Cellular immunological barriers in xenotransplantation: current status and prospects for avoidance of graft destruction

Genetic modification of pigs (e.g. transgenic expression of human complement regulatory molecules or inactivation of α1,3galactosyltransferase) enabled the development of promising strategies to overcome hyperacute rejection after pig‐to‐primate xenotransplantation. However, cellular rejection still remains a hurdle for successful xenograft survival. Cellular rejection of porcine cells in xenotransplantation models is mediated by macrophages, T cells and NK cells. Activation of human monocytes by pig cells is partly due to the incapacity of porcine ligands to bind the inhibitory receptor SIRPα (signal regulatory protein α). Thus, one approach to impair the ability of human macrophages to phagocyte porcine cells is the overexpression of the human ligand for SIRPα in porcine cells. To inhibit human NK cell reactivity after xenotranslantation transgenic expression of HLA‐E in pigs has been shown to be a promising concept. Cells from these pigs were partially protected from lysis by human NK cells. Our group focuses on manipulation of human anti‐pig T cell responses by negative costimulatory signals. Thus, we asked whether overexpression of PD‐L1 on porcine cells can (i) downregulate human anti‐pig cellular responses in vitro, and (ii) inhibit rat anti‐pig cellular immune responses in vivo. Pig cells overexpressing PD‐L1 triggered reduced proliferation and low amounts of IL‐2, IFNγ, TNF‐alpha, IL‐4, and IL‐5 in human CD4⁺ T cells compared to control pig cells. The concentration of IL‐10, however, was increased. In long‐term cultures of human CD4⁺ T cells and PD‐L1 transfectants a high frequency of CD4⁺ CD25^high FoxP3⁺ cells showed up which had the capacity to suppress the activation of conventional CD4⁺ T cells. Cytotoxic CD8⁺ T cells and NK cells lysed pig control cells very efficiently. In contrast, PD‐L1 transfected pig cells were partially protected from lysis by human effector cells. Overexpression of PD‐L1 on porcine cells was not sufficient to prevent rejection after transplantation under the rat kidney capsule. However, in rats that had been grafted with PD‐L1 expressing cells we observed reduced cellular infiltrates in the kidneys and lower antibody responses compared to rats grafted with control cells. Together these observations support the assumption that PD‐1/PD‐Ligand pathways are interesting targets to prevent cellular immune responses after xenotransplantation. PD‐L overexpression might not only impede the initiation of an anti‐pig T cell response by suppressing CD4⁺ T cells but may also protect pig cells from destruction by cytotoxic effectors. Supported by the Deutsche Forschungsgemeinschaft (Transregio Forschergruppe “Xenotransplantation”, FOR 535).     

Xenotransplantation: immunological barriers and strategies to overcome them

Hyperacute and acute vascular rejection of xenografts are well defined barriers to clinical pig‐to‐human xenotransplantation. Enormous progress has been made in recent years to overcome these immunological barriers. For example, transgenic expression of human complement regulatory molecules (e.g. CD46, CD55) in pigs has been shown to be an effective strategy to prevent hyperacute rejection in pre‐clinical models of xenotransplantation. Alpha1,3‐galactosyltransferase knock‐out pigs are available and provide a second possibility to avoid hyperacute rejection mediated by pre‐existing antibodies. Furthermore, transfer of protective genes (e.g. A20, HO‐1) to endothelial cells is expected to reduce their susceptibility to effector mechanisms leading to acute vascular rejection. In addition, the efficiency of strategies to avoid coagulation/thrombosis after pig‐to‐human xenotransplantation (e.g. transgenic expression of human thrombomodulin, CD39) is currently tested. Thus, for further development of clinical xenotransplantation immunological concepts are now required facilitating the control of human anti‐pig cellular immune responses. Our group focuses on the inhibition of human anti‐pig T cell responses by targeting “negative” costimulatory pathways. We tested the hypothesis that overexpression of the human negative costimulatory ligands PD‐L1 and PD‐L2 on pig antigen presenting cells will result in reduced human anti‐pig T cell responses. The data so far show that (i) human CD4⁺ T cells respond with reduced proliferation and cytokine synthesis to PD‐L1/PD‐L2 expressing pig cells, (ii) PD‐L1/PD‐L2 pig transfectants induce human regulatory T cells (T_reg) which suppress the activation of conventional T cells, and (iii) PD‐L1/PD‐L2 expressing pig cells are protected from lysis mediated by CD8⁺ human cells. Together these observations support the assumption that transgenic expression of human PD‐L1 and/or PD‐L2 in pig cells and tissues could be an approach to prevent T cell reactivity after pig‐to‐human xenotransplantation. Supported by the Deutsche Forschungsgemeinschaft (Transregio Forschergruppe “Xenotransplantation”, FOR 535).     

