Hyperacute Xenograft Rejection Research Articles

Walter Brendel and the dawn of transplantation research in Germany.

Walter Brendel was a physiologist who headed the Institut of Experimental Surgery at the University of Munich (LMU) from 1961 until 1989. His legendary career began with the development of an anti-human lymphocyte globulin (ALG) at his Institute during the late 1960s. The initial successful treatment of a small number of patients culminated in the co-treatment of the first successfully heart-transplanted patient in Capetown, South Africa (successful reversal with ALG of an acute allograft rejection). Walter Brendel was a pioneering personality whose work has laid a wide platform for the promotion of interdisciplinarily conducted innovative research programs in various domains of translational science and medicine. Among the many innovative achievements, the most notable are: discovery of involvement of the alternative pathway of complement activation in hyperacute xenograft rejection; induction of immunological tolerance to horse IgG as a means to prevent anaphylactic reactions during ALG therapy; development and clinical implementation of the extracorporeal shock wave lithotripsy for extracorporeal destruction of renal and ureteral calculi. The legacy of Brendel continues with the foundation of the Walter-Brendel Kolleg für Transplantationsmedizin (i.e., the German Transplant School for Transplantation Medicine), which has been held annually since 1994.

Comparison of ICSI, IVF, and in vivo derived embryos to produce CRISPR-Cas9 gene-edited pigs for xenotransplantation

Genome editing in pigs for xenotransplantation has seen significant advances in recent years. This study compared three methodologies to generate gene-edited embryos, including co-injection of sperm together with the CRISPR-Cas9 system into oocytes, named ICSI-MGE (mediated gene editing); microinjection of CRISPR-Cas9 components into oocytes followed by in vitro fertilization (IVF), and microinjection of in vivo fertilized zygotes with the CRISPR-Cas9 system. Our goal was to knock-out (KO) porcine genes involved in the biosynthesis of xenoantigens responsible for the hyperacute rejection of interspecific xenografts, namely GGTA1, CMAH, and β4GalNT2. Additionally, we attempted to KO the growth hormone receptor (GHR) gene with the aim of limiting the growth of porcine organs to a size that is physiologically suitable for human transplantation. Embryo development, pregnancy, and gene editing rates were evaluated. We found an efficient mutation of the GGTA1 gene following ICSI-MGE, comparable to the results obtained through the microinjection of oocytes followed by IVF. ICSI-MGE also showed higher rates of biallelic mutations compared to the other techniques. Five healthy piglets were born from in vivo-derived embryos, all of them exhibiting biallelic mutations in the GGTA1 gene, with three displaying mutations in the GHR gene. No mutations were observed in the CMAH and β4GalNT2 genes. In conclusion, in vitro methodologies showed high rates of gene-edited embryos. Specifically, ICSI-MGE proved to be an efficient technique for obtaining homozygous biallelic mutated embryos. Lastly, only live births were obtained from in vivo-derived embryos showing efficient multiple gene editing for GGTA1 and GHR.

HLA-E and HLA-G Co-expression in Porcine Endothelial Cells Attenuates Human Natural Killer Cells degranulation

