237.8: HLA-E protects genetically engineered porcine endothelial cells from lysis by natural killer cells in 2D and 3D microfluidic systems

Introduction: Natural Killer (NK) cells play a crucial role in the cellular rejection of allo- and xenografts. Expression of human NK cell inhibitory ligands on pig cells represents a preventive strategy to avoid xenogeneic human anti-pig NK cytotoxicity. Human leukocyte antigen-E (HLA-E) is a non-classical major histocompatibility complex class I molecule interacting with the inhibitory C-type lectin receptor NKG2A. Previously, we demonstrated that transgenic expression of HLA-E partially protected porcine aortic endothelial cells (PAECs) from human NK cytotoxicity, and also reduced gamma-interferon secretion. The present study aimed to investigate the protective effects of HLA-E expressed in genetically engineered PAECs using both, 2D and 3D microfluidic systems to better understand the cellular interactions under more physiological conditions. Methods: NK cells were purified from human blood by magnetic negative selection and activated with IL-2 for 5 days. Wild type (WT) and HLA-E/CD46 transgenic (TG) PAECs were isolated from German Landrace pigs. Porcine TNF (pTNF) was used to activate porcine endothelium. For the 2D in vitro method, human NK cells were labeled with a yellow cell tracer and co-cultured with Hoechst-stained PAECs in 96 well plates. Cellular interactions and cytotoxicity were analyzed from video time-lapse data for 2h. Furthermore, we established a live-cell tracking method using Draq7 live/dead staining and a 3D round-section microfluidic chip coated with a monolayer of PAECs to study NK cells’ activity against PAECs in a micro-vessel-like environment under capillary shear stress (2.5 dyn/cm2). Results: Expression of HLA-E/CD46 on PAEC provided significant protection against human NK cell lysis, compared to WT controls. Our 2D live cell tracking data revealed that human NK cells killed PAECs both via apoptosis and necrosis. Membrane-disrupted cells were stained with Draq7 and therefore detected as necrotic cells while cells that had intact membranes but form multiple apoptotic bodies were counted as apoptotic. Cell trajectories displayed three different movements of NK cells on PAEC layers: constrained, random, or directed. NK cells interacting with WT PAECs showed more constrained movements, while NK cells interacting with transgenic HLA-E/CD46 PAECs showed 4 times more directed trajectories. NK cell adhesion to PAEC monolayers and lysis of PAEC was demonstrated under flow conditions for the first time. Conclusion: The development of novel methods allowed us to better analyze and understand interactions between human NK cells and PAEC. Inhibition of NK cytotoxicity by transgenic expression of HLA-E on PAECs was visualized and quantified using 2D live cell tracking data, and a 3D microfluidic system that better mimics the physiological conditions.