Human PD-L1 overexpression decreases xenogeneic human T-cell immune responses towards porcine kidneys.

Xenotransplantation offers a promising alternative to circumvent the lack of donated human organs available for transplantation. Different attempts to improve the survival of xenografts led to the generation of transgenic pigs expressing various combinations of human protective genes or knocked out for specific antigens. Currently, testing the efficiency of porcine organs carrying different genetic modifications in preventing xenogeneic immune responses completely relies on assays, humanized mouse models, or non-human primate transplantation models.

Current Status of Cardiac Xenotransplantation: Report of a Workshop of the German Heart Transplant Centers, Martinsried, March 3, 2023.

This report comprises the contents of the presentations and following discussions of a workshop of the German Heart Transplant Centers in Martinsried, Germany on cardiac xenotransplantation. The production and current availability of genetically modified donor pigs, preservation techniques during organ harvesting, and immunosuppressive regimens in the recipient are described. Selection criteria for suitable patients and possible solutions to the problem of overgrowth of the xenotransplant are discussed.

Unique 40-year survival after heart transplantation with normal graft function and spontaneous operational tolerance.

Unique 40-year survival after heart transplantation with normal graft function and spontaneous operational tolerance.

Expression of viral CD45 ligand E3/49K on porcine cells reduces human anti-pig immune responses.

Transgenic expression of protective molecules in porcine cells and tissues is a promising approach to prevent xenograft rejection. Viruses have developed various strategies to escape the host's immune system. We generated porcine B cells (B cell line L23) expressing the human adenovirus protein E3/49K or the human cytomegalovirus protein pUL11 and investigated how human T, NK and B cell responses are affected by the expression of the viral proteins.

Neonatal islets from human PD-L1 transgenic pigs reduce immune cell activation and cellular rejection in humanized nonobese diabetic-scid IL2rγ mice.

Strong xenorejection limits the clinical application of porcine islet transplantation in type 1 diabetes. Targeting T cell-mediated rejection is one of the main approaches to improve long-term graft survival. Here we study engraftment and survival of porcine islet cells expressing human programmed cell death ligand-1 (hPD-L1) in a humanized mouse model.

Downregulation of Swine Leukocyte Antigen Expression Decreases the Strength of Xenogeneic Immune Responses towards Renal Proximal Tubular Epithelial Cells.

Xenotransplantation reemerged as a promising alternative to conventional transplantation enlarging the available organ pool. However, success of xenotransplantation depends on the design and selection of specific genetic modifications and on the development of robust assays allowing for a precise assessment of tissue-specific immune responses. Nevertheless, cell-based assays are often compromised by low proliferative capacity of primary cells.

Pathways to Clinical Cardiac Xenotransplantation.

Heart transplantation is the only long-lasting lifesaving option for patients with terminal cardiac failure. The number of available human organs is however far below the actual need, resulting in substantial mortality of patients while waiting for a human heart. Mechanical assist devices are used to support cardiac function but are associated with a high risk of severe complications and poor quality of life for the patients.

Generating low immunogenic pig pancreatic islet cell clusters for xenotransplantation.

Xenotransplantation of pancreatic islets offers a promising alternative to overcome the shortage of allogeneic donors. Despite significant advances, either immune rejection or oxygen supply in immune protected encapsulated islets remains major bottlenecks for clinical application. To decrease xenogeneic immune responses, we generated tissue engineered swine leucocyte antigen (SLA)-silenced islet cell clusters (ICC).

Triple (GGTA1, CMAH, B2M) modified pigs expressing an SLA class I phenotype-Effects on immune status and susceptibility to human immune responses.

Porcine xenografts lacking swine leukocyte antigen (SLA) class I are thought to be protected from human T cell responses. We have previously shown that SLA class I deficiency can be achieved in pigs by CRISPR/Cas9-mediated deletion of β -microglobulin (B2M). Here, we characterized another line of genetically modified pigs in which targeting of the B2M locus did not result in complete absence of B2M and SLA class I but rather in significantly reduced expression levels of both molecules.

Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2.

Background: Cell surface carbohydrate antigens play a major role in the rejection of porcine xenografts. The most important for human recipients are α-1,3 Gal (Galactose-alpha-1,3-galactose) causing hyperacute rejection, also Neu5Gc (N-glycolylneuraminic acid) and Sd(a) blood group antigens both of which are likely to elicit acute vascular rejection given the known human immune status. Porcine cells with knockouts of the three genes responsible, GGTA1, CMAH and B4GALNT2, revealed minimal xenoreactive antibody binding after incubation with human serum.

Possible detrimental effects of beta-2-microglobulin knockout in pigs.

Background: Despite major improvements in pig-to-primate xenotransplantation, long-term survival of xenografts is still challenging. The major histocompatibility complex (MHC) class I, which is crucial in cellular immune response, is an important xenoantigen. Abrogating MHC class I expression on xenografts might be beneficial for extending graft survival beyond current limits.

Role of human and porcine MHC DRB1 alleles in determining the intensity of individual human anti-pig T-cell responses.

Background: Differences in quality and strength of immune responses between individuals are mainly due to polymorphisms in major histocompatibility complex (MHC) molecules. Focusing on MHC class-II, we asked whether the intensity of human anti-pig T-cell responses is influenced by genetic variability in the human HLA-DRB1 and/or the porcine SLA-DRB1 locus.

Methods: ELISpot assays were performed using peripheral blood mononuclear cells (PBMCs) from 62 HLA-DRB1-typed blood donors as responder and the porcine B cell line L23 as stimulator cells.

Transient increase of activated regulatory T cells early after kidney transplantation.