Background: Xenotransplantation offers a prospective solution to organ shortage. The elimination of major xenoantigens in pig cells prevents hyperacute xenograft rejection (HXR), driven by preformed antibodies. However, acute xenograft rejection (AXR), driven by immune cell activation,continues to be a barrier in pig-to-human xenotransplantation. Natural killer (NK) cells, with inhibitory and activating receptors, play unique roles in AXR by promoting graft rejection or tolerance. NK cell tolerance occurs naturally in utero where human leukocyte antigen (HLA)-E and HLA-G are present. Expressing HLA-E/G in xenografts may provide immune protection from human NK cell cytotoxicity.
 Methods: This study aims to demonstrate the use of HLA class I molecules in inducing human NK cell tolerance. 5-gene-knock-out (5GKO) porcine endothelial cells (pECs) were transfected with HLA-E and HLA-G genes. Transfected cells were stained with HLA-E and/or HLA-G antibody. HLA class I expressing 5GKO cells were isolated by flow sorter. Genetically modified pECs were co-cultured with human peripheral blood mononuclear cells (PBMCs) for E:T ratio of 10:1 for 2 hours. PBMCs were collected and stained with fluorochrome-conjugated antibodies. NK cell degranulation was accessed by the percentage of CD107a expression in the CD3-CD56+population. Co-localization of HLA-E and HLA-G on pECs was imaged by immunofluorescence microscopy and quantified by Image-Pro. 
 Results: 5GKO pEC lines expressing HLA-E, HLA-G, and co-expressing HLA-E and HLA-G were successfully established. Co-expression of HLA-E and HLA-G in 5GKO pECs reduced human NK cell degranulation by 50% compared to the 21% reduction achieved with 5GKO alone (p<0.0001). Co-localization intersection of 5GKO.HLA-E.HLA-G with stimulated PBMCs was not significantly different than when cultured with media (0.067 vs. 0.059, p=0.57).
 Conclusion and Impacts: Co-expression of HLA-E and HLA-G in 5GKO pECs significantly reduced human NK cell degranulation, compared to 5GKO.HLA-E, 5GKO.HLA-G, and 5GKO pECs. However, co-localization of HLA-E and HLA-G didn’t change when cultured with stimulated PBMCs. This study provides insight into the interactions between HLA molecules that promote an immunotolerant phenotype.

Strategies to induce natural killer cell tolerance in xenotransplantation.

Eliminating major xenoantigens in pig cells has drastically reduced human antibody-mediated hyperacute xenograft rejection (HXR). Despite these advancements, acute xenograft rejection (AXR) remains one of the major obstacles to clinical xenotransplantation, mediated by innate immune cells, including macrophages, neutrophils, and natural killer (NK) cells. NK cells play an ‘effector’ role by releasing cytotoxicity granules against xenogeneic cells and an ‘affecter’ role on other immune cells through cytokine secretion. We highlight the key receptor-ligand interactions that determine the NK cell response to target cells, focusing on the regulation of NK cell activating receptor (NKG2D, DNAM1) and inhibitory receptor (KIR2DL1-4, NKG2A, and LIR-1) signaling pathways. Inhibition of NK cell activity may protect xenografts from cytotoxicity. Recent successful approaches to reducing NK cell-mediated HXR and AXR are reviewed, including genetic modifications of porcine xenografts aimed at improving pig-to-human compatibility. Future directions to promote xenograft acceptance are discussed, including NK cell tolerance in pregnancy and NK cell evasion in viral infection.

Characterization of Mono- and Bi-Transgenic Pig-Derived Epidermal Keratinocytes Expressing Human FUT2 and GLA Genes-In Vitro Studies.