Expression of biologically active human TRAIL in transgenic pigs

BACKGROUND: Xenotransplantation of porcine organs into human recipients is a potential option for overcoming the dramatic shortage of suitable donor organs. To date, transgenic modification of pig organs has achieved partial or temporal reduction of xenograft rejection by inhibition of hyperacute rejection. Expression of human tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) in transgenic pigs might be a strategy for controlling posthyperacute rejection mechanisms mediated by cellular components of the immune system. The objective of this study was generation of a transgenic pig model to evaluate the potential of this strategy for xenotransplantation. METHODS: Transgenic pigs were generated by microinjection of an expression vector for human TRAIL under control of the murine H-2K promoter. Expression of the transgene was analyzed by Western blot and immunohistochemistry. Biologic activity of TRAIL on transgenic porcine lymphocytes was evaluated in co-culture experiments using Jurkat and Hut 78.2 cells as targets. RESULTS: In three lines of transgenic pigs, human TRAIL protein was detected in the membrane fractions of various tissues. Highest expression levels were observed in spleen and lung. Human TRAIL expression on porcine lymphocytes was augmented on activation of cells. Transgenic pig lymphoblasts induced apoptosis in Jurkat and Hut 78.2 cells, which was inhibited by neutralizing anti-TRAIL antibodies, demonstrating a TRAIL-specific effect. CONCLUSIONS: Ubiquitous expression of human TRAIL was achieved in transgenic pigs without detrimental side effects. Pigs expressing biologically active human TRAIL will be used for future xenotransplantation experiments to modulate primate anti-pig cellular immune responses.     

Downregulation of cytolytic activity of human effector cells by transgenic expression of human PD‐ligand‐1 on porcine target cells

Cellular rejection is a relevant hurdle for successful pig‐to‐primate xenotransplantion. We have shown previously that the induction of a human anti‐pig T cell response (in vitro activation of CD4⁺ T cells) can be suppressed by the overexpression of human negative costimulatory ligands (e.g. programmed death receptor ligand, PD‐L1) on pig antigen presenting cells. Here, we asked whether PD‐L1 mediated enhancement of negative signaling might also be efficient during the effector phase of human anti‐pig cellular immune responses. The porcine B‐cell line L23 was transfected with human PD‐L1, and clones were selected stably expressing PD‐L1 with low, medium, or high density. Mock‐transfected L23 cells were effectively lysed by human cytotoxic effector cells (IL‐2 activated CD8⁺ T cells and CD56⁺ cells). The lytic potential of the effectors decreased with increasing levels of PD‐L1 and was reduced by about 50% in L23‐PD‐L^high targets. A proportion of activated CD8⁺ effector cells underwent apoptosis when exposed to PD‐L1 expressing L23 cells. These data suggest that the overexpression of PD‐L1 on target cells may (a) trigger negative signals in effector cells that prevent the release of cytolytic molecules and/or (b) induce apoptosis in the attacking effector cells thereby protecting targets from destruction.     

ITIM‐dependent negative signaling pathways for the control of cell‐mediated xenogeneic immune responses

Xenotransplantation is an innovative field of research with the potential to provide us with an alternative source of organs to face the severe shortage of human organ donors. For several reasons, pigs have been chosen as the most suitable source of organs and tissues for transplantation in humans. However, porcine xenografts undergo cellular immune responses representing a major barrier to their acceptance and normal functioning. Innate and adaptive xenogeneic immunity is mediated by both the recognition of xenogeneic tissue antigens and the lack of inhibition due to molecular cross‐species incompatibilities of regulatory pathways. Therefore, the delivery of immunoreceptor tyrosine‐based inhibitory motif (ITIM)‐dependent and related negative signals to control innate (NK cells, macrophages) and adaptive T and B cells might overcome cell‐mediated xenogeneic immunity. The proof of this concept has already been achieved in vitro by the transgenic overexpression of human ligands of several inhibitory receptors in porcine cells resulting in their resistance against xenoreactivity. Consequently, several transgenic pigs expressing tissue‐specific human ligands of inhibitory coreceptors (HLA‐E, CD47) or soluble competitors of costimulation (belatacept) have already been generated. The development of these robust and innovative approaches to modulate human anti‐pig cellular immune responses, complementary to conventional immunosuppression, will help to achieve long‐term xenograft survival. In this review, we will focus on the current strategies to enhance negative signaling pathways for the regulation of undesirable cell‐mediated xenoreactive immune responses.     

