Porcine Islet Xenograft Rejection Research Articles

106.3: CD40-specific costimulation blockade and rapamycin prevent porcine islet-xenograft rejection in nonhuman primates through regulation of B and T cell xenoreactivity

106.3: CD40-specific costimulation blockade and rapamycin prevent porcine islet-xenograft rejection in nonhuman primates through regulation of B and T cell xenoreactivity

CTLA4Ig/VISTAIg combination therapy selectively induces CD4+ T cell-mediated immune tolerance by targeting the SOCS1 signaling pathway in porcine islet xenotransplantation.

T cell inhibitory receptors can regulate the proliferation or function of T cells by binding to their ligands and present a unique opportunity to manage destructive immune responses during porcine islet xenotransplantation. We applied ex vivo porcine islet xenotransplantation and in vitro mixed lymphocyte-islet reaction models to assess immune checkpoint receptor expression profiles in recipient T cells, investigated whether CTLA4 or VISTA immunoglobulin (Ig) combination therapy alone could suppress porcine islet xenograft rejection and further analyzed its potential immune tolerance mechanism. Recipient T cells expressed moderate to high levels of CTLA4, PD-1, TIGIT and VISTA, and the frequency of CTLA4+ CD4+ , TIGIT+ CD4+ , VISTA+ CD4+ and VISTA+ CD8+ T cells was positively correlated with porcine islet xenograft survival time in xenotransplant recipients. Combined treatment with CTLA4Ig and VISTAIg selectively inhibited recipient CD4+ T cell hyper-responsiveness and proinflammatory cytokine production and significantly delayed xenograft rejection. SOCS1 deficiency in CD4+ T cells stimulated by xenogeneic islets facilitated hyper-responsiveness and abolished the suppressive effect of combination therapy on recipient T cell-mediated porcine islet damage in vivo and in vitro. Further mechanistic studies revealed that combined treatment significantly induced SOCS1 expression and inhibited the Jak-STAT signalling pathway in wild-type recipient CD4+ T cells stimulated by xenogeneic islets, whereas SOCS1 deficiency resulted in Jak-STAT signalling pathway activation in recipient CD4+ T cells. We demonstrated a major role for CTLA4 and VISTA as key targets in CD4+ T cell hyper-responsiveness and porcine islet xenograft rejection. The selective inhibition of CD4+ T cell immunity by CTLA4Ig/VISTAIg is based on SOCS1-dependent signalling.

Early immune mechanisms of neonatal porcine islet xenograft rejection.

Neonatal pigs have the potential to provide an inexhaustible source of islets for the treatment of type 1 diabetes. However, the immunological barriers to islet xenotransplantation still need to be overcome. A better understanding of the xeno-specific immune responses that are involved in neonatal porcine islet (NPI) xenotransplant rejection will help to facilitate the identification of new targets for anti-rejection therapies, and thus enable more specific targeting of the immune cells and molecules involved. In this study, we examined the early events of NPI xenograft rejection in the absence of autoimmunity using an immune-competent B6 mouse transplant model. Immune cells were identified by immunohistochemistry and immune molecules were identified by reverse transcription-PCR and flow cytometry assays. Our results demonstrated that early events in NPI xenograft rejection are characterized by initial infiltration of innate immune cells such as macrophages (M1) and neutrophils. Targeting these cells, which appear early in the rejection process, may provide an opportunity to abort the rejection process prior to activation of T cells. One strategy could be the blockade of chemotactic signals associated with preferential recruitment of immune cells into the graft site. Collectively, our studies demonstrated that early recruitment of immune cells into graft site is controlled by chemotactic activities and suggest a potential target to prevent the early infiltration of immune cells within the graft. Our findings in this study will have significance in improving NPI xenograft acceptance and induce long-term xenograft survival.

Adoptive transfer of xenoantigen‑stimulated T cell receptor Vβ‑restricted human regulatory T cells prevents porcine islet xenograft rejection in humanized mice.

