Introduction: Efforts are underway to modulate acquired immune responses to prevent chronic cell-mediated xenorejection. Our aim was to develop in vitro assays to evaluate the xenospecific responses of CD8+ T cells (CTLs) to monolayers of porcine aortic endothelial cells (PAECs) from wild-type (WT) and transgenic (TG) animals expressing human PD-L1 and knocked-out for beta-2 microglobulin (B2M-KO).
Methods: Human CTLs were purified from PBMC and stimulated weekly for over 14 days with irradiated WT and TG PAECs. CTL phenotype before and after stimulation was assessed by flow cytometry (FC). CTL specificity was tested by conventional non-radioactive cytotoxicity and FC-based degranulation assays against primary PAEC_WT/TG and third-party PEDSV.15 (dd-haplotype) targets in suspension. Live-cell imaging cytotoxicity assays were performed under static (2D, 6 h) and microfluidic (3D, 2 h) co-cultures at an effector-to-target (E:T) ratio of 1:1, mimicking physiological endothelial settings. In the 2D assay, images of the endothelial monolayers were captured to quantify apoptotic bodies and necrotic PAECs. In the 3D microfluidic channels, a shear stress of 2 dyne/cm2 was applied, and readouts included CTL adhesion and Draq7⁺ dead PAECs on images of the PAEC-coated artificial micro-vessels.
Results: Following stimulation with PAECs, CTLs shifted from a naïve to a memory phenotype. Co-culture with PAEC_TG resulted in a reduced percentage of CD3⁺CD8⁺ T cells and lower overall CD8⁺ cell yield compared to PAEC_WT. In suspension assays, CTLs exhibited robust, E:T ratio-dependent cytotoxicity against PAEC_WT. Specific lysis of PAEC_TG was reduced by 18% compared to PAEC_WT, though, the effect was apparent only at E:T ratios ≥ 5:1. In 2D assays, CTLs predominantly induced apoptotic cell death (11.26 ± 0.55%) with lower levels of necrosis (6.02 ± 1.73%) in PAEC_WT monolayers. In contrast, PAEC_TG showed significant resistance, with markedly reduced apoptosis (0.13 ± 0.12%) and necrosis (1.17 ± 0.73%). In the 3D system, CTLs adhesion to PAEC_TG was significantly diminished; however, Draq7⁺ dead PAEC frequencies were similar between PAEC_WT and PAEC_TG.
Conclusion: This study demonstrated the generation of human CTLs with robust cytotoxic activity against porcine endothelial cells. Genetic modifications in PAEC_TG, specifically PD-L1 expression and MHC class I abrogation, partially reduce CTL-mediated cytotoxicity in conventional and 2D assays. Under flow conditions, killing of PAEC_WT/TG was similarly low, though reduced CTL adhesion to PAEC_TG suggests altered immune engagement. These findings underscore the immunomodulatory potential of engineered porcine endothelium and support continued efforts toward developing immune-evasive xenografts. Our in vitro platforms offer valuable tools to dissect cellular immune barriers in xenotransplantation.