New transgenic pigs for xenotransplantation,part 1

The hyperacute rejection response (HAR) after porcine‐to‐human xenotransplantation can now be reliably overcome. The next immunological hurdle is the acute vascular rejection (AVR) primarily caused by endothelial cell activation followed by disseminated intravascular coagulopathy, increased apoptosis and inflammatory symptoms. Several genes have been proposed to show protective effects against AVR, including human heme oxygenase‐I (hHO‐1) and human A20 (hA20) gene. HHO‐1 has primarily anti‐apoptotic and cell protective properties. The hA20 molecule possesses protective features against inflammatory and apoptotic stimuli in endothelial cells. Thus transgenic expression of these genes in pigs may be promising to prolong survival of porcine xenografts. We used somatic cell nuclear transfer (SCNT) for production of transgenic pigs. We produced pigs transgenic for human heme oxygenase 1 (hHO‐1) to evaluate the protective effects of that molecule and to compare it with other transgenes used to control of the hyperacute rejection response (HAR), e.g. the DAF transgenes which gave HAR protection in in vitro cell death assays. Importantly, hHO‐1 transgenic porcine aortic endothelial cells were significantly better protected against TNF‐α mediated apoptosis. In close collaboration with partners at the LMU Munich (Prof. Kupatt et al.) the transgenic pig lines were tested in an ischemia/reperfusion (I/R) circuit. After occlusion of the left anterior descending artery (LAD), hHO‐1 transgenic hearts had significantly smaller infarct lesions and concomitantly significantly better global myocardial function than size‐matched wild‐type controls. In close collaboration with partners at Hannover Medical University (Prof. Winkler et al.), hHO‐1 transgenic porcine kidneys were perfused with pooled human blood for the maximum period of 240 min without addition of C1‐Inhibitor in an ex vivo perfusion circuit. In parallel, we produced and characterized pigs that express hHO‐1 on a Gal^–/– background. Gal^–/–/hHO‐1 pig hearts were tested in the I/R circuit and preliminary results indicate a protective effect shown by decreased infarct size, less inflammation and improved global and regional myocardial function after LAD occlusion. Expression of hA20 from the CAGGS promoter was found in skeletal muscle, heart and PAECs. Cultured human A20‐transgenic PAECs showed significantly reduced apoptosis when compared to their wild type counterparts. Only partial protection of hA20‐transgenic pig hearts was observed after I/R. While infarct size was not different between the two groups after ischemic assault, hA20‐transgenic pig hearts showed a significantly better hemodynamic performance (determined as SES) than the wild type porcine hearts. MPO activity was reduced in transgenic vs. wild type hearts. We also produced pigs carrying shRNA constructs directed against PERV expression. These animals showed significantly reduced PERV‐expression for over 6 months compared to wild‐type and sham controls. This approach could improve the safety of porcine xenografts. We will now produce pigs carrying hHO‐1 on the Gal^–/–/hCD46 background. Tissues and organs from these animals will be tested in the previously established in vitro systems, and when positive results are obtained, hearts and kidneys will be transplanted into baboons. A second line of multi‐transgenic pigs will have both hA20 and shRNA against PERV expression on the Gal^–/–/hCD46/hHO‐1 background. The new somatic cloning protocol developed recently will allow rapid screening of promising transgene combinations and will ensure that we achieve our ambitious goals and move xenotransplantation closer to clinical application. This study was funded by grants from the Deutsche Forschungsgemeinschaft Ni 256/ 22‐1, ‐2, ‐3,‐4.     

The Introduction of Human Heme Oxygenase‐1 and Soluble Tumor Necrosis Factor‐α Receptor Type I With Human IgG1 Fc in Porcine Islets Prolongs Islet Xenograft Survival in Humanized Mice

Apoptosis during engraftment and inflammation induce poor islet xenograft survival. We aimed to determine whether overexpression of human heme oxygenase‐1 (HO‐1) or soluble tumor necrosis factor‐α receptor type I with human IgG₁ Fc (sTNF‐αR‐Fc) in porcine islets could improve islet xenograft survival. Adult porcine islets were transduced with adenovirus containing human HO‐1, sTNF‐αR‐Fc, sTNF‐αR‐Fc/HO‐1 or green fluorescent protein (control). Humanized mice were generated by injecting human cord blood–derived CD34⁺ stem cells into NOD‐scid‐IL‐2Rγ^null mice. Both HO‐1 and sTNF‐αR‐Fc reduced islet apoptosis under in vitro hypoxia or cytokine stimuli and suppressed RANTES induction without compromising insulin secretion. Introduction of either gene into islets prolonged islet xenograft survival in pig‐to‐humanized mice transplantation. The sTNF‐αR‐Fc/HO‐1 group showed the best glucose tolerance. Target genes were successfully expressed in islet xenografts. Perigraft infiltration of macrophages and T cells was suppressed with decreased expression of RANTES, tumor necrosis factor‐α and IL‐6 in treatment groups; however, frequency of pig‐specific interferon‐γ–producing T cells was not decreased, and humoral response was not significant in any group. Early apoptosis of islet cells was suppressed in the treatment groups. In conclusion, overexpression of HO‐1 or sTNF‐αR‐Fc in porcine islets improved islet xenograft survival by suppressing both apoptosis and inflammation. HO‐1 or sTNF‐αR‐Fc transgenic pigs have potential for islet xenotransplantation.     

Transplantation of neonatal islets from LEA29Y‐transgenic pigs restores normoglycemia in streptozotocin‐diabetic NSG‐mice

