Expression of human CD47 in a novel transgenic pig protects porcine endothelial cells partially from NK cell cytotoxicity
Thao Tran1, Elisabeth Kemter2,3, Sarah Grimus2, Mayuko Kurome2, Valeri Zakhartchenko2, Barbara Kessler2, Viktoriia Galdina1, Kaiyuan Yang3,4, Heiko Lickert3,4, Oscar Urquidi5, Eckhard Wolf2,3, Gisella L. Puga Yung1, Jörg D. Seebach1.
1Department of Medicine, Laboratory of Translational Immunology, Geneva University Hospitals and Faculty of Medicine, Geneva, Switzerland; 2Gene Centre and Department of Veterinary Science, Ludwig-Maximilians-Universität München, Munich, Germany; 3German Center for Diabetes Research (DZD), Neuherberg, Germany; 4Institute of Diabetes and Regeneration Research, Helmholtz Center Munich, Neuherberg, Germany; 5Department of Physical Chemistry, Faculty of Science, University of Geneva, Geneva, Switzerland
Introduction: Natural killer (NK) cells pose a major barrier to successful xenotransplantation due to their capacity to recognize and, through the engagement an array of activating receptors, destroy porcine endothelial cells, the first point of contact between donor organ and the recipient immune cells. While the CD47–SIRPα receptor-ligand interaction is a well-established negative regulator of macrophage activity, its capacity in modulating NK cell effector functions in the context of xenograft rejection remains less well defined. Here, we investigated the immunomodulatory effects of human CD47 (hCD47) on porcine aortic endothelial cells (PAECs), derived from new hCD47 transgenic animals on NK cell responses.
Methods: Human NK cells were isolated from healthy donors and activated with interleukin-2 for a minimum of five days. PAEC derived from GGTA1-knockout/human CD47 transgenic pigs were used as target cells. NK cytotoxicity was assessed via live-cell imaging using CellTrace labeling and live/dead staining, under both static conditions (96-well plates) and dynamic flow conditions in 2D Ibidi microfluidic channels, applying a shear stress of 4 dyn/cm2. Co-cultures were performed at an effector-to-target ratio of 1:2 for 2 hours. NK cell dynamics and interactions with PAEC monolayers were evaluated by tracking cell trajectories from time-lapse microscopy recordings.
Results: Expression of hCD47 on PAECs significantly reduced NK cell-mediated lysis, suggesting a protective role for CD47–SIRPα in counterbalancing the activation signals triggered during NK-PAECs interactions. Moreover, under shear stress conditions, NK cells displayed diminished adhesion and motility on hCD47-expressing PAECs compared to their active crawling on wild-type cells, indicating impaired adhesion and surveillance. Blocking of the CD47–SIRPα axis through peptide or antibody inhibition only partially restored NK cytotoxicity. Notably, in the presence of heat-inactivated human plasma, which contains preformed anti-pig antibodies, hCD47 did not protect PAECs from NK cell–mediated antibody-dependent cytotoxicity.
Conclusions: These findings highlight hCD47 as a promising molecular tool for modulating innate immune responses in xenotransplantation. hCD47 expression on donor vasculature may offer a dual strategy to suppress both macrophages and NK cell-mediated graft injury thereby enhancing xenograft survival.