogeneity within the graft and lymphoid organs of tolerant mice. A subpopulation of CD127hi Tregs with memory features were found in lymphoid organs, presented in high proportions within long-surviving islet grafts, and had a transcriptomic and phenotypic profile similar to tissue Tregs. Importantly, these memory-like CD127hi Tregs were better able to prevent rejection by effector T cells, after adoptive transfer into secondary Rag-/- hosts, than naive Tregs or unselected Tregs from tolerant mice. Administration of IL-7 to the CD127hi Treg subset was associated with a strong activation of phosphorylation of STAT5. We proposed that memory-like CD127hi Tregs developed within the draining lymph node and underwent further genetic reprogramming within the graft toward a phenotype that had shared characteristics with other tissue or tumor Tregs. These findings suggested that engineering Tregs with these characteristics either in vivo or for adoptive transfer could enhance transplant tolerance.

Regulatory T cells (Tregs) have potential for the treatment of autoimmune diseases and graft rejection. Antigen specificity and functional stability are considered critical for their therapeutic efficacy. In this study, expansion of human Tregs in the presence of porcine PBMCs (xenoantigen-expanded Tregs, Xn-Treg) allowed the selection of a distinct Treg subset, coexpressing the activation/memory surface markers HLA-DR and CD27 with enhanced proportion of FOXP3+Helios+ Tregs. Compared with their unsorted and HLA-DR+CD27+ double-positive (DP) cell-depleted Xn-Treg counterparts, HLA-DR+CD27+ DP-enriched Xn-Tregs expressed upregulated Treg function markers CD95 and ICOS with enhanced suppression of xenogeneic but not polyclonal mixed lymphocyte reaction. They also had less Treg-specific demethylation in the region of FOXP3 and were more resistant to conversion to effector cells under inflammatory conditions. Adoptive transfer of porcine islet recipient NOD/SCID IL2 receptor γ-/- mice with HLA-DR+CD27+ DP-enriched Xn-Tregs in a humanized mouse model inhibited porcine islet graft rejection mediated by 25-fold more human effector cells. The prolonged graft survival was associated with enhanced accumulation of FOXP3+ Tregs and upregulated expression of Treg functional genes, IL10 and cytotoxic T lymphocyte antigen 4, but downregulated expression of effector Th1, Th2, and Th17 cytokine genes, within surviving grafts. Collectively, human HLA-DR+CD27+ DP-enriched Xn-Tregs expressed a specific regulatory signature that enabled identification and isolation of antigen-specific and functionally stable Tregs with potential as a Treg-based therapy.

The role of Regulatory T cells (Tregs) in tolerance induction post-transplantation is well-established, but Tregs adoptive transfer alone without combined immunosuppressants have failed so far in achieving clinical outcomes. Here we applied a set of well-designed criteria to test the influence of commonly used immunosuppressants (belatacept, tacrolimus, and mycophenolate) on cord blood-derived Tregs (CB-Tregs). Our study shows that while none of these immunosuppressants modulated the stability and expression of homing molecules by CB-Tregs, belatacept met all other selective criteria, shown by its ability to enhance CB-Tregs-mediated in vitro suppression of the allogeneic response without affecting their viability, proliferation, mitochondrial metabolism and expression of functional markers. In contrast, treatment with tacrolimus or mycophenolate led to reduced expression of functional molecule GITR in CB-Tregs, impaired their viability, proliferation and mitochondrial metabolism. These findings indicate that belatacept could be considered as a candidate in Tregs-based clinical immunomodulation regimens to induce transplant tolerance.

Background: A high immunosuppressive burden is required for long-term islet xenograft survival in non-human primates even using genetically modified donor pigs., Aims: We aimed to investigate the capacity of baboon regulatory T cells (Treg) to suppress islet xenograft rejection, thereby developing a potential immunoregulatory or tolerance therapy that could be evaluated in NHP models of xenotransplantation., Materials & Methods: Baboon Treg expanded with stimulation by porcine peripheral blood mononuclear cells (PBMC) were characterized by cell phenotyping and suppressive activity assays in vitro. Their function in vivo was evaluated in neonatal porcine islet cell clusters (NICC) transplanted NOD-SCID IL-2rγ-/- (NSG) mice receiving baboon PBMC alone or with expanded autologous Treg., Results: The majority of expanded Treg coexpressed Foxp3 and CD39 and were highly suppressive of the baboon anti-pig xenogeneic T cell response in vitro. Reconstitution of mice with baboon PBMC alone resulted in NICC xenograft rejection within 35 days. Cotransfer with baboon PBMC and Treg prolonged islet xenograft survival beyond 100 days, correlating with Treg engraftment, intragraft CD39 and Foxp3 gene expression, and reduced graft infiltrating effector T cells and reduced interferon-γ production., Discussion & Conclusion: Our data supports the capacity of ex vivo expanded CD39+ baboon Treg to suppress islet xenograft rejection in primatized mice, suggesting it has potential as an adjunctive immunotherapy in preclinical NHP models of xenotransplantation., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

