Induction of swine major histocompatibility complex class I molecules on porcine endothelium by tumor necrosis factor-alpha reduces lysis by human natural killer cells

BACKGROUND: Natural killer (NK) cells have been implicated in a process of delayed xenograft rejection occurring in pig-to-primate organ transplants. As tumor necrosis factor-a (TNF-a) induces expression of both adhesion receptors and major histocompatibility complex class I molecules on porcine endothelium, we investigated the effects of TNF-alpha on human NK cell adherence to and cytotoxicity of porcine aortic endothelial cell (PAEC) monolayers. METHODS: Adherence of human NK cells was measured after PAEC treatment with increasing concentrations of TNF-alpha. Monoclonal antibodies (mAbs) against adhesion molecules on NK cells and PAEC were used in inhibition studies. Resting or TNF-alpha-treated PAEC were used as targets for NK lysis. Increasing titers of anti-swine leukocyte antigen (SLA) class I antibodies or pooled human immune globulin (IVIg) were used to reverse the effects of TNF-alpha on NK lysis. RESULTS: NK cell adhesion to TNF-a-treated PAEC increased in a dose-dependent manner by a maximum of 44%, and was inhibited by mAbs against CD49d, CD11a, CD11b, CD18, and CD2, as well as porcine vascular cell adhesion molecules. In contrast, TNF-alpha treatment of PAEC reduced human NK lysis in a dose-dependent manner. Preincubation of TNF-a-treated PAEC with increasing concentrations of anti-SLA class I mAb increased NK lysis in a titer-dependent manner, and reversed the protective effect on human NK lysis by 77%. Treatment with IVIg, containing antibodies against an a-helical region of HLA class I molecules, had a similar effect. CONCLUSIONS: These results imply that SLA class I molecules can bind to inhibitory receptors on human NK cells, and that these interactions can be augmented by increasing the level of SLA class I molecule expression on porcine endothelium. Strategies that can increase porcine endothelial cell expression of either swine or human major histocompatibility complex class I molecules may reduce human NK activity against porcine xenografts.     

Monocyte-Induced Endothelial Calcium Signaling Mediates Early Xenogeneic Endothelial Activation

Hallmarks of delayed xenograft rejection include monocyte infiltration, endothelial cell activation and disruption of the endothelial barrier. The monocyte is an important initiator of this type of rejection because monocytes accumulate within hours after xenografting and prior monocyte depletion suppresses the development of this type of rejection. However, the mechanisms that mediate monocyte-induced xenograft injury are unclear at present. Here we report that human monocytes activate xenogeneic endothelial cells through calcium signals. Monocyte contact with porcine but not human endothelium leads to an endothelial calcium transient mediated via a G-protein-coupled receptor (GPCR) that results in up-regulation of porcine VCAM-1 and E-selectin. Although human monocyte adhesion was greater to porcine than to human endothelium, especially when studied under laminar flow, blockade of the xeno-specific endothelial calcium signals did not reduce adhesion of human monocytes to porcine endothelium. Human monocyte contact to porcine endothelium also resulted in reorganization of the F-actin cytoskeleton with a concomitant increase in endothelial monolayer permeability. In contrast to the effect on adhesion, these changes appear to be regulated through endothelial calcium signals. Taken together, these data suggest that human monocytes are capable of activating xenogeneic endothelial cells through calcium transients, as well as other distinct pathways.     

Porcine endothelium supports transendothelial migration of human leukocyte subpopulations: anti-porcine vascular cell adhesion molecule antibodies as species-specific blockers of transendothelial monocyte and natural killer cell migration