Polyclonal expansion of human regulatory T cells (Tregs) prevents xenogeneic rejection by suppressing effector T cell responses in vitro and in vivo. However, a major limitation to using polyclonally expanded Tregs is that they may cause pan-immunosuppressive effects. The present study was conducted to compare the ability of ex vivo expanded human xenoantigen-stimulated Tregs (Xeno-Treg) and polyclonal Tregs (Poly-Treg) to protect islet xenografts from rejection in NOD-SCID interleukin (IL)-2 receptor (IL2r)γ−/− mice. Human cluster of differentiation (CD)4+CD25+CD127lo Tregs, expanded either by stimulating with porcine peripheral blood mononuclear cells (PBMCs) or anti-CD3/CD28 beads, were characterized by immune cell phenotyping, T cell receptor (TCR) Vβ CDR3 spectratyping and performing suppressive activity assays in vitro. The efficiency of adoptively transferred ex vivo human Tregs was evaluated in vivo using neonatal porcine islet cell clusters (NICC) transplanted into NOD-SCID IL-2rγ−/− mice, which received human PBMCs with or without Xeno-Treg or Poly-Treg. Xeno-Treg, which expressed increased levels of human leukocyte antigen-DR and secreted higher levels of IL-10, demonstrated enhanced suppressive capacity in a pig-human mixed lymphocyte reaction. Spectratypes of TCR Vβ4, Vβ10, Vβ18 and Vβ20 in Xeno-Treg showed restriction and expanded clones at sizes of 205, 441, 332 and 196 respectively, compared to those of Poly-Treg. Reconstitution of mice with human PBMCs and Poly-Treg resulted in NICC xenograft rejection at 63 days. Adoptive transfer with human PBMCs and Xeno-Treg prolonged islet xenograft survival beyond 84 days, with grafts containing intact insulin-secreting cells surrounded by a small number of human CD45+ cells. This study demonstrated that adoptive transfer of ex vivo expanded human Xeno-Treg may potently prevent islet xenograft rejection in humanized NOD-SCID IL2rγ−/− mice compared with Poly-Treg. These findings suggested that adoptive Treg therapy may be used for immunomodulation in islet xenotransplantation by minimizing systemic immunosuppression.

Ex vivo-expanded baboon CD39+ regulatory T cells prevent rejection of porcine islet xenografts in NOD-SCID IL-2rγ-/- mice reconstituted with baboon peripheral blood mononuclear cells.

A high immunosuppressive burden is required for long-term islet xenograft survival in non-human primates even using genetically modified donor pigs. 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. 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. 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 35days. Cotransfer with baboon PBMC and Treg prolonged islet xenograft survival beyond 100days, correlating with Treg engraftment, intragraft CD39 and Foxp3 gene expression, and reduced graft infiltrating effector T cells and reduced interferon-γ production. Our data supports the capacity of ex vivo expanded CD39+ baboon Treg to suppress islet xenograft rejection in primatized mice, suggesting it haspotential as an adjunctive immunotherapy in preclinical NHP models of xenotransplantation.

Comment on Yi et al. Adoptive Transfer With In Vitro Expanded Human Regulatory T Cells Protects Against Porcine Islet Xenograft Rejection via Interleukin-10 in Humanized Mice. Diabetes 2012;61:1180–1191

A well-written article by Yi et al. (1) was published in Diabetes in 2012 that demonstrates the ability of human regulatory T cells (Tregs) to prevent T-cell effector function and the importance of interleukin (IL)-10 in this response. Furthermore, the study pointed to a strategy for using Tregs as adjunctive therapy with the potential to reduce the immunosuppressive burden in …

How to Kill Two Birds With One Transgenic Pig

Islet transplantation promises to provide safe, long-lasting insulin independence to individuals with diabetes. Yet the need for lifelong immunosuppression coupled with a vastly insufficient supply of donor tissue continues to prevent the widespread application of this approach to treating diabetes. A study (1) in this issue of Diabetes describes significant progress in addressing both of these issues. Porcine islets have long been touted as a potentially unlimited source of transplantable islet tissue. Porcine and human insulin are highly homologous, and until the late 1970s, with the advent of recombinant protein technology, porcine insulin was routinely injected into humans with diabetes. However, the transplantation of porcine tissue has not proven to be so straightforward. Over the past decade, nonhuman primate studies demonstrated that a strong immunosuppression protocol was required to avoid rejection of porcine islet xenografts (2,3). Consequently, although the use of porcine islets would overcome the issue of tissue supply, their use would be tantamount to patients trading insulin injections for strong immunosuppression—not an attractive clinical solution for many. In the current study, Klymiuk et al. (1) developed transgenic pigs that express an immunomodulatory fusion protein under the control of the insulin promoter. These animals produce the immunosuppressant protein locally upon activation of the insulin promoter within β-cells, but do not appear systemically immunosuppressed, …

Adoptive transfer with in vitro expanded human regulatory T cells protects against porcine islet xenograft rejection via interleukin-10 in humanized mice.