Regulatory T cells (Tregs) have been shown to be important in maintaining immune homeostasis and preventing autoimmune disease, including autoimmune kidney disease. It is also likely that they play a role in limiting kidney transplant rejection and potentially in promoting transplant tolerance. Although other subsets of Tregs exist, the most potent and well-defined Tregs are the Foxp3 expressing CD4(+) Tregs derived from the thymus or generated peripherally. These CD4(+)Foxp3(+) Tregs limit autoimmune renal disease in animal models, especially chronic kidney disease, and kidney transplantation. Furthermore, other subsets of Tregs, including CD8 Tregs, may play a role in immunosuppression in kidney disease. The development and protective mechanisms of Tregs in kidney disease and kidney transplantation involve multiple mechanisms of suppression. Here we review the development and function of CD4(+)Foxp3(+) Tregs. We discuss the specific application of Tregs as a therapeutic strategy to prevent kidney disease and to limit kidney transplant rejection and detail clinical trials in this area of transplantation., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Objective: To establish a method for in vitro expansion of human natural CD4⁺CD25⁺ T regulatory cell (Treg) cells for clinical study and immunotherapy., Methods: Human natural CD4⁺CD25⁺ Treg were isolated from peripheral blood monocyte cells (PBMCs) by magnetic activated cell sorting (MACS) and expanded by CD3/CD28 expansion beads, IL-2 and rapamycin. The number and the viability of the freshly isolated and expanded Treg were detemined by trypan blue staining. The phenotype and the purity of the freshly isolated and expanded Treg were analyzed by FACS. Treg suppression activity was assessed by mixed lymphocyte reaction (MLR) assay., Results: Human natural Treg were expanded up to 2 000 folds after 3 weeks in culture, and the activity was more than 97%. The expanded Treg retained Treg phenotype as shown by their freshly isolated counterparts, and the purity of CD4⁺CD25⁺FoxP3⁺ Treg was (94.22 ± 2.12)%. The expanded Treg demonstrated a similar potent suppression of both proliferating auto- and allo- CD4⁺CD25⁻ effector T cells in vitro in a cell number-dependent manner., Conclusion: An in vitro expansion of human natural Treg was established to obtain large numbers of human Treg with highly suppressive phenotype and function, thereby providing a solution to the availability of sufficient human natural Treg in clinical study and immunotherapy.

Background: Strategies to prevent xenograft rejection while minimizing long-term immunosuppression need to be developed for successful xenotransplantaion. Polyclonally expanded human regulatory T cells (Treg) are known to suppress xenogeneic responses in vivo and in vitro. However, the risk of opportunistic infection and malignancy and the requirement for large numbers of Treg for effective suppression remain drawbacks to their clinical application. This study aimed to expand human Treg with xenoantigen stimulation and to assess their effectiveness at suppressing the xenoimmune response., Methods: Human CD4CD25CD127 Treg were stimulated with anti-CD3/CD28 beads, interleukin (IL)-2, and rapamycin for polyclonal expansion. After 7 days, Treg were further expanded with two subsequent cycles of either polyclonal stimulation or xenoantigen stimulation with irradiated porcine peripheral blood mononuclear cells with or without anti-pig SLA CII monoclonal antibody. Treg phenotype and suppressive capacity were assessed after xenoantigen stimulation., Results: Porcine xenoantigen-stimulated Treg retained Treg phenotype but had an increased expression of human leukocyte antigen-DR, inducible costimulator, and CD45RO when compared with their polyclonally stimulated counterparts. In a pig-human mixed lymphocyte reaction (MLR), xenoantigen-stimulated Treg demonstrated an enhanced suppressive capacity at higher ratios of responder cells:Treg and secreted higher concentrations of IL-10 and IL-35, although they were equally suppressive as polyclonally stimulated Treg in an allogeneic or polyclonal MLR. When Treg expanded in the presence of anti-pig SLA monoclonal antibody were used in the same pig-human MLR, their suppressive capacity was reduced substantially., Conclusions: Xenoantigen-stimulated Treg show enhanced suppressive capacity in the pig-human MLR most likely via an IL-10-mediated pathway.