Regulatory T cells (Tregs) are crucial in controlling allospecific immune responses. However, studies in human kidney recipients regarding the contribution of polyspecific Tregs have provided differing results and studies on alloreactive Tregs are missing completely. In this retrospective study, we specifically analyzed activated CD4CD25FOXP3GARP Tregs in 17 patients of a living donor kidney transplantation cohort longitudinally over 24 months by flow cytometry (FOXP3: forkhead box protein 3, GARP: glycoprotein A repetitions predominant).

Pigs expressing the human inhibitory ligand PD-L1 (CD 274) provide a new source of xenogeneic cells and tissues with low immunogenic properties.

Background: The programmed cell death-1 (PD-1, CD279)/PD-Ligand1 (PD-L1, CD274) receptor system is crucial for controlling the balance between immune activation and induction of tolerance via generation of inhibitory signals. Expression of PD-L1 is associated with reduced immunogenicity and renders cells and tissues to an immune-privileged/tolerogenic state.

Methods: To apply this concept for clinical xenotransplantation, we generated human (h)PD-L1 transgenic pigs and characterized expression and biological function of the transgene at the cellular level.

Strong xenoprotective function by single-copy transgenes placed sequentially at a permissive locus.

Background: Multiple xenoprotective transgenes are best grouped at a single locus to avoid segregation during breeding and simplify production of donor animals.

Methods: We used transgene stacking to place a human CD55 transgene adjacent to a human heme oxygenase 1 construct at the porcine ROSA26 locus. A transgenic pig was analyzed by PCR, RT-PCR, droplet digital PCR, immunohistochemistry, immunofluorescence, and flow cytometry.

Inhibition of B-cell activation and antibody production by triggering inhibitory signals via the PD-1/PD-ligand pathway.

Background: The development of donor-reactive antibodies is regarded to be an important barrier limiting long-term outcome of allo- and xenografts. We asked whether enhanced signaling via the co-inhibitory receptor programmed cell death-1 (PD-1; CD279) can downregulate human B-cell activation.

Methods: Proliferation of human purified CD19(+) B cells was induced by in vitro stimulation with CpG oligodeoxynucleotides (CpG-B).

Kidneys From α1,3-Galactosyltransferase Knockout/Human Heme Oxygenase-1/Human A20 Transgenic Pigs Are Protected From Rejection During Ex Vivo Perfusion With Human Blood.

Unlabelled: Multiple modifications of the porcine genome are required to prevent rejection after pig-to-primate xenotransplantation. Here, we produced pigs with a knockout of the α1,3-galactosyltransferase gene (GGTA1-KO) combined with transgenic expression of the human anti-apoptotic/anti-inflammatory molecules heme oxygenase-1 and A20, and investigated their xenoprotective properties.

Methods: The GGTA1-KO/human heme oxygenase-1 (hHO-1)/human A20 (hA20) transgenic pigs were produced in a stepwise approach using zinc finger nuclease vectors targeting the GGTA1 gene and a Sleeping Beauty vector coding for hA20.

Efficient production of multi-modified pigs for xenotransplantation by 'combineering', gene stacking and gene editing.

Xenotransplantation from pigs could alleviate the shortage of human tissues and organs for transplantation. Means have been identified to overcome hyperacute rejection and acute vascular rejection mechanisms mounted by the recipient. The challenge is to combine multiple genetic modifications to enable normal animal breeding and meet the demand for transplants.

Ubiquitous LEA29Y Expression Blocks T Cell Co-Stimulation but Permits Sexual Reproduction in Genetically Modified Pigs.

We have successfully established and characterized a genetically modified pig line with ubiquitous expression of LEA29Y, a human CTLA4-Ig derivate. LEA29Y binds human B7.1/CD80 and B7.

The c.503T>C Polymorphism in the Human KLRB1 Gene Alters Ligand Binding and Inhibitory Potential of CD161 Molecules.

Studying genetic diversity of immunologically relevant molecules can improve our knowledge on their functional spectrum in normal immune responses and may also uncover a possible role of different variants in diseases. We characterized the c.503T>C polymorphism in the human KLRB1 gene (Killer cell lectin-like receptor, subfamily B, member 1) coding for the cell surface receptor CD161.

Pig to rat cell transplantation: reduced cellular and antibody responses to xenografts overexpressing PD-L1.

Background: Programmed death-1 (PD-1) costimulation acts as a negative regulator of T-cell responses to allografts. However, the role of the PD-1 pathway in xenotransplantation is not well defined yet. We have shown previously that human in vitro T-cell responses to porcine transfectants overexpressing PD-Ligand1 (L23-PD-L1 cells) are remarkably weak.

Pre-transplant immune state defined by serum markers and alloreactivity predicts acute rejection after living donor kidney transplantation.

Acute rejection (AR) remains a major cause for long-term kidney allograft failure. Reliable immunological parameters suitable to define the pre-transplant immune state and hence the individual risk of graft rejection are highly desired to preferably adapt the immunosuppressive regimen in advance. Donor and third party alloreactivities were determined by mixed lymphocyte cultures.

Regulatory sequences of the porcine THBD gene facilitate endothelial-specific expression of bioactive human thrombomodulin in single- and multitransgenic pigs.

Background: Among other mismatches between human and pig, incompatibilities in the blood coagulation systems hamper the xenotransplantation of vascularized organs. The provision of the porcine endothelium with human thrombomodulin (hTM) is hypothesized to overcome the impaired activation of protein C by a heterodimer consisting of human thrombin and porcine TM.

Methods: We evaluated regulatory regions of the THBD gene, optimized vectors for transgene expression, and generated hTM expressing pigs by somatic cell nuclear transfer.