Pig-to-human xenotransplantation seems to be the response to the contemporary shortage of tissue/organ donors. Unfortunately, the phylogenetic distance between pig and human implies hyperacute xenograft rejection. In this study, we tested the hypothesis that combining expression of human α1,2-fucosyltransferase (hFUT2) and α-galactosidase A (hGLA) genes would allow for removal of this obstacle in porcine transgenic epidermal keratinocytes (PEKs). We sought to determine not only the expression profiles of recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) proteins, but also the relative abundance (RA) of Galα1→3Gal epitopes in the PEKs stemming from not only hFUT2 or hGLA single-transgenic and hFUT2×hGLA double-transgenic pigs. Our confocal microscopy and Western blotting analyses revealed that both rhα1,2-FT and rhα-Gal A enzymes were overabundantly expressed in respective transgenic PEK lines. Moreover, the semiquantitative levels of Galα1→3Gal epitope that were assessed by lectin fluorescence and lectin blotting were found to be significantly diminished in each variant of genetically modified PEK line as compared to those observed in the control nontransgenic PEKs. Notably, the bi-transgenic PEKs were characterized by significantly lessened (but still detectable) RAs of Galα1→3Gal epitopes as compared to those identified for both types of mono-transgenic PEK lines. Additionally, our current investigation showed that the coexpression of two protective transgenes gave rise to enhanced abrogation of Galα→3Gal epitopes in hFUT2×hGLA double-transgenic PEKs. To summarize, detailed estimation of semiquantitative profiles for human α-1,2-FT and α-Gal A proteins followed by identification of the extent of abrogating the abundance of Galα1→3Gal epitopes in the ex vivo expanded PEKs stemming from mono- and bi-transgenic pigs were found to be a sine qua non condition for efficiently ex situ protecting stable lines of skin-derived somatic cells inevitable in further studies. The latter is due to be focused on determining epigenomic reprogrammability of single- or double-transgenic cell nuclei inherited from adult cutaneous keratinocytes in porcine nuclear-transferred oocytes and corresponding cloned embryos. To our knowledge, this concept was shown to represent a completely new approach designed to generate and multiply genetically transformed pigs by somatic cell cloning for the needs of reconstructive medicine and dermoplasty-mediated tissue engineering of human integumentary system.

New Insights into Xenotransplantation for Cartilage Repair: Porcine Multi-Genetically Modified Chondrocytes as a Promising Cell Source.

Transplantation of xenogenic porcine chondrocytes could represent a future strategy for the treatment of human articular cartilage defects. Major obstacles are humoral and cellular rejection processes triggered by xenogenic epitopes like α-1,3-Gal and Neu5Gc. Besides knockout (KO) of genes responsible for the biosynthesis of respective epitopes (GGTA1 and CMAH), transgenic expression of human complement inhibitors and anti-apoptotic as well as anti-inflammatory factors (CD46, CD55, CD59, TNFAIP3 and HMOX1) could synergistically prevent hyperacute xenograft rejection. Therefore, chondrocytes from different strains of single- or multi-genetically modified pigs were characterized concerning their protection from xenogeneic complement activation. Articular chondrocytes were isolated from the knee joints of WT, GalTKO, GalT/CMAH-KO, human CD59/CD55//CD46/TNFAIP3/HMOX1-transgenic (TG), GalTKO/TG and GalT/CMAHKO/TG pigs. The tissue-specific effectiveness of the genetic modifications was tested on gene, protein and epitope expression level or by functional assays. After exposure to 20% and 40% normal human serum (NHS), deposition of C3b/iC3b/C3c and formation of the terminal complement complex (TCC, C5b-9) was quantified by specific cell ELISAs, and generation of the anaphylatoxin C5a by ELISA. Chondrocyte lysis was analyzed by Trypan Blue Exclusion Assay. In all respective KO variants, the absence of α -1,3-Gal and Neu5Gc epitope was verified by FACS analysis. In chondrocytes derived from TG animals, expression of CD55 and CD59 could be confirmed on gene and protein level, TNFAIP3 on gene expression level as well as by functional assays and CD46 only on gene expression level whereas transgenic HMOX1 expression was not evident. Complement activation in the presence of NHS indicated mainly effective although incomplete protection against C3b/iC3b/C3c deposition, C5a-generation and C5b-9 formation being lowest in single GalTKO. Chondrocyte viability under exposure to NHS was significantly improved even by single GalTKO and completely preserved by all other variants including TG chondrocytes without KO of xenoepitopes.

Generation of GGTA1-/-β2M-/-CIITA-/- Pigs Using CRISPR/Cas9 Technology to Alleviate Xenogeneic Immune Reactions.