T cell-mediated rejection remains a barrier to the clinical application of islet xenotransplantation. Regulatory T cells (Treg) regulate immune responses by suppressing effector T cells. This study aimed to determine the ability of human Treg to prevent islet xenograft rejection and the mechanism(s) involved. Neonatal porcine islet transplanted NOD-SCID IL2rγ(-/-) mice received human peripheral blood mononuclear cells (PBMC) with in vitro expanded autologous Treg in the absence or presence of anti-human interleukin-10 (IL-10) monoclonal antibody. In addition, human PBMC-reconstituted recipient mice received recombinant human IL-10 (rhIL-10). Adoptive transfer with expanded autologous Treg prevented islet xenograft rejection in human PBMC-reconstituted mice by inhibiting graft infiltration of effector cells and their function. Neutralization of human IL-10 shortened xenograft survival in mice receiving human PBMC and Treg. In addition, rhIL-10 treatment led to prolonged xenograft survival in human PBMC-reconstituted mice. This study demonstrates the ability of human Treg to prevent T-cell effector function and the importance of IL-10 in this response. In vitro Treg expansion was a simple and effective strategy for generating autologous Treg and highlighted a potential adoptive Treg cell therapy to suppress antigraft T-cell responses and reduce the requirement for immunosuppression in islet xenotransplantation.

Naturally occurring CD4+CD25+ regulatory T cells (nTregs) that express high level of Foxp3 actively suppress pathological and physiological immune responses, contributing to the maintenance of immunological self-tolerance and immune homeostasis. Although Foxp3 is required for nTreg development and appears to be necessary for mature murine Treg function, the precise role of Foxp3 in regulating natural human Treg function in alloimmune response is unclear. In this study, we used siRNA-mediated gene silencing to knockdown Foxp3 expression in natural human Tregs and investigated the importance of Foxp3 in maintaining human nTreg suppressive function. We showed that Foxp3 knockdown resulted in impaired phenotype and nonresponsiveness, downregulated expression of function molecules, and reduced production of suppressive cytokines in nTregs. These changes correlated with diminished nTreg activity in suppressing proliferation of effector CD4+CD25- T cells, their cytotoxicity against allogeneic target cells and production of effector cytokines in response to allogeneic stimulation. Thus, this study shows that ongoing Foxp3 expression is required for natural human Tregs to maintain their phenotype and suppressive function in the alloimmune response., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Cellular rejection of xenografts is predominantly mediated by CD4(+) T cells. Human CD4(+)CD25(+) regulatory T cells (Tregs) are capable of suppressing the CD4(+) T cell-mediated xenogeneic response in vitro. However, the precise mechanisms that are involved remain to be identified. In this study, we analyzed whether interleukin-10 (IL-10) is required for Tregs to suppress xenogeneic responses in vitro by small interfering RNA (siRNA)-mediated IL-10 knockdown. After siRNA transfection, Tregs were analyzed for IL-10 gene and protein expression and their phenotype. Mixed lymphocyte reactions (MLRs) were performed by stimulating human CD4(+)CD25(-) T cells with allogeneic or pig peripheral blood mononuclear cells (PBMCs) in the presence or absence of Tregs in a coculture or transwell system. The production of effector cytokines by xeno- or alloreactive CD4(+)CD25(-) T cells, or suppressive cytokines by Tregs, was examined using enzyme-linked immunosorbant assay (ELISA). We showed that IL-10 knockdown resulted in a substantially reduced IL-10 production by Tregs, leading to impaired Treg-mediated suppression of xeno- but not alloreactive CD4(+) CD25(-) T-cell proliferation. However, IL-10 knockdown had no effect on Treg phenotype, their suppression of effector cytokine production by xeno- or alloreactive T cells and the production of the Treg-suppressive cytokine, transforming growth factor-beta (TGF-beta). This study shows that IL-10 is required for human Tregs to suppress xenogeneic but not allogeneic proliferation in vitro.

