Reinhard Schwinzer

Porcine endogenous retroviruses (PERVs), their transmembrane envelope protein p15E and peptides corresponding to a domain in p15E that is highly conserved among retroviruses, the immunosuppressive (isu) domain, demonstrated immunosuppressive properties. They inhibited in vitro immune reactions, and induced release of IL-6 and IL-10 in human peripheral blood mononuclear cells (PBMCs). We recently showed that p15E of PERV expressed on 293T cells reduced MHC expression, induced cytokine release in co-incubated human PBMCs, and inhibited cytotoxic cells. The cell-surface expression of p15E was chosen to simulate an artificially introduced expression on a transplant. Here, a new construct with a higher expression of p15E and consequently higher effects on cytokine release was designed and used. An intracellular cytokine assay was newly developed and applied, whereas a commercial cytokine array was used to analyze the release of 105 cytokines into the supernatant. The differential gene expression was analyzed by sequencing the RNA of PBMCs incubated with p15E-expressing and wild-type 293T cells. PERV p15E induced an elevated expression of IL-10, IL-6 and 24 other cytokines and modulated the expression of hundreds of genes. Furthermore, coating porcine L23 cells with the synthetic isu peptide of the PERV p15E protein and subsequently co-incubating them with human PBMCs induced IL-10 production, whereas direct addition of the peptide to PBMCs alone did not induce cytokine production. These results demonstrate that the PERV p15E protein can modulate cytokine release by human immune cells and suppress their cytotoxic activity. This immunomodulatory property may have potential applications in protecting transplanted tissues from immune-mediated rejection.

Porcine endogenous retroviruses (PERVs), their transmembrane envelope protein p15E and peptides corresponding to a highly conserved among all retroviruses domain in p15E, the immunosuppressive (isu) domain, demonstrated immunosuppressive properties. They inhibited in vitro immune reactions, and induced release of IL-6 and IL-10 in human peripheral blood mononuclear cells (PBMCs). We recently showed that p15E of PERV expressed on 293T cells reduced the MHC expression and induced cytokine release in co-incubated human PBMCs. Furthermore, expression of p15E inhibited cytotoxic cells. Here, a new construct with a higher expression of p15E and consequently higher effects on cytokine release was designed and used. Furthermore, a newly developed cytokine assay measuring intracellular cytokine production and a cytokine array to analyze the release of 105 cytokines into the supernatant was developed. The differential gene expression was analyzed by sequencing the RNA of PBMCs incubated with p15E-expressing and wild-type 293T cells. PERV p15E induced an elevated expression of IL-10, IL-6 and 24 other cytokines and modulated the expression of hundreds of genes. Furthermore, coating porcine L23 cells with the synthetic isu peptide of the PERV p15E protein and subsequently co-incubating them with human PBMCs induced IL-10 production, whereas direct addition of the peptide to PBMCs alone did not induce cytokine production. These results indicate that the PERV p15E protein is capable of modulating cytokine release by human PBMCs and inhibiting their cytotoxic activity.

Retroviruses are immunosuppressive, and there is evidence that a highly conserved immunosuppressive domain (isu domain) in their transmembrane envelope protein contributes to this activity. Studies have shown that inactivated retroviruses, their purified transmembrane envelope proteins, and synthetic peptides corresponding to the isu domain inhibit mitogen-triggered proliferation of peripheral blood mononuclear cells (PBMCs) and modulate their cytokine and gene expression. This has been demonstrated for human immunodeficiency virus type 1 (HIV-1), as well as for beta- and gammaretroviruses and for both exogenous and endogenous retroviruses, including syncytins. In the case of HIV-1, homopolymers of its isu peptide stimulated an increased release of IL-10, IL-6, and other cytokines from human PBMCs. Up-regulated genes included IL-6, IL-8, and IL-10, as well as MMP-1, TREM-1, and IL-1β. In vivo, in a mouse tumor model, tumor cells that were unable to induce tumors in immunocompetent animals gained the ability to do so when expressing the transmembrane envelope protein or the isu domain of various retroviruses on their surface. Here, we demonstrate that the transmembrane envelope protein p15E of PERV can modulate cytokine expression in human PBMCs. Human 293 cells were transfected with four constructs that express a portion of p15E, including the isu domain, and were cultured in the presence of a selection medium containing hygromycin. The p15E-expressing cells were co-cultured with human PBMCs, leading to the release of IL-6 and IL-10 protein and the modulation of multiple cytokines and other markers, including IL-6, IL-10, IFN-α, TNF-α, MMP1, and SEPP1. Similar, but more pronounced, effects were observed when PERV-producing 293 and pig cells were used in parallel; both expressed higher levels of p15E. Additionally, p15E expression reduced MHC class I expression, and preliminary data indicate that p15E expression could have a protective effect against cellular cytotoxicity. This finding underscores the need for further research to elucidate the dynamics of p15E expression and its immunosuppressive activity. It also contributes to the understanding of the immunosuppressive properties of pathogenic retroviruses. Furthermore, expressing the immunosuppressive p15E of PERV on the surface of a pig xenotransplant may reduce the need for pharmaceutical immunosuppressants.

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. Proximal tubular epithelial cells (PTECs) play a crucial role in kidney function. Here, we generated immortalized PTECs (imPTECs) by overexpression of simian virus 40 T large antigen. ImPTECs not only showed typical morphology and phenotype, but, in contrast to primary PTECs, they maintained steady cell cycling rates and functionality. Furthermore, swine leukocyte antigen (SLA) class I and class II transcript levels were reduced by up to 85% after transduction with lentiviral vectors encoding for short hairpin RNAs targeting β2-microglobulin and the class II transactivator. This contributed to reducing xenogeneic T-cell cytotoxicity (p < 0.01) and decreasing secretion of pro-inflammatory cytokines such as IL-6 and IFN-γ. This study showed the feasibility of generating highly proliferative PTECs and the development of tissue-specific immunomonitoring assays. Silencing SLA expression on PTECs was demonstrated to be an effective strategy to prevent xenogeneic cellular immune responses and may strongly support graft survival after xenotransplantation.