BACKGROUND: In cases where hyperacute rejection has been prevented, pig to primate organ transplantation results in a delayed rejection mediated by graft-infiltrating leukocytes. The migration of human leukocytes across porcine endothelium is poorly characterized, but may offer targets for species-specific antirejection therapy. METHODS: Transwell tissue culture inserts with endothelial cells growing on polycarbonate filters were used to characterize the migration of peripheral blood monocuclear cells and purified leukocyte subpopulations across pig and human endothelial cells and cell lines. Endothelial cell morphology was evaluated by scanning and transmission electron microscopy, and the contribution of different adhesion receptor pairs to transendothelial migration was evaluated by antibody blocking experiments. RESULTS: There were no evident quantitative or qualitative differences in the capacity of human and porcine endothelium to support transendothelial migration of human leukocytes [T, B, and natural killer (NK) cells, monocytes, and neutrophils]. Monocytes and large granular CD3+ lymphocytes migrated most efficiently across the endothelium. Antiporcine vascular cell adhesion molecule-1 antibodies blocked transendothelial migration of human monocytes and NK cells across tumor necrosis factor-alpha stimulated pig endothelium by at least 60%. Anti-CD18 antibodies had no effect on the migration of human NK cells across pig endothelium, whereas they partly blocked migration of NK cells across human endothelium and migration of monocytes across porcine endothelium. Interleukin-2 stimulated, but not unstimulated, T and NK cells were cytotoxic to porcine endothelium. CONCLUSIONS: Porcine endothelium supports transendothelial migration of human leukocyte subpopulations as efficiently as human endothelium. Incompatibilities in some adhesion receptor pairs may be compensated for by other adhesion receptor pairs, as exemplified by human NK cells whose migration across human, but not pig, endothelium was blocked by anti-CD18 antibodies. Antiporcine vascular cell adhesion molecule-1 antibodies may be used as species-specific blockers of transendothelial NK cell and monocyte migration, and as such may prove to be useful inhibitors of cellular organ xenograft rejection.     

Cloning of porcine intercellular adhesion molecule-1 and characterization of its induction on endothelial cells by cytokines

BACKGROUND: The transplantation of pig organs into humans requires a detailed knowledge of similarities and differences between the two species in the molecular physiology of host defense mechanisms. We therefore set out to identify porcine intercellular adhesion molecule (ICAM)-1 and to characterize its expression by endothelial cells. METHODS: Porcine ICAM-1 cDNA was isolated from an endothelial cell cDNA library. An anti-pig ICAM-1 monoclonal antibody was generated and used to investigate the regulation by cytokines of ICAM-1 expression by porcine aortic endothelial cells (PAEC), using flow cytometry. RESULTS: We found that porcine ICAM-1 was similar in primary structure to human ICAM-1, with five Ig-like domains. COS-7 cells transfected with porcine ICAM-1 supported beta2 but not alpha4 integrin-dependent adhesion of human T lymphoblasts. There was a low-level surface expression of ICAM-1 on unstimulated PAEC and increased expression after stimulation with tumor necrosis factor (TNF)-alpha. However expression of ICAM-1 seemed to be significantly lower than that of vascular cell adhesion molecule-1, both on unstimulated and TNF-alpha-activated PAEC. Recombinant porcine interferon-gamma weakly stimulated ICAM-1 expression when incubated alone with PAEC but had an inhibitory effect on the increase in ICAM-1 due to TNF-alpha, both at 8 and 24 hr. CONCLUSIONS: Our observations confirm the existence of ICAM-1 in the pig and provide novel insights into how porcine and human endothelial cells differ in terms of adhesion molecule expression and cytokine responsiveness. Such differences are potentially important in interpreting models of inflammation in the pig and also in understanding the process of rejection of porcine xenografts.     

Activation of human dendritic cells by porcine aortic endothelial cells: transactivation of na?ve T cells through costimulation and cytokine generation.