Beta cell replacement therapy represents the only way towards complete restoration of the physiological glucose homeostasis in patients with Type 1 diabetes. Xenotransplantation of transgenic pig islets expressing immunomodulatory molecules might represent a promising approach to overcome the problems of lacking organ donors, adverse effects of the required systemic immunosuppression, and disappointing transplantation outcomes. LEA29Y, a second generation CTLA‐4‐Ig fusion protein exerts potent immunosuppressive effects by selective blockade of the B7 costimulatory pathway. To assess the impact of high local LEA29Y concentrations on xenogeneic islet graft rejection, a transgenic pig model with islet‐specific LEA29Y transgene expression was generated. Islet‐like clusters from neonatal pigs expressing LEA29Y under the control of the porcine insulin promoter and from wild‐type pigs were isolated by collagenase digestion and subsequent in vitro culture. After 6 days 2500 clusters/mouse were transplanted under the kidney capsule of streptozotocin‐diabetic NOD‐scidIL2Rgamma^null (NSG) mice. After an initial period of insulin dependency, mice developed stable normoglycemia within 8 weeks after transplantation. Transplanted mice of both groups (Tx, wt; Tx, LEA29Y) exhibited normal and as compared to non‐transplanted normoglycemic NSG mice even improved fasting glucose and glucose tolerance resulting from glucose‐responsive graft‐derived porcine insulin secretion. Beta cell‐specific expression of LEA29Y was detectable in neonatal porcine pancreas, in the grafts of transplanted mice, and was also measurable in the plasma of normoglycemic mice. Furthermore, mice with LEA29Y transgenic grafts exhibited a glucose‐dependent LEA29Y release during glucose tolerance testing. The present study demonstrates that LEA29Y transgenic islet‐like clusters display normal beta cell function and have the same potential as wild‐type islet clusters to restore normoglycemia in streptozotocin‐diabetic NSG mice after an in vivo maturation period towards a functional endocrine tissue. NSG mice with LEA29Y islet grafts may therefore represent a promising model to study the modulation of human‐anti‐pig xenograft rejection in detail. In ongoing experiments xenograft rejection is analyzed in “humanized” mice after the transfer of human PBMCs or isolated CD34⁺ stem cells.     

Gene transfer of the adaptor Lnk (SH2B3) prevents porcine endothelial cell activation and apoptosis: implication for xenograft's cytoprotection

Chatelais M, Devallière J, Galli C, Charreau B. Gene transfer of the adaptor Lnk (SH2B3) prevents porcine endothelial cell activation and apoptosis: implication for xenograft’s cytoprotection.
Xenotransplantation 2011; 18: 108–120. © 2011 John Wiley & Sons A/S. Abstract: Background: Targeting protective gene expression to porcine endothelium by genetic modification of the donor could improve xenograft survival by controlling cell activation and death. We previously found that, in endothelial cells (EC), the molecular adaptor Lnk (SH2B3) is a negative regulator of cytokine signaling. We also have shown that Lnk is upregulated in pig EC (PAEC) in response to tumor necrosis factor‐α (TNF) and xenoreactive natural antibodies (XNA) binding. The present study investigated whether ectopic expression of human Lnk using gene transfer may be efficient to control signaling pathways associated with inflammation and apoptosis in porcine aortic endothelial cells (PAEC). Methods: Endothelial cells cultures were established from WT and Gal^?/? pigs and transduced with a recombinant adenovirus encoding human Lnk. Phenotype and functions of transduced PAEC expressing Lnk were analyzed by flow cytometry, western blot and XNA and complement‐dependent assays. The regulatory functions of Lnk toward inflammation were assessed in TNF‐activated EC, and the protective functions were tested toward TNF‐induced apoptosis and anoïkis. Apoptosis assays included DNA content analysis and caspase‐3/7 activity. Results: First, we found that as a result of adenoviral transduction, human Lnk was efficiently and similarly expressed in EC from WT or Gal^?/? pigs. Lnk expression or EC transduction caused no significant change in the binding of XNA (IgG and IgM) to PAEC and has no effect on complement activation and C5b‐9 formation. We demonstrated that expression of human Lnk efficiently inhibits TNF signaling in PAEC and decreases VCAM‐1 induction by 46.3 ± 1.2% compared to controls (n = 6, **P < 0.01). Furthermore, expression of Lnk was associated with a significant decrease in the percentage of caspase‐3/7‐dependent apoptosis caused by TNF in the presence of actinomycin D and also reduces cell death by anoïkis by 25.0 ± 1.9% compared to controls (n = 5, **P < 0.01). Conclusions: Together, these findings indicate that the signaling adaptor Lnk is effective to reduce PAEC activation and apoptosis. Thus, Lnk is a potential candidate for the modulation of signaling pathways to protect vascular EC from inflammation in xenotransplantation.     

Pig islet xenograft rejection in a mouse model with an established human immune system

Abstract: Background: Xenotransplantation from pigs provides a potential solution to the severe shortage of human pancreata, but strong immunological rejection prevents its clinical application. A better understanding of the human immune response to pig islets would help develop effective strategies for preventing graft rejection. Methods: We assessed pig islet rejection by human immune cells in humanized mice with a functional human immune system. Humanized mice were prepared by transplantation of human fetal thymus/liver tissues and CD34⁺ fetal liver cells into immunodeficient mice. Islet xenograft survival/rejection was determined by histological analysis of the grafts and measurement of porcine C‐peptide in the sera of the recipients. Results: In untreated humanized mice, adult pig islets were completely rejected by 4 weeks. These mice showed no detectable porcine C‐peptide in the sera, and severe intra‐graft infiltration by human T cells, macrophages, and B cells, as well as deposition of human antibodies. Pig islet rejection was prevented by human T‐cell depletion prior to islet xenotransplantation. Islet xenografts harvested from T‐cell‐depleted humanized mice were functional, and showed no human cell infiltration or antibody deposition. Conclusions: Pig islet rejection in humanized mice is largely T‐cell‐dependent, which is consistent with previous observations in non‐human primates. These humanized mice provide a useful model for the study of human xenoimmune responses in vivo.     