Xenogeneic organ transplantation has been proposed as a potential approach to fundamentally solve organ shortage problem. Xenogeneic immune responses across species is one of the major obstacles for clinic application of xeno-organ transplantation. The generation of glycoprotein galactosyltransferase α 1, 3 (GGTA1) knockout pigs has greatly contributed to the reduction of hyperacute xenograft rejection. However, severe xenograft rejection can still be induced by xenoimmune responses to the porcine major histocompatibility complex antigens swine leukocyte antigen class I and class II. We simultaneously depleted GGTA1, β2-microglobulin (β2M), and major histocompatibility complex class II transactivator (CIITA) genes using clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins technology in Bamma pig fibroblast cells, which were further used to generate GGTA1β2MCIITA triple knockout (GBC-3KO) pigs by nuclear transfer. The genotype of GBC-3KO pigs was confirmed by polymerase chain reaction and Sanger sequencing, and the loss of expression of α-1,3-galactose, SLA-I, and SLA-II was demonstrated by flow cytometric analysis using fluorescent-conjugated lectin from bandeiraea simplicifolia, anti-β2-microglobulin, and swine leukocyte antigen class II DR antibodies. Furthermore, mixed lymphocyte reaction assay revealed that peripheral blood mononuclear cells from GBC-3KO pigs were significantly less effective than (WT) pig peripheral blood mononuclear cells in inducing human CD3CD4 and CD3CD8 T-cell activation and proliferation. In addition, GBC-3KO pig skin grafts showed a significantly prolonged survival in immunocompetent C57BL/6 mice, when compared with wild-type pig skin grafts. Taken together, these results demonstrate that elimination of GGTA1, β2M, and CIITA genes in pigs can effectively alleviate xenogeneic immune responses and prolong pig organ survival in xenogenesis. We believe that this work will facilitate future research in xenotransplantation.

Xenotransplantation: Current Status in Preclinical Research.

The increasing life expectancy of humans has led to a growing numbers of patients with chronic diseases and end-stage organ failure. Transplantation is an effective approach for the treatment of end-stage organ failure; however, the imbalance between organ supply and the demand for human organs is a bottleneck for clinical transplantation. Therefore, xenotransplantation might be a promising alternative approach to bridge the gap between the supply and demand of organs, tissues, and cells; however, immunological barriers are limiting factors in clinical xenotransplantation. Thanks to advances in gene-editing tools and immunosuppressive therapy as well as the prolonged xenograft survival time in pig-to-non-human primate models, clinical xenotransplantation has become more viable. In this review, we focus on the evolution and current status of xenotransplantation research, including our current understanding of the immunological mechanisms involved in xenograft rejection, genetically modified pigs used for xenotransplantation, and progress that has been made in developing pig-to-pig-to-non-human primate models. Three main types of rejection can occur after xenotransplantation, which we discuss in detail: (1) hyperacute xenograft rejection, (2) acute humoral xenograft rejection, and (3) acute cellular rejection. Furthermore, in studies on immunological rejection, genetically modified pigs have been generated to bridge cross-species molecular incompatibilities; in the last decade, most advances made in the field of xenotransplantation have resulted from the production of genetically engineered pigs; accordingly, we summarize the genetically modified pigs that are currently available for xenotransplantation. Next, we summarize the longest survival time of solid organs in preclinical models in recent years, including heart, liver, kidney, and lung xenotransplantation. Overall, we conclude that recent achievements and the accumulation of experience in xenotransplantation mean that the first-in-human clinical trial could be possible in the near future. Furthermore, we hope that xenotransplantation and various approaches will be able to collectively solve the problem of human organ shortage.