BACKGROUND: Dendritic cells (DC) are the most potent antigen-presenting cells in the immune system. To define the role of human DC in human anti-porcine immune responses, we defined the interaction of human DC with porcine aortic endothelial cells (PAEC). METHODS: To determine the immune responses, both monocyte-derived and peripheral blood DC were cultured with porcine and human endothelial cells. We analyzed the role of CD11a, CD11b, and CD54 in a cell-to-cell adhesion assay using antibodies against these molecules. The expression pattern of costimulatory molecules (CD40, CD80, CD86), adhesion molecules (CD54), and intracellular cytokines (interleukin-12p70 and tumor necrosis factor [TNF]-alpha) in DC after interaction with endothelial cells was determined by immunofluorescence. RESULTS: Human DC significantly adhered to PAEC (38-40%), and this adhesion was augmented (>50%) upon treatment with either recombinant swine interferon-gamma or recombinant human TNF-alpha. Addition of human DC to PAEC was blocked by pretreatment of DC with antibodies specific to human leukocyte function-associated antigen-1 or CD54. Adhesion of DC to PAEC also resulted in the activation of DC, which was manifested by up-regulation of costimulatory molecules (CD40, CD80, CD86), adhesion molecules (CD54), and HLA-DR. PAEC-activated human DC provided proliferative signals to the na?ve autologous CD4+ T cells and synthesized interleukin-12p70 and TNF-alpha. However, activated DCs failed to lyse PAEC in such interaction. CONCLUSION: Human DC effectively adhered to PAEC and were activated by xenoantigen, resulting in highly efficient antigen presentation and proliferation of CD4+ T cells. Further, this interaction of human DC to PAEC is regulated by the participation of costimulatory and adherence molecules and cytokines.     

Role of porcine P-selectin in complement-dependent adhesion of human leukocytes to porcine endothelial cells

BACKGROUND: Rapid leukocyte adherence to donor organ vasculature is a hallmark of hyperacute xenograft rejection. However, the molecular interactions required for leukocyte binding to vascular endothelium have not been characterized. METHODS AND RESULTS: Binding assays performed between human neutrophils and porcine aortic endothelial cells (PAEC) after exposure to human complement demonstrated that adhesion was mediated by both surface-bound C3b and C5b-9 activity. C5b-9-dependent adhesion was blocked by neuraminidase treatment of the neutrophils, suggesting that this binding was mediated by porcine P-selectin. Porcine P-selectin was isolated from a PAEC cDNA library. The porcine P-selectin primary sequence contained an open reading frame encoding 646 amino acids with 82% identity to human P-selectin. Recombinant soluble porcine P-selectin specifically bound to human neutrophils and HL-60 cells. Transfection of COS cells with the full-length porcine P-selectin cDNA resulted in surface expression of the protein and markedly increased the binding of human neutrophils to these cells. The binding of both soluble and COS-expressed porcine P-selectin to human neutrophils was blocked by pretreatment of the neutrophils with neuraminidase or the addition of EDTA. Finally, treatment of PAEC with human thrombin or normal human serum but not purified human C5a- or C8-deficient human serum resulted in the rapid expression of porcine P-selectin on the cell surface. CONCLUSIONS: This report establishes that porcine P-selectin supports the binding of human neutrophils to PAEC in vitro. Further, these data suggest that sublytic deposition of C5b-9 during hyperacute rejection results in the expression of porcine P-selectin, which may contribute to the rapid adhesion of neutrophils to porcine xenografts.     

Human lymphocyte adhesion to xenogeneic porcine endothelial cells: modulation by human TNF-α and involvement of VLA-4 and LFA-1

Considering that in the allogeneic situation the adhesion of recipient lymphocytes to donor endothelial cells initiates the cellular rejection, we questioned the possible occurrence of a similar process in the xenogeneic situation. The adhesion of human peripheral blood lymphocytes (PBL) to porcine aortic endothelial cells (PAEC) was thus studied in an in vitro porcine-to-human xenogeneic model. It was found that 25.9% of human PBL adhered to resting PAEC. Furthermore, this adhesion increased significantly when the PAEC were stimulated by the human cytokine TNF-α (tumor necrosis factor-α). The effect of human TNF-α was concentration- and time-dependent and was maximal (from 25.9% to 35.6%) with 100 U/ml during 6 h. Moreover, blocking experiments with monoclonal antibody (mAb) demonstrated the role of the PBL adhesion molecules LFA-1 and especially VLA-4. Indeed, an anti-CDl 1a mAb decreased PBL adhesion to resting PAEC by 17.1% and to TNF-α stimulated PAEC by 16.9%, whereas an anti-CD49d mAb decreased dramatically PBL adhesion to resting PAEC by 53.1% and to TNF-α stimulated PAEC by 41.0%. Finally, phenotypic analysis of the adherent PBL showed that 50.5% of adherent cells to resting PAEC were NK (natural killer) cells, whereas 50.7% of adherent cells to TNF-α stimulated PAEC were T lymphocytes, showing the preferential adhesion of NK cells to resting PAEC, and that the stimulation of the PAEC with human TNF-α affects predominantly T lymphocyte adhesion. These results indicate that human PBL could bind to xenogeneic PAEC and that this interaction could be a first step of xenogeneic cellular rejection.     

