Wiebke Baars

Transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys

Petersen B, Ramackers W, Lucas‐Hahn A, Lemme E, Hassel P, Queißer A‐L, Herrmann D, Barg‐Kues B, Carnwath JW, Klose J, Tiede A, Friedrich L, Baars W, Schwinzer R, Winkler M, Niemann H. Transgenic expression of human heme oxygenase‐1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys. Xenotransplantation 2011; 18: 355–368.

Suppression of human T-cell activation and expansion of regulatory T cells by pig cells overexpressing PD-ligands.

Background. 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. This report tested the hypothesis that overexpression of human negative costimulatory PD-Ligands (PD-L) in pig antigen presenting cells might be an approach to prevent human anti-pig T-cell responses. Methods. The pig B-cell line L23 was transfected with the pIRES-AcGFP vector containing human PD-L1 or PD-L2. Stable transfectants (L23-PD-L1, L23-PD-L2 cells) were established and used for in vitro stimulation of purified human CD4+ T cells. Results. Human CD4+ T cells responded with significantly reduced proliferation to L23-PD-L1 or L23-PD-L2 cells and produced less IL-2, IFN&ggr;, TNFα, IL-4, and IL-5 than cells stimulated with mock-transfected B cells. The concentration of IL-10, however, was increased in CD4+ T cells responding to stimulation with PD-L1 or PD-L2 transfectants. Furthermore, in cultures of CD4+ T cells stimulated for 3 weeks with PD-L1 or PD-L2 transfectants a CD4+CD25highFoxp3+ subset showed up that effectively suppressed the activation of conventional CD4+ T cells. Conclusions. These findings imply that PD-1/PD-Ligand pathways are interesting targets to prevent human anti-pig T-cell responses after xenotransplantation, and also suggests that PD-1/PD-Ligand interactions may play a role in the control of the activity and/or homeostasis of regulatory T cells.

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. Genetically modified pigs were characterized at the molecular, cellular, histological, and physiological levels. Results A 7.6-kb fragment containing the entire upstream region of the porcine THBD gene was found to drive a high expression in a porcine endothelial cell line and was therefore used to control hTM expression in transgenic pigs. The abundance of hTM was restricted to the endothelium, according to the predicted pattern, and the transgene expression of hTM was stably inherited to the offspring. When endothelial cells from pigs carrying the hTM transgene—either alone or in combination with an aGalTKO and a transgene encoding the human CD46—were tested in a coagulation assay with human whole blood, the clotting time was increased three- to four-fold (P<0.001) compared to wild-type and aGalTKO/CD46 transgenic endothelial cells. This, for the first time, demonstrated the anticoagulant properties of hTM on porcine endothelial cells in a human whole blood assay. Conclusions The biological efficacy of hTM suggests that the (multi-)transgenic donor pigs described here have the potential to overcome coagulation incompatibilities in pig-to-primate xenotransplantation.

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. We used two methods to colocate xenoprotective transgenes at one locus, sequential targeted transgene placement - ‘gene stacking’, and cointegration of multiple engineered large vectors - ‘combineering’, to generate pigs carrying modifications considered necessary to inhibit short to mid-term xenograft rejection. Pigs were generated by serial nuclear transfer and analysed at intermediate stages. Human complement inhibitors CD46, CD55 and CD59 were abundantly expressed in all tissues examined, human HO1 and human A20 were widely expressed. ZFN or CRISPR/Cas9 mediated homozygous GGTA1 and CMAH knockout abolished α-Gal and Neu5Gc epitopes. Cells from multi-transgenic piglets showed complete protection against human complement-mediated lysis, even before GGTA1 knockout. Blockade of endothelial activation reduced TNFα-induced E-selectin expression, IFNγ-induced MHC class-II upregulation and TNFα/cycloheximide caspase induction. Microbial analysis found no PERV-C, PCMV or 13 other infectious agents. These animals are a major advance towards clinical porcine xenotransplantation and demonstrate that livestock engineering has come of age.

