The immune profile of donor pigs with ubiquitous expression of LEA29Y
Andrea Bähr1, Tobias Käser2, Wilhelm Gerner3, Kristi Witter4, Annegret Wünsch1, Nadja Herbach5, Barbara Kessler1, Mayuko Kurome1, Reinhard Schwinzer6, Armin Saalmüller3, Bruno Reichart7, Eckhard Wolf1, Nikolai Klymiuk1.
1Molecular Animal Breeding and Biotechnology, LMU Munich, Munich, Germany; 2VIDO-Intervac, University of Saskatchewan, Saskatoon, SK, Canada; 3Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria; 4Institute of Anatomy, Histology and Embryology, Department of Pathobiology , University of Veterinary Medicine Vienna, Vienna, Austria; 5Institute for Pathology, LMU Munich, Munich, Germany; 6Transplantation Laboratory MH Hanover, MH Hanover, Hanover, Germany; 7Walter Brendel Centre of Experimental Medicine, LMU Munich, Munich, Germany
Recent studies suggest that survival of vascularized pig-to-non-human primate xenografts can be achieved when complement-mediated rejection and coagulation incompatibilities are overcome by a combination of suitable genetic modifications. Blockade of B-cell and T-cell activation, however, still requires systemic administration of specific compounds. Local expression of immune modulatory proteins in transgenic donor organs is an attractive approach to reduce systemic immunosuppression in the recipient.
Here, we describe the establishment and characterization of a transgenic pig line strongly expressing the T-cell co-stimulation blocker LEA29Y, a CTLA-4Ig variant optimized to bind human CD80/CD86 with high affinity, in multiple organs. To achieve this, we designed a vector that places the LEA29Y coding sequence under the transcriptional control of the strong and ubiquitous CAG-promoter, transfected pig primary cells with the vector and generated founder animals by somatic cell nuclear transfer. Characterization of 3 founder boars at an age of 3 months revealed expression of LEA29Y transcripts and protein in all tissues examined. Serum from the pigs was used to demonstrate binding of the transgene product to antigen presenting cells as well as the inhibitory potential on human T-cell proliferation in vitro. The founder animal with the highest expression was initially re-produced by cloning and animals were observed for a longer period of time under conventional housing conditions. All clones showed repeated episodes of acute and chronic infection, indicating an inhibitory effect of LEA29Y on the porcine immune system and leading to retarded growth and impaired sexual maturation of the pigs. At an age of 10 months, we isolated epididymal sperm from one cloned boar and generated F1 offspring by in vitro fertilization of oocytes and embryo transfer to synchronized recipients. Southern blot analysis revealed an identical integration pattern in all transgenic offspring, which had LEA29Y in their serum and showed impaired development when housed under conventional conditions.
In concordance with the clinical phenotype, the T-cell population in the expanded CAG-LEA line was dramatically affected. At the age of 6 months, resting T-cells in lymphoid tissue were present, but we did not observe activated or memory T-cells. Moreover, peripheral blood mononuclear cells also completely lacked regulatory T-cells, B-cells and NK-cells. Thus, the immune profile of these 6-month-old animals resembled that of a 2 to 4 week old piglet. This was confirmed by the further analysis of their lymph nodes that were populated with resting T-cells like wild-type lymph nodes, but showed distinctly smaller B cell follicles with no reaction centres, indicating a lack of stimulation upon antigen challenge.
Previous studies have demonstrated that by local LEA29Y transgene expression can prevent porcine islet and skin xenografts in mouse models with humanized immune systems. The new CAG-LEA29Y transgenic pigs are a potential source of vascularized organs that may be protected against T-cell-mediated rejection. Although CAG-LEA29Y transgenic pigs are immunocompromised, they can be maintained under optimized hygienic conditions and reproduced by assisted reproduction techniques and likely also by conventional breeding. Currently the CAG-LEA29Y transgene is being combined with additional genetic modifications encountering hyperacute and acute vascular rejection pathways.
This work was funded by the German Research Foundation (Transregio CRC 127)..
15:30 - 17:00
|Genetic Engineering; Methodology||The immune profile of donor pigs with ubiquitous expression of LEA29Y||Room 109|