Generation of GGTA1 <sup>-/-</sup>β2M <sup>-/-</sup>CIITA <sup>-/-</sup> Pigs Using CRISPR/Cas9 Technology to Alleviate Xenogeneic Immune Reactions

Background: The generation of glycoprotein galactosyltransferase alpha 1, 3 (GGTA1) knockout pigs has greatly contributed to the reduction of hyperacute xenograft rejection. However, severe xenograft rejection can still be induced by xenoimmune responses to the porcine major histocompatibility complex (MHC) antigens SLA-I and SLA-II. Methods: We simultaneously depleted GGTA1, β 2M, and CIITA genes using CRISPR-Cas9 technology to generate GGTA1-/-β2M-/-CIITA-/- triple knockout (GBC-3KO) pigs. Mixed lymphocyte reaction (MLR) and BrdU incorporating assays were utilized to quantify the potency of pig cells to stimulate human T cell responses. Skin grafts were determined the immunogenicity of pig tissues in xenogeneic hosts in vivo. Findings: The genotype of GBC-3KO pigs was confirmed by PCR and Sanger sequencing, and the loss of expression of α-Gal, SLA-I, and SLA-II was demonstrated by flow cytometric analysis. Mixed lymphocyte reaction (MLR) assay revealed that splenocytes from GBC-3KO pigs were significantly less effective than WT pig splenocytes in inducing human CD3+CD4+ and CD3+CD8+ T cell activation (measured by CD25 expression) and proliferation. Furthermore, GBC-3KO pig skin grafts showed a significantly prolonged survival (with a median survival time (MST) of 16 days) in immunocompetent C57BL/6 mice, when compared to WT pig skin grafts (MST = 13.5 days). Interpretation: These results demonstrate that elimination of GGTA1, β2M, and CIITA genes in pigs can effectively alleviate xenogeneic immune responses. Funding Statement: This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030000 and XDA16030303), the National Key Research and Development Program (2017YFA0104400, 2016YFA0100202 and 2017YFA0104402), the National Natural Science Foundation of China (31621004, 81570145, 81870091), and the Key Research Projects of the Frontier Science of the Chinese Academy of Sciences (QYZDB-SSW-SMC022). Declaration of Interests: The authors stated: None. Ethics Approval Statement: Bama miniature pigs were raised and cloned at the Beijing Farm Animal Research Center. Human blood was obtained from healthy volunteers. Protocols related to the use of animals and human tissues were approved by the Institutional Review Board and Institutional Animal Care and Use Committee of the First Hospital of Jilin University and the Institute of Zoology of Chinese Academy of Sciences. All the experiments were performed in accordance with the protocols.

What's Hot, What's New From the 2016 American Transplant Congress.

From June 11-15, 2016 the American Transplant Congress, the joint meeting of the American Society of Transplantation and the American Society of Transplant Surgeons, held its annual meeting in Boston, MA. The meeting, attended by 5200 registrants, included pre-meeting conferences, focused topic sessions, and hundreds of high-quality presentations from the transplant field. This meeting report highlights key findings from specific basic science, translational, and clinical research presentations deemed to have notable impact in thematic areas. In particular, there were a number of transformative studies indicating important advances in the understanding of alloimmunity, chronic rejection, tolerance, and organ-specific outcomes. Many of these results are discussed in the context of the published literature to showcase rapid advances in the transplant field.

Abstract 4862: AGI-134: a fully synthetic alpha-Gal glycolipid that prevents the development of distal lesions and is synergistic with an anti-PD-1 antibody in a mouse melanoma model

