HLA E CD94 NKG2A pathway confers superior NK cell resistance compared to extensive multitransgenic modifications in porcine endothelial cells

Introduction: Natural Killer (NK) cells are key mediators of innate immunity and represent a major barrier to xenotransplantation due to their ability to induce cytotoxicity without prior sensitization. HLA-E, a non-classical MHC class I molecule, engages the inhibitory CD94/NKG2A receptor on NK cells, providing inhibitory signals that suppress NK cell activation. This study investigates whether HLA-E expression on minimally modified porcine aortic endothelial cells (PAECs) confers superior NK cell resistance compared to extensively engineered cells lacking HLA-E, and validates the underlying mechanism.
Methods: Human NK cells were isolated from peripheral blood and activated with IL-2. Three PAEC variants were compared: (i) GalKO controls, (ii) 10GE cells (triple carbohydrate knockouts, seven human transgenes including hCD46, hCD55, hTBM, hEPCR, hHO-1, hCD47, and porcine GHR-KO — but NO HLA-E), and (iii) 4GE cells (GalTKO, B4GalTKO, hCD46, PLUS HLA-E). Cytotoxicity was measured by LDH release assay (E:T 10:1, 2.5h), degranulation by CD107a flow cytometry, and cell death by fluorescence microscopy (DiOC6/PI). To validate the mechanism, anti-CD94 blocking antibody experiments were performed.
Results: Despite carrying 10 genetic modifications, 10GE cells showed NO significant NK resistance compared to GalKO (fold-change 0.828, p=0.3031). In contrast, 4GE cells with HLA-E demonstrated a 51.5% reduction in NK cytotoxicity (fold-change 0.485, p=0.0028 vs. GalKO), significantly outperforming 10GE cells (p=0.0336). Degranulation (CD107a) was lowest in 4GE co-cultures (11.75%) compared to GalKO (27.28%) and 10GE (15.91%). Microscopy confirmed that 4GE cells had significantly less cell death (48.5% PI+) than GalKO (70.5%, p=0.0019), while 10GE showed only a non-significant reduction (60.0%, p=0.0874). Mechanistically, CD94 blocking reversed 4GE protection: cytotoxicity increased from 0.365 to 0.665 fold-change, and CD107a rose from 11.75% to 16.20%. Anti-CD94 alone had no effect on NK activation (2.49% CD107a), ruling out non-specific effects.
Conclusion: HLA-E expression on a minimal genetic background (4GE) provides superior NK resistance compared to extensive multi-transgenic modifications lacking HLA-E (10GE). CD94 blocking experiments mechanistically confirm that this protection is mediated via the HLA-E– CD94/NKG2A pathway. These findings establish HLA-E as the dominant genetic modification for NK cell evasion in xenotransplantation. HLA-E's absence cannot be compensated by other immunomodulatory strategies, underscoring its essential inclusion in next-generation xenograft donor pig designs — with direct implications for future clinical pig-to-human transplantation.