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.

Anticarbohydrate Antibody Repertoires in Patients Transplanted with Fetal Pig Islets Revealed by Glycan Arrays

Ten patients with type I diabetes were transplanted with porcine fetal islet-like cell clusters (ICC) between 1990 and 1993. A significant rise in the anti-α-Gal antibody titers was seen posttransplant, but also non-α-Gal-specific antibodies were detected in some patients. We have reanalyzed the carbohydrate specificity of antibodies in the sera from seven of these patients taken before transplantation, 1, 6 and 12 months posttransplantation using a glycan array with 200 structurally defined glycans. The main findings were: (i) prepig ICC transplantation patients had antibodies reactive with terminal α-GalNAc (e.g. the Forssman antigen, but not the blood group A determinant in blood group A patients), α-Gal (except blood group B determinants in B individuals), β3-linked Gal especially Galβ1,3GlcNAc even if terminally sulfated or sialylated, β-GlcNAc except if β1,3-linked and oligomannosyl compounds; (ii) the titers of all carbohydrate-specific antibodies detected before transplantation rose after transplantation; (iii) the kinetics of the antibody responses differed between patients; (iv) in some patients antibodies reacting with Galα1,3Lex and several structures terminated with Neu5Gc appeared after transplantation. In conclusion, anti-α-Gal antibodies are the predominant anticarbohydrate antibodies detected after porcine ICC transplantation, with some patients also developing Neu5Gc-specific antibodies. Their clinical significance needs to be established.

Selective rejection of porcine islet xenografts by macrophages

Our previous study has shown that porcine antigen-primed and CD4+ T cell-activated macrophages are capable of recognition and rejection of porcine xenografts after adoptive transfer. However, whether this is an absolute xenograft specific rejection remains to be confirmed. Mouse islet allografts and neonatal porcine islet cell cluster (NICC) xenografts were admixed and transplanted under the left kidney capsule, and NICC xenografts alone were transplanted under the right kidney capsule of strepotozotocin-induced diabetic NOD-SCID mice. After achievement of normoglycemia, the NOD-SCID recipients were transferred with macrophages purified from NICC transplant NOD-SCID mice reconstituted with CD4+ T cells. Five weeks after macrophage transfer the left kidney with the admixed grafts were removed. Graft survival and function following macrophage transfer was assessed by blood glucose measurement and immunohistochemistry. Adoptive transfer with activated macrophages did not affect the normalized blood glucose levels in NOD-SCID recipients of admixed grafts until left nephrectomy 5 weeks post-macrophage transfer. Insulin-positive and porcine C-peptide-negative mouse islets were detected in the admixed grafts. The surviving mouse islets in the admixed grafts were surrounded but not infiltrated by macrophages. The nephrectomized recipients demonstrated sustained hyperglycemia and completely destroyed NICC xenografts in their remaining right kidneys 8 weeks after macrophage transfer. Taken together, these data provide direct evidence of porcine islet xenograft specific rejection by activated macrophages.

Rejection of porcine islet xenografts mediated by CD4+ T cells activated through the indirect antigen recognition pathway.

We have previously demonstrated that human T cells responding to porcine islets are primarily CD4+ and recognized porcine major histocompatibility complex class I molecules through the indirect pathway of antigen presentation. To determine whether this mechanism is responsible for rejection of adult porcine islets xenografts, porcine islets from adult pigs were transplanted under the kidney capsule of streptozotocin-treated CD4-knockout (KO), CD8-KO, Ig-KO and normal C57BL/6 mice. Islet xenografts were acutely rejected with similar kinetics when transplanted into normal C57BL/6 (MST=17.6 +/- 3.5 days) and Ig-KO (MST=19.0 +/- 1.7 days) mice. Interestingly, islet xenografts were rejected significantly earlier when transplanted into CD8-KO mice as compared with normal C57BL/6 (MST=7.0 +/- 0.01 days, P=2 x 10-4). Histopathological analysis revealed classical acute cellular rejection with severe diffuse interstitial cellular infiltrates in all rejected islet xenografts. In contrast, islet xenografts were not rejected when transplanted into CD4-KO mice (MST >/= 100 days, P=1 x 10-9). Histopathological analysis revealed no cellular infiltrates and intact islet xenografts. CD4+ T cells from both normal C57BL/6 and CD8-KO xenograft recipients showed detectable proliferative responses to porcine islets in the presence but not in the absence of syngeneic antigen-presenting cells. In addition, the anti-islet proliferative responses observed in normal C57BL/6 mice were significantly lower than those observed in CD8-KO mice. IgG anti-porcine antibodies were readily detected in C57BL/6 and CD8-KO xenograft recipients but not in Ig-KO or CD4-KO recipients. These results indicate that indirectly activated CD4+ T cells mediate acute rejection of adult porcine islet xenografts and that xenoreactive CD8+ T cells and antibodies are not necessary in this process.