Activation of Natural Killer Cells and Macrophages by Porcine Endothelial Cells Augments Specific T-Cell Xenoresponse

The rejection of xenografts is characterized by infiltration of monocytes and natural killer (NK) cells into the graft, suggesting an important role for the innate immune system in xenorecognition. In this study, purified human NK or T cells were cocultured with porcine endothelial cells, and cytokines were analyzed by ELISA and intracellular FACS. We demonstrated a vigorous human anti-porcine xenoresponse that was associated with a strong T-cell proliferation against porcine endothelial cells. Limiting dilution cloning and T-cell receptor (TCR) Vbeta gene usage revealed a low number of xenoreactive T-cell precursors. We demonstrated that xenogeneic porcine but not allogeneic human endothelial cells induced the early production of interferon (IFN)-gamma by human NK cells but not by CD3+ T cells. Porcine xenoantigen-induced IFN-gamma production was only partially dependent on IL-12. Blocking IL-12 with neutralizing antibodies or by depletion of human macrophages partially decreased IFN-gamma production by CD56+ NK cells. Three-color flow cytometry revealed that IL-12 was produced through a species-specific activation of human macrophages by porcine endothelial cells. Our results indicate that the direct activation of NK cells and macrophages by porcine endothelial cells provides a unique pathway of xenorecognition that augments downstream specific T-cell immunity and represents a powerful effector mechanism in xenograft rejection.     

Different mechanisms mediate the rejection of porcine neurons and endothelial cells transplanted into the rat brain

In order to investigate the early cellular responses mediating xenograft rejection in the brain, porcine aortic endothelial cells (PAEC) or porcine fetal mesencephalic neurons (PNEU) were transplanted into the striatum of LEW.1A rats. PAEC were detected with a specific anti-beta1 integrin antibody, and PNEU with an anti-porcine neurofilament antibody, or an antibody recognizing the NeuN antigen. PAEC grafts were massively infiltrated within 24 h by OX42-positive cells, which may correspond to polymorphonuclear (PMN) cells or macrophages. At that moment, the graft contained numerous cells expressing the inducible isoform of NO-synthase (iNOS). Infiltration by ED1-positive macrophages was effective after three days. The beta1-integrin labeling decreased from that time-point to day 7 post-implantation, and vanished after 11 days. Although some OX8-positive cells were present around the graft as soon as 3 days after transplantation, cells expressing the T-cell receptor (TCR)-beta chain infiltrated the graft after 7 days and their number remained low. A strong, diffuse OX8-and ED1-positive immunoreactive material remained in the scar up to the third week. In striking contrast, PNEU grafts remained poorly infiltrated by OX42- or ED1-positive cells during the first two weeks. A massive infiltration by macrophages and TCRbeta-positive lymphocytes occurred after 3 weeks. Natural killer (NK) cells were more scarce. The inflammation territory enlarged, and blood vessels were overloaded with macrophages or lymphocytes. Nevertheless, the graft contained NeuN-positive nuclei and neurites harbouring the porcine neurofilament protein. Hence, rejection was not completed at this time-point. These results suggest that the rapid rejection of PAEC is mainly driven by macrophages and possibly PMN cells, unlike PNEU, whose rejection is delayed and also involves lymphocytes. Differences in immunogenicity of grafted cells and/or patterns of production of pro-inflammatory cytokines may account for these contrasted rejection kinetics.     