Abstract Background: AGI-134 is a fully synthetic glycolipid, composed of an alpha-Gal (Galá1-3Galâ1-4GlcNAc-R) sugar epitope attached via a linker to a lipid tail. Natural antibodies to the alpha-Gal epitope are responsible for the hyperacute rejection of xenografts in humans. It is proposed that intratumorally administered AGI-134 will incorporate into the cell membranes of the tumor cells, presenting the alpha-Gal epitope for binding of anti-Gal antibodies to the tumor cells. This will initiate an immune response that attacks the injected tumor and, through uptake of immune-complexed tumor antigens by antigen presenting cells, will create a patient-specific, systemic anti-tumor response against distant metastases. Results: We demonstrate that AGI-134 incorporates into tumor cell membranes in vitro and that the exposed alpha-Gal epitope binds anti-Gal IgG and IgM antibodies from human serum to the tumor cell surface. Using flow cytometry and a complement-dependent cytotoxicity assay we show that tumor cell opsonization with anti-Gal antibodies leads to deposition of complement proteins C3b and C5b-9, which ultimately leads to tumor cell lysis. Furthermore, we demonstrate that AGI-134-labeled tumor cells opsonized with human serum proteins are phagocytosed by professional APCs. Using the B16-F10 melanoma model in anti-Gal producing á1,3-galactosyltransferase knockout (GT KO) mice we present data to demonstrate that AGI-134 injection into a primary tumor provides significant dose-dependent protection from the development of established distant lesions. Using GT KO mouse serum we demonstrate in vitro that deposition of complement on AGI-134-labeled mouse tumor cells is both alpha-Gal and anti-Gal dependent. In vivo, we demonstrate that the effect of AGI-134 is due to the alpha-Gal moiety by replacing it with human blood group antigens. The protection from secondary lesions conferred by AGI-134 is long lasting in the GT KO mouse melanoma model (monitored up to 90 days). Importantly, when sub-optimal concentrations of AGI-134 were tested in vivo in combination with an anti-PD-1 antibody (RMP1-14), a significant enhancement in efficacy over either of the agents administered alone was observed. Citation Format: Stephen Shaw, Sascha Kristian, Kim Wigglesworth, Jenny Middleton, Mel Glossop, Giles Whalen, Robert Old, Mike Westby, Chris Pickford. AGI-134: a fully synthetic alpha-Gal glycolipid that prevents the development of distal lesions and is synergistic with an anti-PD-1 antibody in a mouse melanoma model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4862.

Double Transgenic Pigs with Combined Expression of Human α1,2-Fucosyltransferase and α-Galactosidase Designed to Avoid Hyperacute Xenograft Rejection

Hyperacute rejection (HAR) depends on the response of xenoreactive antibodies principally against porcine α-Gal epitope. Methods eliminating HAR include GGTA1 inactivation, regulation of the complement system and modification of the oligosaccharide structure of surface proteins in donor’s cells. Transgenic animals designed for the purpose of xenotransplantation with single modification do not display full reduction of the α-Gal epitope level, which means that a accumulation of several modifications in one transgenic individual is needed. The aim of the study was to create a molecular and cytogenetic profile of a double transgenic animal with α1,2-fucosyltransferase and α-galactosidase expression. As a result of interbreeding of an individual with α1,2-fucosyltransferase expression with an individual with α-galactosidase expression 12 living piglets were obtained. PCR revealed the pCMVFUT gene construct was present in four individuals and pGAL-GFPBsd in three, including one with a confirmed integration of both the gene constructs. Fluorescence in situ hybridization confirmed the site of transgene integration, which corresponded to the mapping site of the transgenes which occurred in the parental generations. Karyotype analysis did not show any changes in the structure or the number of chromosomes (2n = 38, XX). As for the results pertaining to the single transgenic individuals, expression analysis demonstrated a high extent of α-Gal epitope level reduction on the surface of cells, whereas human serum cytotoxicity tests revealed the smallest decrease in longevity of cells in the obtained double transgenic individual (4.35 %). The tests suggest that the co-expression of both the transgenes leads to a considerable reduction of the α-Gal antigen level on the surface of cells and a decrease of xenotransplant immunogenicity.