The role of indirect antigen recognition in islet xenograft rejection

PORCINE pancreatic islets are considered a viable source of insulin-producing tissue for the treatment of Type 1 diabetes mellitus. For this therapy to become a reality, a detailed characterization of human immune responses to porcine islets should be carried out. Our recent studies characterized the mechanism of porcine islet xenograft rejection by human CD41 T cells generated against porcine islets both by in vitro stimulation and in vivo using severe combined immunodeficient mice reconstituted with human peripheral blood leukocytes and transplanted with porcine islets. These studies concluded that a majority of human CD41 T cells recognized porcine MHC molecules as xenoantigens presented by self-antigen presenting cells (APC). To further define the mechanisms of islet xenograft rejection, we transplanted porcine islets under the kidney capsules of streptozotocin-treated C57BL/6 (BL/6), CD4 Knockout (KO), and CD8KO mice, and islet rejection was analyzed. Similarly, BL/6, CD4KO, and CD8KO mice were treated with anti-CD4 or anti-CD8 depleting monoclonal antibodies (mAb), transplanted, and monitored for rejection. To further document the role of CD41 T cells in the response to porcine islets, we studied specific T-cell proliferation to islet antigens in vitro and its requirement of APC for maximum stimulation. In addition, antibody response to porcine xenoantigens in each group of transplanted mice was measured.

The importance of the Gal??1-3Gal antigen in islet xenotransplantation: Immunosuppression that inhibits porcine islet xenograft rejection in Gal+ mice is equally effective in Gal- mice

726 The presence of the Galα1-3Gal antigen in porcine tissues is regarded as the major immunological barrier to clinical transplantation of porcine organs. The aim of this study was to investigate the importance of this antigen in transplantation of porcine islets to immunosuppressed mice.Methods: Fetal porcine islet like cell clusters (ICC's) were transplanted under the kidney capsule in SV129 mice with a targeted disruption of the α1-3 galactosyl transferase gene. These mice do not express the Galα1-3Gal antigen (Gal-) but have anti-Gal antibodies. Normal mice expressing the antigen (Gal+) served as controls. After transplantation, the mice were left untreated or were immunosuppressed with FK-506 (FK, 2 mg/kg BW, s.c.) single-drug therapy or cyclosporine A (CsA, 10 mg/kg BW, s.c.) combined with 15-deoxyspergualin (DSG, 5 mg/kg BW, s.c.). All drugs were administered once daily. There were 5 animals in each group. Evaluation was performed morphologically and immunohistochemically at 12 days after transplantation. Results: In non-immunosuppressed Gal+ mice, the ICC xenografts were completely destroyed by a massive cellular infiltration dominated by macrophages. In non-immunosuppressed Gal- mice the results were similar. Immunosuppression with FK had no or only a marginal effect on the rejection process, and there was no difference between Gal+ and Gal- mice. No deposits of IgG, IgM or C3 could be detected in any of the non-immunosuppressed mice or in mice treated with FK. Immunosuppression with CsA+DSG partially inhibited rejection with no difference between Gal+ and Gal- mice. However, parts of these ICC xenografts were infiltrated by large numbers of T cells and B cells in equal numbers. Among the T cells, there was a slight dominance of CD4+ cells over CD8+ cells. In the non-infiltrated parts of these xenografts, many morphologically intact ICC's could be found. In all these animals, a sharp border was seen between the infiltration and the morphologically intact parts of the xenografts. In animals treated with CsA+DSG, large numbers of lymphocytes staining positive for IgM were found. Only occasional IgG+ cells were detected. No deposits of IgM, IgG or C3 could be detected on remaining morphologically intact ICC's.Conclusions: The results of the present study in the pig-to-mouse model indicate that the Galα1-3Gal antigen is of minor importance in islet xenotransplantation. Furthermore, immunosuppression that inhibits islet xenograft rejection in Gal+ mice is equally effective also in islet xenotransplantation across the Galα1-3Gal-barrier.