Human T-cell-porcine endothelial cell interactions induce human Th1 cytokines and porcine activation markers

BACKGROUND: Since the future of clinical transplantation will undoubtedly include xenotransplantation, there is a need to examine human anti-pig cellular reactions. The objective of this study is to use human anti-porcine mixed lymphocyte endothelial cell culture (MLEC) to investigate cell interactions, cross-species molecular compatibilities, and the induction of human cytokines and porcine activation markers. METHODS: Human peripheral blood mononuclear cells or enriched CD4+ T cells depleted of professional antigen-presenting cells were cultured with resting pig aortic endothelial cells in the absence of exogenous cytokines. T-cell proliferative responses were measured and PAEC were monitored for cell surface markers by flow cytometry. Culture supernatants were assayed for human TNF-alpha and IFN-gamma by ELISA. RESULTS: Human T cells proliferated strongly in response to PAEC (median stimulation index = 75), even in serum-free cultures. High levels of the human Th1 cytokines TNF-alpha (20-350 pg/ml) and IFN-gamma (200-3800 pg/ml) were detected only in cultures containing PAEC, with levels peaking on Day 4. CD4+ T-cell-enriched, APC-depleted responders maintained proliferative anti-PAEC responses and cytokine release. By Day 3, MHC Class II and VCAM expression was induced in 92-96% PAEC: mean fluorescence intensity (MFI) increased from 5 to 83 +/- 12 and 166 +/- 74, respectively, and MHC Class I was increased from MFI 31 to 965 +/- 269. CONCLUSIONS: These results indicate that MLEC is an excellent in vitro model in which to study human anti-porcine cellular responses. Human T cells are activated in response to direct antigen presentation by PAEC, which are also activated in this system. Specific cytokines, receptors, and adhesion molecules appear to cross the xenograft barrier and play a critical role in T-cell - PAEC interactions. Such interactions are likely to affect VEC activation and immune responses to porcine xenografts in vivo.     

HMGB1 release in co-cultures of porcine endothelial and human T cells

High mobility group box-1 (HMGB1) protein, primarily from the nucleus, is released into the extracellular milieu either passively by necrotic or damaged cells, or actively by secretion from monocytes/macrophages. Extracellular HMGB1 acts as a potent inflammatory stimulator by promoting cytokine (for example, tumor necrosis factor-alpha) production, and also has pro-coagulant activity. The signaling pathway initiated by receptor for advanced glycation end-product (RAGE), which is the HMGB1 receptor, also induces complement activation. Recent studies have implicated HMGB1 in acute cardiac allograft rejection, and have identified infiltrating T cells and other damaged cells as its main sources. HMGB1 blockade using the anti-HMGB1 antibody HMGB1 box-A (amino-terminal region) and soluble RAGE rescues mice from acute rejection. We therefore studied the release of HMGB1 in co-cultures of porcine aortic endothelial cells (PAEC) and human leukocytes. Human T cells, but not B cells, monocytes or neutrophils, stimulated significant HMGB1 release in culture with PAEC; this activity required cell-cell contact and was dose-dependent, as determined by Western blotting. The released HMGB1 originated from both cell types, as immunofluorescent microscopy showed that it was present in the cytosol of PAEC in contact with T cells, and had disappeared from the T-cell nuclei. These results demonstrate that direct interactions between PAEC and T cells might be a key factor in triggering HMGB1 release, which suggests that HMGB1 is associated with graft rejection in the early phase.     