The 77C→G Mutation in the Human CD45 (PTPRC) Gene Leads to Increased Intensity of TCR Signaling in T Cell Lines from Healthy Individuals and Patients with Multiple Sclerosis

The 77C→G mutation in exon A of the human CD45 gene occurs with low frequency in healthy individuals. An enhanced frequency of 77C→G individuals has been reported in cohorts of patients suffering from multiple sclerosis, systemic sclerosis, autoimmune hepatitis, and HIV-1. To investigate the mechanisms by which the variant allele may contribute to disease susceptibility, we compared T cell reactivity in heterozygous carriers of the mutation (healthy individuals and multiple sclerosis patients) and wild-type controls. In vitro-generated T cell lines and freshly isolated CD4+CD45R0+ primed/memory T cells from 77C→G individuals aberrantly expressed CD45RA isoforms and showed enhanced proliferation and IL-2 production when stimulated with anti-TCR/CD3 mAb or Ag. Mutant T cell lines contained a more active pool of p56lck tyrosine kinase and responded with increased phosphorylation of Zap70 and TCR-ζ and an enhanced Ca2+ flux to TCR/CD3 stimulation. These data suggest that 77C→G may act as a risk factor for certain diseases by increasing the intensity of TCR signaling.

Down‐regulation of human alloimmune responses by genetically engineered expression of CD95 ligand on stimulatory and target cells

Transgenic expression of apoptosis‐inducing molecules could be a strategy to protect cells and tissues from destruction by apoptosis‐susceptible effector T cells. Some evidence for the potencyof this approach has been obtained in mouse and rat transplantation models. However, limited data are available on the capacity of apoptosis‐inducing molecules to modulate human alloimmune responses. In this study we analyzed the in vitro consequences of an interaction of human T cells with allogeneic 293 cells and 293 transfectants stably expressing high levels of the apoptosis‐inducing CD95 ligand (CD95L). Both, CD95L– and CD95L+ 293 cells were able to activate allogeneic T cells as demonstrated by comparable CD25 expression at day 2 of culture. The analysisof viable T cells at day 7, however, revealed anti‐293 cytotoxic activity only in cultures that had been stimulated with CD95L– 293 cells. Alloactivated effector T cells lysed CD95L– and CD95L+ 293 targets with similar efficiency when tested in a 4‐h 51Cr‐release assay. Prolongation of the effector phase to 20 h resulted in a further increase in the destruction of CD95L– target cells, whereas lysis of CD95L+ targets remained low. These data suggest that genetically engineered expression of CD95L on cells or tissues could be an approach to control human T cell reactivity towards allografts. During the induction of an alloimmune response depletion of cytotoxic precursor cells may be obtained by overexpressing CD95L on stimulatory cells; CD95L expression on graft tissue might limit T cell‐mediated destruction of the transplant during the effector phase of the response.

Human TNF-related apoptosis-inducing ligand-expressing dendritic cells from transgenic pigs attenuate human xenogeneic T cell responses

Kemter E, Lieke T, Kessler B, Kurome M, Wuensch A, Summerfield A, Ayares D, Nagashima H, Baars W, Schwinzer R, Wolf E. Human TNF‐related apoptosis‐inducing ligand‐expressing dendritic cells from transgenic pigs attenuate human xenogeneic T cell responses. Xenotransplantation 2012; 19: 40–51.

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 B7.2/CD86 with high affinity and is thus a potent inhibitor of T cell co-stimulation via this pathway. We have characterized the expression pattern and the biological function of the transgene as well as its impact on the porcine immune system and have evaluated the potential of these transgenic pigs to propagate via assisted breeding methods. The analysis of LEA29Y expression in serum and multiple organs of CAG-LEA transgenic pigs revealed that these animals produce a biologically active transgenic product at a considerable level. They present with an immune system affected by transgene expression, but can be maintained until sexual maturity and propagated by assisted reproduction techniques. Based on previous experience with pancreatic islets expressing LEA29Y, tissues from CAG-LEA29Y transgenic pigs should be protected against rejection by human T cells. Furthermore, their immune-compromised phenotype makes CAG-LEA29Y transgenic pigs an interesting large animal model for testing human cell therapies and will provide an important tool for further clarifying the LEA29Y mode of action.