The effect of Gal expression on pig cells on the human T-cell xenoresponse

Wilhite T, Ezzelarab C, Hara H, Long C, Ayares D, Cooper DKC, Ezzelarab M. The effect of Gal expression on pig cells on the human T cell xenoresponse. Xenotransplantation 2012; 19: 56–63. © 2012 John Wiley & Sons A/S. Abstract: Background: Lack of Gal expression on pig cells is associated with a reduced primate humoral immune response as well as a reduction in cytokine production by human cells in vitro. We investigated whether lack of Gal expression is associated with reduced human T‐cell response in vitro. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from healthy humans and naïve baboons. Human CD4⁺ and CD8⁺ T cells were isolated. Porcine aortic endothelial cells (pAECs) were isolated from wild‐type (WT) and α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs. WT pAECs were treated with α‐galactosidase, reducing Gal expression. Swine leukocyte antigen (SLA) class I and II expression on pAECs was measured, as was T‐cell proliferation and cytokine production in response to pAECs. Results: Reduced Gal expression on WT pAECs after α‐galactosidase treatment was associated with reduced human PBMC proliferation (P < 0.005). SLA class I and II expression on WT and GTKO pAECs was comparable. Human CD4⁺ and CD8⁺ T‐cell proliferation was less against GTKO pAECs before (P < 0.001) and after (P < 0.01 and P < 0.05, respectively) activation. Human and baboon PBMC proliferation was less against GTKO pAECs before (P < 0.05) and after (P < 0.01 and P < 0.05, respectively) activation. Human PBMCs produced a comparable cytokine/chemokine response to WT and GTKO pAECs. However, there was less production of IFN‐γ/TNF‐α by CD4⁺ and IFN‐γ/granzyme B/IP‐10 by CD8⁺ T cells in response to GTKO pAECs. Conclusions: The absence of Gal on pig cells is associated with reduced human T‐cell proliferation (and possibly selected cytokine production). Adaptive primate T‐cell responses are likely to be reduced in GTKO xenograft recipients.     

The potential of local immunomodulation and tolerance induction in porcine islet xenotransplantation

Transplantation of pancreas or isolated islet cells is currently the only option to cure type 1 diabetes. The success of islet transplantation is still limited by the requirement of large numbers of high quality islets and the shortage of organ donors. Porcine islets are a promising cell source, but the intensive immunosuppressive regimen required to suppress rejection prevents the translation into clinical practice. We aimed to develop a novel method to inhibit the human‐anti‐pig immune reaction by the expression of immunomodulatory molecules in porcine beta cells. Thus, a transgenic pig was generated expressing LEA29Y – a second generation human CTLA4‐Ig fusion protein, which inhibits activation of T cells by CD80/CD86‐CD28 costimulation – under the control of the porcine insulin promotor. Islet‐like clusters (ICC) from neonatal pigs were isolated and transplanted under the kidney capsule of diabetic NOD‐scid‐IL2γnull (NSG) mice. After an in vivo maturation period mice transplanted with wildtype (wt) as well as with LEA29Y transgenic (tg) ICCs developed normal glucose homeostasis. Within 30 days after the transfer of human PBMCs 80% of NSG mice transplanted with wt‐ICCs developed diabetes indicating xenograft rejection. By contrast, LEA‐tg ICCs were completely protected from rejection in all animals (1). Immunohistochemistry revealed a massive intra‐islet T cell infiltration, which was absent in the LEA‐tg ICCs. This proof of principle study suggests that specific expression of immunomodulatory molecules in beta cells does not disturb beta cell function and may have the potential to modulate immune response locally at the transplantation site without systemic immunosuppression. To overcome the strong xenogeneic barrier of the human and cellular immune system a combination of LEA29Y with additional immunomodulatory factors may be required. Recently, Yi and coworkers demonstrated that the treatment with in vitro expanded regulatory T cells (Treg) prevents porcine islet rejection in humanized NSG mice by the suppression of the T cell‐mediated graft destruction (2). Other potential candidates to induce a state of tolerance against porcine islets currently under investigation are molecules targeting innate immunity and factors that prevent the reoccurrence of autoimmunity. Recent advances in xenotransplantation suggest that it may be possible to start with clinical trials using porcine neonatal or adult islets within the near future. References: 1. KLYMIUK N, VAN BÜRCK L, BÄHR Aet al. Xenografted islet‐cell‐clusters from INSLEA29Y transgenic pigs rescue diabetes and prevent immune rejection in humanized mice. Diabetes 2012; 61:1527–1532. 2. YI S, JI M, WU J 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.     