Background Porcine islet transplantation is a potential treatment for type 1 diabetes and greatly limited by powerful xenorejection. Manipulation of T cell co-inhibitory signals is one of the many approaches to induce immune tolerance. Here we studied the grafting and survival of genetic modified porcine islet expressing human programmed death-ligand 1 (hPD-L1) in a humanized mouse model of type 1 diabetes. Methods We isolated neonatal porcine islet-like clusters from transgenic pigs overexpressing hPD-L1 and from wild type pigs. We transplanted clusters into streptozotocin-induced diabetic NOD-scid IL2Rγnull mice stably reconstituted with human immune cells (hPD-L1 group, n=10; wild type group, n=6). Our primary endpoint was development of normoglycemia (random non-fasting blood glucose levels<180 mg/dl) at week 16 after transplantation. Results Compared to wild type group, more mice in the hPD-L1 group achieved normoglycemia (50% versus 0%, p<0.05). hPD-L1 expressing grafts exhibited less infiltrating hCD45+ cells and significant higher plasma C-peptide. In situ hybridization staining showed that infiltrating cells in hPD-L1 group expressed significant less human IFN-gamma as compared to the wild type group. Conclusions We here demonstrate for the first time that overexpression of hPD-L1 in porcine islets provided a strong porcine islet xenograft protection without administration of immunosuppressive drugs. These findings support the hypothesis that hPD-L1 has the capacity to control cellular rejection and therefore represents one promising transgene candidate for clinical porcine islet xenotransplantation. Article published online: 02 May 2023 © 2023. Thieme. All rights reserved. Georg Thieme Verlag Rüdigerstraße 14, 70469 Stuttgart, Germany

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. However, human leucocyte antigen (HLA) antibodies cross‐reacted with swine leucocyte antigen class I (SLA‐I). We previously demonstrated efficient generation of pigs with multiple xeno‐transgenes placed at a single genomic locus. Here we wished to assess whether key xenoreactive antigen genes can be simultaneously inactivated and if combination with the multi‐transgenic background further reduces antibody deposition and complement activation.Methods: Multiplex CRISPR/Cas9 gene editing and somatic cell nuclear transfer were used to generate pigs carrying functional knockouts of GGTA1, CMAH, B4GALNT2 and SLA class I. Fibroblasts derived from one‐ to four‐fold knockout animals, and from multi‐transgenic cells (human CD46, CD55, CD59, HO1 and A20) with the four‐fold knockout were used to examine the effects on human IgG and IgM binding or complement activation in vitro.Results: Pigs were generated carrying four‐fold knockouts of important xenoreactive genes. In vitro assays revealed that combination of all four gene knockouts reduced human IgG and IgM binding to porcine kidney cells more effectively than single or double knockouts. The multi‐transgenic background combined with GGTA1 knockout alone reduced C3b/c and C4b/c complement activation to such an extent that further knockouts had no significant additional effect.Conclusion: We showed that pigs carrying several xenoprotective transgenes and knockouts of xenoreactive antigens can be readily generated and these modifications will have significant effects on xenograft survival.

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.Methods: In this study, we employed the CRISPR/Cas9 system to target exon 2 of the porcine beta‐2‐microglobulin (B2M) gene to abrogate SLA class I expression on porcine cells. B2M‐KO cells served as donor cells for somatic cell nuclear transfer, and cloned embryos were transferred to three recipient sows. The offspring were genotyped for mutations at the B2M locus, and blood samples were analyzed via flow cytometry for the absence of SLA class I molecules.Results: Pregnancies were successfully established and led to the birth of seven viable piglets. Genomic sequencing proved that all piglets carried biallelic modifications at the B2M locus leading to a frameshift, a premature stop codon, and ultimately a functional knockout. However, survival times of these animals did not exceed 4 weeks due to unexpected disease processes.Conclusion: Here, we demonstrate the feasibility of generating SLA class I knockout pigs by targeting the porcine beta‐2‐microglobulin gene using the CRISPR/Cas9 system. Additionally, our findings indicate for the first time that this genetic modification might have a negative impact on the viability of the animals. These issues need to be solved to unveil the real value for xenotransplantation in the future.

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. Based on the frequency of IFN‐γ‐secreting cells, groups of weak, medium, and strong responder individuals were defined. Mixed lymphocyte reaction (MLR) assays were performed to study the stimulatory capacity of porcine PBMCs expressing different SLA‐DRB1 alleles.Results: Concerning the MHC class‐II configuration of human cells, we found a significant overrepresentation of HLA‐DRB1*01 alleles in the medium/strong responder group as compared to individuals showing weak responses to stimulation with L23 cells. Evaluation of the role of MHC class‐II variability in porcine stimulators revealed that cells expressing SLA‐DRB1*06 alleles triggered strong proliferation in approximately 70% of humans. Comparison of amino acid sequences indicated that strong human anti‐pig reactivity may be associated with a high rate of similarity between human and pig HLA/SLA‐DRB1 alleles.Conclusion: Variability in human and porcine MHC determines the intensity of individual human anti‐pig T‐cell responses. MHC typing and cross‐matching of prospective recipients of xenografts and donor pigs could be relevant to select for donor‐recipient combinations with minimal anti‐porcine immunity.