The importance of the Galα1-3Gal antigen in islet xenotransplantation: Immunosuppression that inhibits porcine islet xenograft rejection in Gal+ mice is equally effective in Gal- mice

726 The presence of the Galα1-3Gal antigen in porcine tissues is regarded as the major immunological barrier to clinical transplantation of porcine organs. The aim of this study was to investigate the importance of this antigen in transplantation of porcine islets to immunosuppressed mice.Methods: Fetal porcine islet like cell clusters (ICC's) were transplanted under the kidney capsule in SV129 mice with a targeted disruption of the α1-3 galactosyl transferase gene. These mice do not express the Galα1-3Gal antigen (Gal-) but have anti-Gal antibodies. Normal mice expressing the antigen (Gal+) served as controls. After transplantation, the mice were left untreated or were immunosuppressed with FK-506 (FK, 2 mg/kg BW, s.c.) single-drug therapy or cyclosporine A (CsA, 10 mg/kg BW, s.c.) combined with 15-deoxyspergualin (DSG, 5 mg/kg BW, s.c.). All drugs were administered once daily. There were 5 animals in each group. Evaluation was performed morphologically and immunohistochemically at 12 days after transplantation.Results: In non-immunosuppressed Gal+ mice, the ICC xenografts were completely destroyed by a massive cellular infiltration dominated by macrophages. In non-immunosuppressed Gal- mice the results were similar. Immunosuppression with FK had no or only a marginal effect on the rejection process, and there was no difference between Gal+ and Gal- mice. No deposits of IgG, IgM or C3 could be detected in any of the non-immunosuppressed mice or in mice treated with FK. Immunosuppression with CsA+DSG partially inhibited rejection with no difference between Gal+ and Gal- mice. However, parts of these ICC xenografts were infiltrated by large numbers of T cells and B cells in equal numbers. Among the T cells, there was a slight dominance of CD4+ cells over CD8+ cells. In the non-infiltrated parts of these xenografts, many morphologically intact ICC's could be found. In all these animals, a sharp border was seen between the infiltration and the morphologically intact parts of the xenografts. In animals treated with CsA+DSG, large numbers of lymphocytes staining positive for IgM were found. Only occasional IgG+ cells were detected. No deposits of IgM, IgG or C3 could be detected on remaining morphologically intact ICC's. Conclusions: The results of the present study in the pig-to-mouse model indicate that the Galα1-3Gal antigen is of minor importance in islet xenotransplantation. Furthermore, immunosuppression that inhibits islet xenograft rejection in Gal+ mice is equally effective also in islet xenotransplantation across the Galα1-3Gal-barrier.

Human peripheral blood lymphocyte-reconstituted, severe combined immunodeficient mice as a model for porcine islet xenograft rejection in humans.

Previously we established human peripheral blood lymphocyte-reconstituted severe combined immunodeficiency (SCID) (hu-PBL-SCID) mice as a model for human islet allograft rejection. The function of xenografted hu-PBL was confirmed to reject human alloislets in hu-PBL-SCID mice. In this study, we modified this model as a porcine islet xenograft to study porcine islet rejection in humans. Chimeric mice were used as the recipients of porcine islets to reveal the mechanisms of xenograft rejection in humans. SCID mice were reconstituted with 30 x 10(6) of hu-PBL initially, and 10 x 10(6) of antihuman CD3-primed PBL was injected intraperitoneally 2 days later as a booster. An additional booster injection provided greater possibility (86.7%, n = 15) of chimera establishment as well as a higher human immunoglobulin concentration in SCID mice than the single injection group. In an in vitro assay, sera from hu-PBL-SCID mice were found to recognize porcine islets by FACS staining. In an in vivo study, immunofluorescent analysis of a frozen section showed that human immunoglobulins adhered to the xenografted porcine islet under the kidney capsule of hu-PBL-SCID mice. Although no mouse immunoglobulins were detected on sections, mouse complement (C3) was shown to adhere to the xenografted porcine islet. Thus, hu-PBL-SCID mice provide a useful model for investigating the real-life situation of porcine islet xenograft rejection in humans.