Genetically-modified pig mesenchymal stromal cells: xenoantigenicity and effect on human T-cell xenoresponses

Ezzelarab M, Ezzelarab C, Wilhite T, Kumar G, Hara H, Ayares D, Cooper DKC. Genetically‐modified pig mesenchymal stromal cells: xenoantigenicity and effect on human T‐cell xenoresponses. Xenotransplantation 2011; 18: 183–195. © 2011 John Wiley & Sons A/S. Abstract: Background: Mesenchymal stromal cells (MSC) are being investigated as immunomodulatory therapy in the field of transplantation, particularly islet transplantation. While MSC can regenerate across species barriers, the immunoregulatory influence of genetically modified pig MSC (pMSC) on the human and non‐human primate T‐cell responses has not been studied. Methods: Mesenchymal stromal cells from wild‐type (WT), α1,3‐galactosyltransferase gene knockout (GTKO) and GTKO pigs transgenic for the human complement‐regulatory protein CD46 (GTKO/CD46) were isolated and tested for differentiation. Antibody binding and T‐cell responses to WT and GTKO pMSC in comparison with GTKO pig aortic endothelial cells (pAEC) were investigated. The expression of swine leukocyte antigen (SLA) class II (SLA II) was tested. Costimulatory molecules CD80 and CD86 mRNA levels were measured. Human T‐cell proliferation and the production of pro‐inflammatory cytokines in response to GTKO and GTKO/CD46 pMSC in comparison with human MSC (hMSC) were evaluated. Results: α1,3‐galactosyltransferase gene knockout and GTKO/CD46 pMSC isolation and differentiation were achieved in vitro. Binding of human antibodies and T‐cell responses were lower to GTKO than those to WT pMSC. Human and baboon (naïve and sensitized) antibody binding were significantly lower to GTKO pMSC than to GTKO pAEC. Before activation, <1% of GTKO pMSC expressed SLA II, compared with 2.5% of GTKO pAEC. After pig interferon‐gamma (pIFN‐γ) activation, 99% of GTKO pAEC upregulated SLA II expression, compared with 49% of GTKO pMSC. Only 3% of GTKO pMSC expressed CD80 compared with 80% of GTKO pAEC without activation. After pIFN‐γ activation, GTKO pAEC upregulated CD86 mRNA level stronger than GTKO pMSC. The human CD4⁺ T‐cell response to GTKO pMSC was significantly weaker than that to GTKO pAEC, even after pIFN‐γ activation. More than 99% of GTKO/CD46 pMSC expressed hCD46. Human peripheral blood mononuclear cells and CD4⁺T‐cell responses to GTKO and GTKO/CD46 pMSC were comparable with those to hMSC, and all were significantly lower than to GTKO pAEC. GTKO/CD46 pMSC downregulated human T‐cell proliferation as efficiently as hMSC. The level of proinflammatory cytokines IL‐2, IFN‐γ, TNF‐α, and sCD40L correlated with the downregulation of T‐cell proliferation by all types of MSC. Conclusion: Genetically modified pMSC is significantly less immunogenic than WT pMSC. GTKO/CD46 pMSC downregulates the human T‐cell responses to pig antigens as efficiently as human MSC, which can be advantageous for therapeutic cell xenotransplantation.     

Successful cross‐breeding of cloned pigs expressing endo‐β‐galactosidase C and human decay accelerating factor

Yazaki S, Iwamoto M, Onishi A, Miwa Y, Suzuki S, Fuchimoto D, Sembon S, Furusawa T, Hashimoto M, Oishi T, Liu D, Nagasaka T, Kuzuya T, Maruyama S, Ogawa H, Kadomatsu K, Uchida K,
Nakao A, Kobayashi T. Successful cross‐breeding of cloned pigs expressing endo‐β‐galactosidase C and human decay accelerating factor. Xenotransplantation 2009; 16: 511–521. © 2009 John Wiley & Sons A/S. Abstract: Background: For successful organ xenotransplantation, genetically engineered pigs have been actively produced. Our attention has focused on (i) reduction of αGal expression by its digestion enzyme, endo‐β‐galactosidase C (EndoGalC), and (ii) inhibition of complement activation by human decay accelerating factor (hDAF). Cell sorting and nuclear transfer enabled the effective production of cloned pigs expressing transgene at high levels. We report the successful cross‐breeding of pigs expressing EndoGalC and hDAF. Methods: After hDAF and EndoGalC genes were transfected into pig fibroblasts from the fetus of Landrace × Yorkshire and Meishan, respectively, transfected cells expressing transgenes effectively were collected using a cell sorter. Cloned pigs were produced using the technology of somatic cell nuclear transfer. After cross‐breeding of cloned pigs, kidneys expressing both EndoGalC and hDAF were transplanted into baboons to examine the efficacy of gene transduction. Results: Well‐designed cloned pigs were produced by cross‐breeding. αGal expression levels in cloned pigs were reduced up to 2 to 14%, compared to that in wild‐type pigs. hDAF expression reached about 10‐ to 70‐fold, compared to that in human umbilical vein endothelial cells. No congenital deformity was observed. There was no problem of increased stillbirth rate or growth retardation. Hyperacute rejection could be avoided in such a cloned pig to baboon kidney transplantation without any treatment for anti‐pig antibody removal. However, grafts suffered from fibrin deposition as early as 1 h after transplantation, and were rejected after 1 week. Conclusions: Using a cell sorting system for effective collection of transfected cells, two types of cloned pigs were produced with a very high level of hDAF expression and a low level of αGal expression. Such genetic modification was effective in preventing hyperacute rejection, but there was an immediate lapse into procoagulation after transplantation, resulting in acute vascular rejection. Effective suppression of antibody binding to the graft would be necessary, even if a high level of hDAF is expressed.     

