Anita T. Patel

T Cell-Activated Macrophages Are Capable of Both Recognition and Rejection of Pancreatic Islet Xenografts

Macrophages have been proposed as the major effector cell in T cell-mediated xenograft rejection. To determine their role in this response, NOD-SCID mice were transplanted with fetal pig pancreas (FPP) before reconstitution with CD4+ T cells from BALB/c mice. Twelve days after CD4+ T cell reconstitution, purified macrophages (depleted of T cells) were isolated from CD4+ T cell-reconstituted FPP recipient mice and adoptively transferred to their nonreconstituted counterparts. After adoptive macrophage transfer, FPP recipient mice transferred with macrophages from CD4+ T cell-reconstituted mice demonstrated xenograft destruction along with massive macrophage infiltration at day 4 and complete graft destruction at day 8 postmacrophage transfer. By contrast, FPP recipients that received macrophages from nonreconstituted mice showed intact FPP xenografts with few infiltrating macrophages at both days 4 and 8 after macrophage transfer. The graft-infiltrating macrophages showed increased expression of their activation markers. Depletion of endogenous macrophages or any remaining CD4+ T cells did not delay graft rejection in the macrophage-transferred FPP recipients, whereas depletion of transferred macrophages with clodronate liposomes prevented graft rejection. Our results show that macrophages primed by FPP and activated by CD4+ T cells were attracted from the peripheral circulation and were capable of specific targeting and destruction of FPP xenografts. This suggests that in xenograft rejection, there are macrophage-specific recognition and targeting signals that are independent of those received by T cells.

Characterization of the swine major histocompatibility complex alleles at eight loci in Westran pigs

Abstract:  Background:  Pigs are an important large animal model for transplantation and a potential source of xenografts. Swine leukocyte antigen (SLA) molecules are strong mediators of alloreactive and xenoreactive immune responses. We have characterized the SLA alleles of a new pig line bred for transplantation research, the Westran (Westmead Hospital transplantation) pig, described in a companion paper.

CD4+ T cells initiate pancreatic islet xenograft rejection via an interferon-gamma-dependent recruitment of macrophages and natural killer cells.

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.

CD8+ T cells are capable of rejecting pancreatic islet xenografts

BACKGROUND In this study, the capacity of CD8+ T cells to act as a potential effector mechanism in pancreatic xenograft rejection was examined. METHODS The fate of pancreatic islet xenografts was studied in mice deficient in MHC class II molecules and CD4+ T cells. Fetal pig pancreas (FPP) or Wistar rat islets (RI) were transplanted into nondiabetic or streptozotocin-induced diabetic I-A knock-out (CII K/O) mice. RESULTS CII K/O mice were capable of rejecting both RI and FPP grafts. RI graft survival was not prolonged compared with wild type C57BL/6 controls. However, FPP grafts did survive longer in CII K/O recipients than in C57BL/J6 mice. Both RI and FPP graft rejection were CD8+ T-cell phenomena in CII K/O mice, as anti-CD8 monoclonal antibody prolonged graft survival, there were increased CD8+ T cells in the grafts and spleens of CII K/O recipients, and cell-mediated cytotoxicity was a CD8+ T-cell phenomenon associated with activation of the perforin/granzyme B system. By contrast, RI and FPP graft rejection was a CD4+ T cell-dependent phenomenon in wild type C57BL/6 mice with graft survival prolonged by anti-CD4 monoclonal antibody. There were increased numbers of CD4+ T cells, and cell-mediated cytotoxicity was a CD4+ T-cell phenomenon associated with activation of the Fas/FasL lytic pathway. CONCLUSIONS The results demonstrate that, in the absence of CD4+ T cells, CD8+ T cells were capable of rejecting both rat and pig pancreatic islet xenografts.

Tirofiban and Activated Protein C Synergistically Inhibit the Instant Blood Mediated Inflammatory Reaction (IBMIR) from Allogeneic Islet Cells Exposure to Human Blood

Instant blood mediated inflammatory reaction (IBMIR) occurs when islets are exposed to blood and manifests clinically as portal vein thrombosis and graft failure. The aim of this study was to determine the impact of recombinant human activated protein C (rhAPC) and platelet inhibition on IBMIR in order to develop a better targeted treatment for this condition. Five thousand human islet cell equivalents (IEQ) were mixed in a PVC loop system with 7 mL of ABO compatible human blood and incubated with rhAPC, either alone or in combination with tirofiban. Admixing human islets and blood caused rapid clot formation, consumption of platelets, leukocytes, fibrinogen, coagulation factors and raised d‐dimers. Islets were encased in a fibrin and platelet clot heavily infiltrated with neutrophils. Tirofiban monotherapy was ineffective, whereas rhAPC monotherapy prevented IBMIR in a dose‐dependent manner, preserving islet integrity while maintaining platelet and leukocyte counts, fibrinogen and coagulation factor levels, and reducing d‐dimer formation. The combination of tirofiban and low‐dose rhAPC inhibited IBMIR synergistically with an efficacy equal to high dose rhAPC. Tirofiban and rhAPC worked synergistically to preserve islets, suggesting that co‐inhibition of the platelet and coagulation pathways’ contribution to thrombin generation is required for the optimal anti‐IBMIR effect.

Genetic and functional evaluation of the level of inbreeding of the Westran pig: a herd with potential for use in xenotransplantation

Abstract:  Background:  The Westran pig has been purposely inbred for use in xenotransplantation. The herd originated in the wild from a limited gene pool and has been inbred by repeated full‐sib matings for nine generations.

Chemokine and toll‐like receptor signaling in macrophage mediated islet xenograft rejection

Abstract:  Background:  Adoptive transfer of antigen‐primed T‐cell‐activated macrophages into NOD‐SCID mice within 14 days of foetal porcine pancreatic fragment (FPP) or foetal porcine skin (FPS) transplantation had been shown to cause xenograft rejection. In the present study, it was proposed that signaling between the graft and macrophages promoted specific graft recognition and destruction in this setting.

Involvement of CCR5 signaling in macrophage recruitment to porcine islet xenografts.

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.

Subcapsular fetal pig pancreas fragment transplantation provides normal blood glucose control in a preclinical model of diabetes.

Objective. Identifying a limitless source of &bgr;-cells that survive transplantation into a neovascularised site and provide normal blood glucose control remains an important goal in the development of pancreatic islet xenotransplantation. It was our hypothesis that fetal porcine pancreas fragments could achieve these objectives, and this was tested in a large preclinical animal model. Research Design and Methods. Inbred “Westran Pig” fetal porcine pancreas fragments were transplanted beneath the splenic capsule into syngeneic Westran Pig recipients without immunosuppression, and 3 months later, a total native pancreatectomy was performed to demonstrate function. Results. Histologic analysis showed appropriate development of islet-like structures up to and beyond 120 days after transplantation. After native pancreatectomy, recipients survived more than 100 days without exogenous insulin and with normal glucose homeostasis as assessed by normal glucose tolerance tests, K values, and normal glucagon secretion. Conclusions. This study confirms that fetal pig islet tissue has the potential to mature and function normally in a neovascularised site, hence, avoiding the innate immune destruction that occurs when islet tissue is exposed directly to the circulation.

Pre-clinical model of composite foetal pig pancreas fragment/renal xenotransplantation to treat renal failure and diabetes

Hawthorne WJ, Simond DM, Stokes R, Patel AT, Walters S, Burgess J, O’Connell PJ. Pre‐clinical model of composite foetal pig pancreas fragment/renal xenotransplantation to treat renal failure and diabetes. Xenotransplantation 2011; 18: 390–399.