Development of a Convenient Competitive ELISA for the Detection of the Free and Protein-Bound Nonhuman Galactosyl-α-(1,3)-Galactose Epitope Based on Highly Specific Chicken Single-Chain Antibody Variable-Region Fragments

The presence of the nonhuman galactosyl-α-(1,3)-galactose (Gal-α-(1,3)-Gal) carbohydrate epitope on a number of recombinant therapeutic proteins has recently been reported, renewing interest in this immunogenic carbohydrate epitope. It is well-known that this motif is the primary contributing factor in hyperacute rejection of porcine organ xenograft, due to the existence of natural antibodies against this epitope in human serum. Though the number of epitopes on recombinant glycoproteins may be low when compared directly to whole tissue, circulating anti-Gal-α-R immunoglobulins can still induce anaphylaxis. Therefore, there is a need for rapid and convenient methods for detection and monitoring of this epitope in biopharmaceuticals produced in recombinant mammalian systems. To this end, we have generated immune-challenged chicken single-chain antibody variable-region fragment (scFv) libraries targeting the Gal-α-(1,3)-Gal motif and have selected a panel of scFv's that bind the target. We have used one of these antibodies to develop a competitive ELISA for both free and protein-bound Gal-α-(1,3)-Gal and have demonstrated that the ELISA is specific for the target and can be used to determine the loading of the target on glycoproteins. This competitive ELISA will provide a convenient method of detecting and quantifying Gal-α-(1,3)-Gal on therapeutic glycoproteins.

The Role of Macrophages in Transplant Rejection

Macrophage accumulation has been recognized as a feature of allograft rejection, however, the role of macrophages in rejection remains underappreciated. Macrophages are present within graft tissues throughout the lifespan of the graft, including acute rejection episodes. Recent advances in macrophage biology have demonstrated that different types of macrophages in grafts serve a range of functions, including promotion or attenuation of inflammation, participation in innate and adaptive immune responses, and mediation of tissue injury, fibrosis, and tissue repair. Macrophages contribute to both the innate and acquired arms of the alloimmune response, and, thus, may be involved in all aspects of acute and chronic allograft rejection. Macrophages are also involved in hyperacute and acute vascular rejection of xenografts. A deeper understanding of how macrophages accumulate within grafts and of the factors that control differentiation and function of these cells could lead to identification of novel therapeutic targets in transplantation.

T‐cell‐mediated immunological barriers to xenotransplantation

Xenotransplantion remains the most viable option for significant expansion of the donor organ pool in clinical transplantation. With the advent of nuclear transfer technologies, the production of transgenic swine has become a possibility. These animals have allowed transplant investigators to overcome humoral mechanisms of hyperacute xenograft rejection in experimental pig-to-non-human primate models. However, other immunologic barriers preclude long-term acceptance of xenografts. This review article focuses on a major feature of xenogeneic rejection: xenogeneic T cell responses. Evidence obtained from both small and large animal models, particularly those using either islet cells or kidneys, have demonstrated that T cell responses play a major role in xenogeneic rejection, and that immunosuppression alone is likely incapable of completely suppressing these responses. Additionally, both the direct and indirect pathway of antigen presentation appear to be involved in these anti donor processes. Enhanced understanding of (i) CD47 and its role in transduced xeno-bone marrow (ii) CD39 and its role in coagulation dysregulation and (iii) thymic transplantation have provided us with encouraging results. Presently, experiments evaluating the possibility of xenogeneic tolerance are underway.

Human Immune Reactivity against Liver Sinusoidal Endothelial Cells from GalTα(1,3)GalT-Deficient Pigs