Purpose of Review: Although much has been written about the biology of regulatory T cells (Tregs), their characterization and role in xenotransplantation has been limited. This review summarizes the recent literature supporting a role for Tregs in the cell-mediated xenoimmune response., Recent Findings: Studies in in-vitro assays systems have focused on the biology of natural Tregs, whereas in-vivo rodent models of induced tolerance to xenografts have implied an important role for CD4CD8 double-negative T cells. Although it was not unexpected that natural Tregs would have the capacity to modulate the T-cell-mediated response to xenografts, there are features that are different to what is seen in alloimmunity. The Tregs response to pig xenografts is more dependent on immunomodulatory cytokines such as IL-10, whereas in-vitro Tregs function in alloimmunity is focused strongly on cell contact mechanisms. In the acquired immune response, the majority of published findings have highlighted the properties of CD4CD8 double-negative Tregs, whereas little if anything has been written about the more familiar CD4CD25 Tregs, which has been well described with in-vivo models of acquired alloimmune tolerance., Summary: Although many investigators feel there are unique opportunities for using cell-based immunomodulatory therapies in xenotransplantation, our current understanding of the biology of Treg response remains incomplete. These gaps in our understanding need to be overcome before Tregs can be realized therapeutically to modulate the strong cellular xenoimmune response.

Backgrounds: Cellular rejection of xenografts is predominantly mediated by CD4+ T cells. Foxp3 expressing human naturally occurring CD4+CD25+ regulatory T cells (nTregs) have been shown to suppress pathological and physiological immune responses, including the CD4+ T-cell-mediated anti-pig xenogeneic response in vitro. Although Foxp3 is required for nTreg development and their function, the precise role of Foxp3 in regulating Treg suppressive function in xenoimmune response remains to be identified., Methods: In vitro expanded human nTregs were transfected with fluorescein isothiocyanate -conjugated Foxp3 small interfering RNA (siRNA) by Lipofectamine 2000. Transfected nTregs were sorted by fluorescence-activated cell sorting, and then analyzed for Foxp3 gene and protein expression as well as their phenotypic characteristics. Human CD4+CD25- T cells were stimulated with xenogeneic pig peripheral blood mononuclear cell in the presence or absence of nTregs in a coculture or transwell system for evaluation of nTreg suppressive activity. The production of effector cytokines by xenoreactive CD4+CD25- T cells as well as suppressive cytokine by nTregs in their cocultures was examined by ELISA., Results: The siRNA-mediated Foxp3 knockdown resulted in impaired nTreg anergic state, downregulated expression of nTreg function associated molecules, and reduced production of suppressive cytokines by nTregs, which together leading to impaired nTreg-mediated suppression of CD4+CD25- T-cell proliferation and their effector cytokine production in response to xenogeneic stimulation., Conclusions: This study demonstrates that Foxp3 expression is required for human nTregs to maintain their suppressive function in the xenoimmune response.

Background: Macrophages are important effector cells in T cell-mediated xenograft rejection. The aim of this study was to determine whether CD4+CD25+ regulatory T cells (Tregs) were capable of suppressing macrophage activation in vitro., Methods: Porcine cell or xenoantigen-primed human peripheral blood mononuclear cells, CD4+ T cell-depleted peripheral blood mononuclear cells, or CD14+ macrophages plus autologous CD4+CD25- T cells were cultured with or without expanded autologous Tregs. Transwell cultures were used to separate the various components to determine the need for cell-cell contact., Results: Pig cell primed CD14+ macrophages required the presence of CD4+CD25- T cells for activation and increased expression of CD40, interleukin-12, and tumor necrosis factor-alpha. This up-regulated expression of macrophage activation markers was reduced substantially in the presence of autologous Tregs. Coculture with Tregs did not alter macrophage viability but reduced the capacity of macrophages to stimulate proliferation of responder T cells. Tregs required direct contact with CD4+CD25- T cells to inhibit macrophage activation but activated macrophage phenotype was not altered by separating the stimulated human peripheral blood mononuclear cells or CD14+ macrophages from Tregs in a transwell system. Macrophages did not require direct cell contact with porcine stimulator cells for full activation by CD4+CD25- T cells., Conclusions: Human Tregs were able to suppress xenoantigen-primed and CD4+ T-cell-mediated macrophage activation and antigen-presenting cell function. However, Tregs had no direct effect on macrophages in vitro.

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.