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.

Background 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 &agr;1,3-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. Two piglets were analyzed by quantitative reverse-transcription polymerase chain reaction, flow cytometry, and sequencing. The biological function of the genetic modifications was tested in a 51Chromium release assay and by ex vivo kidney perfusions with human blood. Results Disruption of the GGTA1 gene by deletion of few basepairs was demonstrated in GGTA1-KO/hHO-1/hA20 transgenic pigs. The hHO-1 and hA20 mRNA expression was confirmed by quantitative reverse-transcription polymerase chain reaction. Ex vivo perfusion of 2 transgenic kidneys was feasible for the maximum experimental time of 240 minutes without symptoms of rejection. Conclusions Results indicate that GGTA1-KO/hHO-1/hA20 transgenic pigs are a promising model to alleviate rejection and ischemia-reperfusion damage in porcine xenografts and could serve as a background for further genetic modifications toward the production of a donor pig that is clinically relevant for xenotransplantation.

Combining Enhancement of Inhibitory Signals and Blockade of Co-Stimulation to Modulate Human Anti-Pig Immune Responses

Introduction Transgenic expression of human modulatory molecules in pigs is a promising approach to prevent human anti-porcine immune responses and thus, an important step on the way to clinical xenotransplantation. We have previously shown that porcine cells overexpressing the human inhibitory ligand PD-L1 (CD274) trigger inhibitory signals in PD-1+ (CD279) human T, B, and NK cells thereby attenuating their activation (Buermann A, et al. Xenotransplantation. 2016: 23: 347). Furthermore, blocking of CD28-CD80/86-mediated co-stimulation by transgenic expression of CTLA4.Ig (CD152.Ig) in pigs decreases human T cell activation (Baehr A, et al. PLoS One. 2016: 11: e0155676). It is not yet clear, which modulatory effects could be expected when the two concepts are combined. Thus, it was the aim of this study to establish and characterize porcine model cell lines which overexpress hPD-L1 together with hCTLA4.Ig. Methods The porcine B cell line L23 as an antigen presenting cell (APC) was used as the model cell line. L23 cells were transfected with either hPD-L1, LEA29Y (a more potent derivate of human CTLA4.Ig, provided by Dr. N. Klymiuk, Munich), or both. L23-hPD-L1, -LEA29Y, and -hPD-L1/LEA29Y transfectants were tested for their potential to stimulate human immune cells in different in vitro cell culture assays. Results The different transgenic cell lines were characterized using flow cytometry analyses. L23-hPD-L1 cells strongly expressed the PD-L1 molecule on their cell surface. L23-LEA29Y cells produced LEA29Y which was detectable in the supernatant as well as bound to CD80/86 on the cell surface of transgenic L23 cells. Measurement of cell surface-bound-LEA29Y over time showed that in fresh medium not all CD80/CD86 epitopes of L23 transfectants were occupied by LEA29Y but after five days in culture complete saturation of epitope binding was obtained. Characterization of double transgenic L23-hPD-L1/LEA29Y cells revealed both, strong expression of hPD-L1 and production of LEA29Y. Co-culturing of L23-hPD-L1 or L23-LEA29Y cells with human T cells showed a reduced potential to stimulate proliferation for about 30-50% and 20-30% respectively, compared to stimulation with mock-transfected control L23 cells. No further reduction of proliferation was observed when the double transgenic L23-hPD-L1/LEA29Y cells were used as stimulators for human T cells. Conclusion Porcine APC genetically engineered to provide strong inhibitory signals (overexpression of hPD-L1) or prevent CD28-CD80/86 costimulatory interactions (production of hCTLA4.Ig) are weak stimulators of human T cell proliferation. So far, no synergistic inhibition of proliferation was observed by the combination of both approaches. It remains to be determined whether other effector functions of human T cells are modified by simultaneous enhancement of inhibition and blockade of co-stimulation. SFB-TRR 127 (“Xenotransplantation”) of the German Research Foundation (DFG).