Xenotransplantation: role of natural immunity

Hyperacute rejection, mediated by natural anti-Galalpha1,3Galbeta1,4GlcNAc (alphaGal) antibodies and the classically activated complement pathway, was identified as the first major barrier to the survival of porcine organs in humans. Subsequently, discordant pig-to-nonhuman primate and concordant rodent models revealed key roles for T and B lymphocytes in the second form of rejection, acute vascular rejection (AVR) or delayed xenograft rejection (DXR). As significant progress was made in strategies to circumvent or suppress xenoreactivity of the adaptive immune system, it became clear that, apart from natural antibodies, other innate immune system elements actively participate in AVR/DXR and represent a barrier to xenograft acceptance that may be particularly difficult to overcome. Observations in pig-to-primate and semi-discordant and concordant rodent models indicate that Natural Killer (NK) cells play a more prominent role in xenograft than in allograft rejection. Several mechanisms through which human NK cells recognize porcine endothelial cells have been elucidated and these appear to be more diverse than those involved in NK cell alloreactivity. Further, it has been demonstrated that human macrophages and neutrophils can directly recognize pig derived cells and can mediate direct xenograft damage. Here, we review the recent progress in the understanding of the xenoreactivity of the natural immune system, focussing on preclinical pig-to-(non)human primate systems, and discuss the proposed strategies to overcome these barriers.     

Porcine endothelium activated by anti-alpha-GAL antibody binding mediates increased human neutrophil adhesion under flow

BACKGROUND: Neutrophils participate in acute vascular rejection (AVR) of organ xenografts. Induced antibodies (Abs), including anti-Galalpha1,3Gal (alpha-Gal) Abs, have been suggested to cause AVR. We investigated the adhesion of naive human neutrophils to porcine aortic endothelial cells (PAECs) stimulated with anti-alpha-Gal Abs under conditions of flow. In addition, the ability of human neutrophils to adhere to human and porcine endothelium under static and flow conditions was evaluated. METHODS AND RESULTS: In a flow-adhesion assay, a significant increase in adhesion of human neutrophils to PAECs, but not to human aortic endothelial cells (HAECs), was detected 6 hours after anti-alpha-Gal Ab-binding. After Ab stimulation, PAECs expressed CD62E and increased levels of CD106, indicating an activated endothelial cell (EC) phenotype. In a migration assay, supernatants from Ab-stimulated PAECs induced migration of human neutrophils, which was partially blocked by anti-porcine (p) interleukin (IL)-8 Abs and an antagonist to platelet-activating factor (PAF). In static and flow-adhesion assays, no difference in adhesion of human neutrophils to unstimulated or tumor necrosis factor (TNF)-alpha-stimulated HAECs and PAECs could be detected. CONCLUSIONS: Our data suggest that anti-alpha-Gal Abs play an important role in the initiation of AVR by mediating adhesion and recruitment of neutrophils within an organ xenograft. In contrast with previous investigations, our data argues against a differential recognition of PAECs and HAECs by human neutrophils. Thus, to prevent AVR and accomplish long-term xenograft survival, it will be important to remove anti-alpha-Gal Abs before and after pig-to-human transplantation.     

Inhibition of monocyte/endothelial cell interactions and monocyte adhesion molecule expression by the immunosuppressant mycophenolate mofetil

STUDY DESIGN: In vitro study on the effects of mycophenolate mofetil (MMF) on isolated human monocytes and endothelial cells. OBJECTIVES: Haematogenous macrophages play an essential role in the development of secondary damage following spinal cord injury (SCI), and there is evidence that the use of immunosuppressants such as MMF can reduce monocyte invasion and neuronal damage. SETTING: University Hospital for Orthopaedic Surgery, Frankfurt am Main, Germany. METHODS: The effects of MMF on the adhesion of human monocytes to human umbilical vein endothelial cells (HUVEC), monocyte binding to immobilised E-selectin, and monocyte expression of intercellular adhesion molecule (ICAM)-1, sialyl Lewis X (sLeX) and major histocompatibility complex (MHC)-II were studied. The binding of monocytes to E-selectin was examined by using purified and immobilised E-selectin fusion protein. Adhesion molecule expression was investigated by flow cytometry. RESULTS: The binding of monocytes to HUVEC was significantly reduced by 30.1% after treatment of monocytes with MMF (10 microg/ml), whereas the pretreatment of HUVEC with MMF did not result in significant changes in monocyte adhesion. MMF forcefully inhibited monocyte binding to immobilised E-selectin by 55.7%. Furthermore, MMF significantly inhibited the upregulation of ICAM-1- and MHC-II-expression on monocytes stimulated with either lipopolysaccharide or interferon-gamma, whereas the expression of sLeX was not impaired. Toxic effects were excluded by propidium-iodide staining and measurement of fluorescein-diacetate metabolism. CONCLUSION: MMF can downregulate important monocytic adhesion molecules and inhibits monocyte adhesion to endothelial cells, thus indicating that treatment with MMF could be beneficial after SCI. SPONSORSHIP: This study was supported by the DFG (Ha 2721/1-3), the Paul und Ursula Klein-Stiftung and the Stiftung Friedrichsheim.     