Ex vivo perfusion of HLA‐E/CD46 transgenic pig limbs with human blood: evaluation of NK cell recruitment

In vitro, interactions between human NK cells and porcine endothelial cells (pEC) are characterized by NK cell recruitment and cytotoxicity. NK cells play a role in allo‐ and xenotransplantation due to the incompatibility of the MHC class I molecules expressed on transplanted cells and tissues on one hand, and the MHC class I specific inhibitory NK receptors of the recipient on the other hand. In this context we published previously that: (i) the expression of human HLA‐E on pEC partially protects from polyclonal huNK cytotoxicity and completely protects from killing by NKG2Abright NK clones in vitro; but does not affect the adhesion of huNK cells to pEC or the heteroconjugate formation between huNK and porcine cells (1, 2); (ii) lymphoblasts and pEC derived from HLA‐E/human beta2microglobulin transgenic pigs are effectively protected against huNK cell‐mediated cytotoxicity and inhibit the secretion of IFN‐gamma by co‐cultured huNK cells in vitro, depending on CD94/NKG2A expression on the NK cells (3); (iii) HLA‐G expression on pEC protects partially against direct huNK cytotoxicity but not against ADCC (4); (iv) HLA‐G expression inhibits rolling adhesion of activated huNK cells on pEC (5, 6); (v) HLA‐Cw3 expression on pEC protects against NK cell‐mediated cytotoxicity (7), HLA‐Cw4 reduced NK cell adhesion and cytotoxicity; HLA‐Cw4 and HLA‐Cw3 co‐expression is not sufficient to completely overcome NK cytotoxicity via recognition of the KIR CD158a (KIR2DL1) and CD158b (KIR2DL2/3) receptors (8). The aim of this study was to evaluate NK cell recruitment and infiltration of porcine tissues using an ex vivo perfusion system of HLA‐E transgenic pig legs with human blood. Over the past year, a pilot study has been set up in order to establish an experimental protocol that would allow for the study and the evaluation of the rejection mechanisms occurring during xenotransplantation and especially following reperfusion of pig organs with human blood (9). Amputated pig legs were perfused with heparinised human blood over a period of 12h. Blood samples were collected at several time points and blood cell populations were analyzed by flow cytometry (FACS) analysis. Two wild type and 2 HLA‐E/CD46 transgenic pig legs were perfused ex vivo. A strong diminution of NK cells was observed after 7 h of perfusion in both the wild type and the transgenic legs. However, this decrease occurred earlier in the wild type leg than in the transgenic. In addition, the apparition of a SLA‐I+ cells population of pig origin of higher granularity than the lymphocyte population over time was observed. We hypothesized that these cells could be pig endothelial cells detached from the vessel wall due to xenorejection, or alternatively originating from the pig bone marrow. These results confirmed previous in vitro studies demonstrating that pEC damage was mediated by human NK cells. Moreover, the expression of HLA‐E/CD46 provided a partial protection with regard to NK cell recruitment and tissue infiltration. From these preliminary experiments the following conclusions were drawn: Pig leg perfusion with human blood was feasible for up to 12 h with stable perfusion parameters thus extending the observation time compared to previously described pig kidney, lung and beating heart perfusion systems. pH and potassium were maintained at normal levels and muscle stimulation resulted in contraction throughout the entire perfusion. Thus the pig leg reperfusion system appears to be a stable and optimized system to study rejection mechanisms over several hours in the pig‐to‐human xenotransplantation setting. This system allows for the study of the dynamics of the different cell populations in the blood. Indeed, a strong diminution of NK, NKT and T cells was observed. In addition, this system enables to study the apparition of cells of pig origin in the circulating blood. In conclusion, this system represents a powerful tool to study the basic molecular mechanisms taking place in a setting of xenotransplantation, and in particular to evaluate the protection from early cell‐mediated rejection mechanisms in tissues originating from transgenic pigs. References: Forte P, Lilienfeld BG, Baumann BC, Seebach JD. Human NK cytotoxicity against porcine cells is triggered by NKp44 and NKG2D. J Immunol 2005; 175: 5463–5470. Lilienfeld BG, Crew MD, Forte Pet al.Transgenic expression of HLA‐E single chain trimer protects porcine endothelial cells against human natural killer cell‐mediated cytotoxicity. Xenotransplantation 2007; 14: 126–134. Weiss EH, Lilienfeld BG, Muller Set al. HLA‐E/human beta2‐microglobulin transgenic pigs: protection against xenogeneic human anti‐pig natural killer cell cytotoxicity. Transplantation 2009; 87: 35–43. Seebach JD, Pazmany L, Waneck GLet al. HLA‐G expression on porcine endothelial cells protects partially against direct human NK cytotoxicity but not against ADCC. Transplant Proc 1999; 31: 1864–1865. Forte P, Matter‐ Reissmann UB, Strasser Met al.Porcine aortic endothelial cells transfected with HLA‐G are partially protected from xenogeneic human NK cytotoxicity. Hum.Immunol 2000; 61:1066–1073. Forte P, Pazmany L, Matter‐ Reissmann UBet al. HLA‐G inhibits rolling adhesion of activated human NK cells on porcine endothelial cells. J Immunol. 2001; 167: 6002–6008. Seebach JD, Comrack C, Germana Set al. HLA‐Cw3 expression on porcine endothelial cells protects against xenogeneic cytotoxicity mediated by a subset of human NK cells. J Immunol 1997; 159: 3655–3661. Forte P, Baumann BC, Schneider MKet al. HLA‐Cw4 expression on porcine endothelial cells reduces cytotoxicity and adhesion mediated by CD158a+ human NK cells. Xenotransplantation 2009; 16: 19–26. Constantinescu MA, Knall E, Xu Xet al. Preservation of amputated extremities by extracorporeal blood perfusion; a feasibility study in a porcine model. J Surg.Res 2010.     