Elimination of galactose-α(1,3)galactose (Gal) expression in pig organs has been previously shown to prevent hyperacute xenograft rejection. However, naturally present antibodies to non-Gal epitopes activate endothelial cells, leading to acute humoral xenograft rejection. Still, it is unknown whether xenogeneic pig liver sinusoidal endothelial cells (LSECs) from α(1,3)galactosyltransferase (GalT)-deficient pigs are damaged by antibody and complement-mediated mechanisms. The present study examined the xeno-antibody response of LSECs from GalT-deficient and wild pigs. Isolated LSEC from wild-type and GalT pigs were expose to human and baboon sera; IgM and IgG binding was analyzed by flow cytometry. Complement activation (C3a and CH50) was quantified in vitro from serum-exposed LSEC cultures using Enzyme-Linked ImmunoSorbent assay (ELISA). Levels of complement-activated cytotoxicity (CAC) were also determined by a fluorescent Live-Dead Assay and by the quantification of LDH release. IgM binding to GalT knockout (KO) LSECs was significantly lower (80% human and 87% baboon) compare to wild-type pig LSEC. IgG binding was low in all groups. Moreover, complement activation (C3a and CH50) levels released following exposure to human or baboon sera were importantly reduced (42% human and 52% baboon), CAC in GalT KO LSECs was reduced by 60% in human serum and by 72% in baboon serum when compared to wild-type LSECs, and LDH release levels were reduced by 37% and 57%, respectively. LSECs from GalT KO pigs exhibit a significant protection to humoral-induced cell damage compared to LSECs from wild pigs when exposed to human serum. Although insufficient to inhibit xenogeneic reactivity completely, transgenic GalT KO expression on pig livers might contribute to a successful application of clinical xenotransplantation in combination with other protective strategies.

Research of xenograft hyperacute rejection by perfusing pig kidneys with human blood in vitro

Objective Ex vivo studies by perfusing pig kidneys with differents blood to evaluate human antiporcine xenograft hyperacute rejection. Methods Pig kidneys were perfused for 30 minutes with pig blood (group 1; n =3),human blood (group 2; n =5),complement heat innactivated human blood (group 3; n =5), and xenoreactive natural antibodies (XNA) -depleted human blood (group 4; n=5). Renal blood flow rate and vascular resistance were caculated very 5 minutes. Histologic and immunohistologic examinations were carried out after perfusion. Results Pig kidneys perfused with human blood, group 2, showed HAR, higher vascular resistance and lower blood flow rate. Kidneys in Group 1,3,4 had no histologic evidence of HAR. Blood flow rate and vasular resistance kept steady. Conclusion The depletion of complement or XNA in human blood protected pig kidneys form HAR. Key words: Xenotransplant; Rejection; Kidney

Can hypothermia and cardioplegic solutions prevent hyperacute xenograft rejection by influencing the α-Gal epitopes on porcine aortic endothelial cells?

Can hypothermia and cardioplegic solutions prevent hyperacute xenograft rejection by influencing the α-Gal epitopes on porcine aortic endothelial cells?

In silico analysis of antibody–carbohydrate interactions and its application to xenoreactive antibodies

Antibody–carbohydrate interactions play central roles in stimulating adverse immune reactions. The most familiar example of such a process is the reaction observed in ABO-incompatible blood transfusion and organ transplantation. The ABO blood groups are defined by the presence of specific carbohydrates expressed on the surface of red blood cells. Preformed antibodies in the incompatible recipient (i.e., different blood groups) recognize cells exhibiting host-incompatible ABO system antigens and proceed to initiate lysis of the incompatible cells. Pig-to-human xenotransplantation presents a similar immunological barrier. Antibodies present in humans recognize carbohydrate antigens on the surface of pig organs as foreign and proceed to initiate hyperacute xenograft rejection. The major carbohydrate xenoantigens all bear terminal Galα(1,3)Gal epitopes (or αGal). In this study, we have developed and validated a site mapping technique to investigate protein–ligand recognition and applied it to antibody–carbohydrate systems. This site mapping technique involves the use of molecular docking to generate a series of antibody–carbohydrate complexes, followed by analysis of the hydrogen bonding and van der Waals interactions occurring in each complex. The technique was validated by application to a series of antibody–carbohydrate crystal structures. In each case, the majority of interactions made in the crystal structure complex were able to be reproduced. The technique was then applied to investigate xenoantigen recognition by a panel of monoclonal anti-αGal antibodies. The results indicate that there is a significant overlap of the antibody regions engaging the xenoantigens across the panel. Likewise, similar regions of the xenoantigens interact with the antibodies.

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 (Treg) 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).