Rejection of xenogeneic porcine islets in humanized mice is characterized by graft‐infiltrating Th17 cells and activated B cells

Xenogeneic porcine islet transplantation is a promising potential therapy for type 1 diabetes (T1D). Understanding human immune responses against porcine islets is crucial for the design of optimal immunomodulatory regimens for effective control of xenogeneic rejection of porcine islets in humans. Humanized mice are a valuable tool for studying human immune responses and therefore present an attractive alternative to human subject research. Here, by using a pig‐to‐humanized mouse model of xenogeneic islet transplantation, we described the human immune response to transplanted porcine islets, a process characterized by dense islet xenograft infiltration of human CD45⁺ cells comprising activated human B cells, CD4⁺CD44⁺IL‐17⁺ Th17 cells, and CD68⁺ macrophages. In addition, we tested an experimental immunomodulatory regimen in promoting long‐term islet xenograft survival, a triple therapy consisting of donor splenocytes treated with ethylcarbodiimide (ECDI‐SP), and peri‐transplant rituximab and rapamycin. We observed that the triple therapy effectively inhibited graft infiltration of T and B cells as well as macrophages, promoted transitional B cells both in the periphery and in the islet xenografts, and provided a superior islet xenograft protection. Our study therefore indicates an advantage of donor ECDI‐SP treatment in controlling human immune cells in promoting long‐term islet xenograft survival.     

Cloning and potential utility of porcine Fas ligand: overexpression in porcine endothelial cells protects them from attack by human cytolytic cells

Tsuyuki S, Kono M, Bloom ET. Cloning and potential utility of porcine Fas ligand: overexpression in porcine endothelial cells protects them from attack by human cytolytic cells. Xenotransplantation 2002; 9:410–421. © Blackwell Munksgaard, 2002
Endothelial cells (EC) are primary targets of the recipient's immune response to transplanted organs and constitutively express Fas (CD95) ligand (FasL) on their surface. We investigated the role of porcine FasL in the generation of the human anti-pig response in vitro. Porcine aortic endothelial cells (PAEC) lysed a Fas⁺ human T-cell line, Jurkat. Anti-human Fas monoclonal antibody (mAb) specifically inhibited this killing in a dose-dependent manner, suggesting that porcine FasL recognizes and binds human Fas to induce apoptosis of human Fas⁺ cells. We next cloned porcine FasL, identifying an open reading frame of 849 base pairs predicting a protein of 282 amino acids. The predicted amino acid sequence was 85, 76, and 75% homologous to the predicted amino acid sequences of human, mouse, and rat, respectively, and found that PAEC expressed both FasL mRNA and protein. Transient transfection was used to increase or induce porcine FasL expression in PAEC or COS-7 cells. Transfection of PAEC with a plasmid encoding porcine FasL increased their ability to induce apoptosis in Jurkat cells, fresh human T cells activated with IL-2 and anti-CD3, and fresh IL-2-activated human (natural killer) NK cells. Moreover, porcine Fas L -transfected COS-7 cells induced significant apoptosis in Jurkat cells compared with that induced by mock-transfected COS-7 cells. Finally, the overexpression of porcine FasL in PAEC reduced their susceptibility as target cells to lysis by activated human NK or T cells. These findings suggest that porcine FasL overexpression in EC of vascularized xenografts may provide protection from cellular xenograft rejection.     