Human T‐cell proliferation in response to thrombin‐activated GTKO pig endothelial cells

Ezzelarab C, Ayares D, Cooper DKC, Ezzelarab MB. Human T‐cell proliferation in response to thrombin‐activated GTKO pig endothelial cells. Xenotransplantation 2012; 19: 311–316. © 2012 John Wiley & Sons A/S. Abstract: Background: Thrombin formation is a key feature in the activation of coagulation in pig xenograft recipients. As thrombin is known to activate endothelial and immune cells, we explored whether thrombin activation of pig endothelial cells (EC) was associated with an increased human T‐cell response. Methods: α1,3‐galactosyltransferase gene‐knockout (GTKO) pig aortic EC (pAEC) were activated by porcine interferon‐gamma (pIFNγ), human (h)IFN‐γ, or thrombin. Swine leukocyte antigen (SLA) class I and class II expression were measured. Human peripheral blood mononuclear cells (PBMC) and CD4⁺ T‐cell proliferation in response to activated pAEC, the effect of thrombin on pig CD80/CD86 mRNA, and the effect of thrombin inhibition by hirudin were evaluated. Results: After pAEC activation, SLA I expression did not change, and only pIFNγ upregulated SLA II expression. PBMC proliferation to pIFNγ‐ and thrombin‐activated pAEC was significantly higher (P < 0.001 and P < 0.01) than to non‐activated pAEC. CD4⁺ T‐cell proliferation to pIFNγ‐ and thrombin‐activated pAEC was significantly higher (P < 0.001 and P < 0.01) than to non‐activated pAEC. Thrombin inhibition by hirudin reduced thrombin‐induced upregulation of pAEC CD86 mRNA, and significantly reduced human PBMC proliferation to pAEC in comparison with thrombin alone (P < 0.05). Conclusions: Thrombin upregulates CD86 mRNA on pAEC, which is associated with increased human T‐cell proliferation against pAEC. Hirudin reduces CD86 mRNA in thrombin‐activated pAEC and is associated with downregulation of the human T‐cell proliferative response. The transplantation of organs from GTKO pigs transgenic for human thrombomodulin, and/or endothelial protein C receptor, in addition to therapeutic regulation of thrombin activation may reduce the cellular response to a pig xenograft and thus reduce the need for intensive immunosuppressive therapy.     

Production and characterization of transgenic pigs expressing porcine CTLA4‐Ig

Phelps CJ, Ball SF, Vaught TD, Vance AM, Mendicino M, Monahan JA, Walters AH, Wells KD, Dandro AS, Ramsoondar JJ, Cooper DKC, Ayares DL. Production and characterization of transgenic pigs expressing porcine CTLA4‐Ig.
Xenotransplantation 2009; 16: 477–485. © 2009 John Wiley & Sons A/S. Abstract: Background: Inhibition of the T‐cell‐mediated immune response is a necessary component of preventing rejection following xenotransplantation with pig α1,3‐galactosyltransferase gene‐knockout (GTKO) organs. Cytotoxic T lymphocyte‐associated antigen (CTLA4) is a co‐stimulatory molecule that inhibits T‐cell activity and may be useful in prolonging graft rejection. Methods: An expression vector was built containing the extracellular coding region of porcine (p) CTLA4 fused to the hinge and CH2/CH3 regions of human IgG1 (pCTLA4‐Ig). Pigs transgenic for pCTLA4‐Ig, on either a GTKO or wild‐type (WT) genetic background, were produced by nuclear transfer and characterized using Western blot analysis, immunofluorescence, ELISA, and necropsy. Results: Fifteen pCTLA4‐Ig‐transgenic piglets resulted from five pregnancies produced by nuclear transfer. All transgenic pigs exhibited robust expression of the pCTLA4‐Ig protein and most expressed the transgene in all organs analyzed, with significant levels in the blood as well. Despite initial good health, these pigs exhibited diminished humoral immunity, and were susceptible to infection, which could be managed for a limited time with antibiotics. Conclusions: Viable pigs exhibiting robust and ubiquitous expression of pCTLA4‐Ig were produced on both a WT and GTKO background. Expression of pCTLA4‐Ig resulted in acute susceptibility to opportunistic pathogens due at least in part to a significantly compromised humoral immune status. As this molecule is known to have immunosuppressive activity, high levels of pCTLA4‐Ig expression in the blood, as well as defective development related to exposure to pCTLA4‐Ig in utero, may contribute to this reduced immune status. Prophylactic treatment with antibiotics may promote survival of disease‐free transgenic pigs to a size optimal for organ procurement for transplantation. Additional genetic modifications and/or tightly regulated expression of pCTLA4Ig may reduce the impact of this transgene on the humoral immune system.