CD86 blockade in genetically modified porcine cells delays xenograft rejection by inhibiting T-cell and NK-cell activation

Porcine xenografts transplanted into primates are rejected in spite of immunosuppression. Identification of the triggering mechanisms and the strategies to overcome them is crucial to achieve long-term graft survival. We hypothesized that porcine CD86 (pCD86) contributes to xenograft rejection by direct activation of host T cells and NK cells. Formerly, we designed the human chimeric molecule hCD152-hCD59 to block pCD86 in cis. To test the efficacy in vivo, we have utilized a pig-to-mouse xenotransplant model. First, we showed that hCD152-hCD59 expression prevents the binding of murine CD28Ig to pCD86 on porcine aortic endothelial cells (PAEC) and dramatically reduces IL-2 secretion by Con A-stimulated mouse splenocytes in coculture. Moreover, IFN-gamma secretion by IL-12-stimulated mouse NK cells was averted after coculture with hCD152-hCD59 PAEC. In vivo, control PAEC implanted under the kidney capsule were rapidly rejected (2-4 weeks) in BALB/c and BALB/c SCID mice. Rejection of hCD152-hCD59 PAEC was significantly delayed in both cases. Signs of immune modulation in the hCD152-hCD59-PAEC BALB/c recipients were identified such as early hyporesponsiveness and diminished antibody response. Thus, simply modifying the donor xenogeneic cell can diminish both T cell and NK cell immune responses. We specifically demonstrate that pCD86 contributes to rejection of porcine xenografts.     

Xenograft rejection of fetal porcine islet-like cell clusters in the rat: effects of active and passive immunization

The process of islet xenograft rejection is still poorly understood. To elucidate further possible mechanism(s) involved in xenograft rejection, the effect of different immunization protocols was investigated. Fetal porcine islet-like cell clusters (ICCs) were transplanted under the kidney capsule in otherwise untreated rats, rats pre-immunized by s.c. injections of ICCs and in rats passively immunized with immune serum. The rejection process was evaluated with regard to antibody and complement deposition in the graft, as well as to morphology and phenotype of the infiltrating cells. In otherwise untreated animals, a moderate perigraft mononuclear cell infiltrate was seen after 3 days. Graft destruction became evident on day 6 with marked intragraft infiltration by macrophages (ED1 positive), whereas T cells were in the minority and mainly located in the perigraft area. In contrast to the findings in non-immunized rats, the rejection process in pre-immunized rats was characterized by marked intragraft infiltration by macrophages 3 days after transplantation. Moreover, both T cells and macrophages heavily infiltrated the adjacent kidney parenchyma, and major histocompatibility complex (MHC) class II expression in surrounding kidney tubular cells was concomitantly enhanced. Syngeneic rat islets mixed with porcine ICCs escaped the rejection process in non-immunized rats but were affected in pre-immunized animals. Thus, the specificity of the rejection process in non-immunized animals seems to be lost in pre-immunized animals. The early macrophage infiltration was also accelerated in rats passively immunized with immune serum, but no early switch from perigraft to intragraft infiltration or subsequent cellular infiltration in the adjacent kidney parenchyma was seen. Circulating xenoreactive antibodies of the IgG isotype increased after transplantation in normal and otherwise untreated rats. No distinct IgG deposition in the ICC xenografts was observed until day 12 after transplantation in untreated rats, whereas perigraft deposition of IgG was found 1 day after transplantation in pre-immunized rats and in rats given immune serum. No deposition of complement was observed within the ICC xenograft in any of the groups during the observation period. The dependence on T cells, the massive infiltration of macrophages with a unique phenotype, the cellular distribution, and the loss of specificity (bystander killing) of the rejection process in immunized rats suggest that ICC xenograft rejection shares some of its main characteristics with a delayed type hypersensitivity-like (DTH) immune response.