Introduction: Natural Killer cells are critical mediators of innate immunity and a major barrier to xenotransplantation. HLA-E, a non-classical MHC class I molecule, binds the inhibitory CD94/NKG2A receptor on human NK cells (hNK), suppressing their activation. This study evaluates the impact of HLA-E expression on genetically engineered (GE) porcine aortic endothelial cells (PAECs) in reducing human NK cell-mediated cytotoxicity and degranulation.
Methods: hNK were isolated from peripheral blood and cultured with IL-2. GE PAECs included Gal-knockout (GalKO/WT) cells, 10GE triple-knockout (GalKO, β4GalKO, CMAHKO) cells with human genes (hCD46, hCD55, hTBM, hEPCR, hHO1, hCD47, GHR knockout), and HLA-E-expressing 11GE cells (10GE+HLA-E=11GE cells). Cytotoxicity was measured via an LDH release assay with hNK co-cultured at an effector-to-target (E:T) ratio of 10:1 for 2.5 hours. Degranulation was quantified by CD107a flow cytometry after a 2-hour co-culture. To probe the HLA-E pathway, an anti-CD94 blocking antibody was added to hNK before incubation with 11GE cells. Additionally, a static co-culture fluorescent microscopy assay using DiOC6 (live cells) and PI (dead cells) was performed to visualize and quantify NK cell-mediated cytotoxicity.
Results: HLA-E expression significantly modulated hNK cell responses toward porcine endothelial cells. In the LDH cytotoxicity assay, 11GE PAECs exhibited a markedly reduced cytotoxicity level (52.18%, n=5), which was statistically significant compared to both GalKO PAECs (p=0.0030) and 10GE PAECs (86.5%, n=4, p=0.0329). No significant difference was observed between 10GE and GalKO groups (p=0.5004). In the degranulation assay (CD107a), NK cells co-cultured with 11GE targets showed reduced activation (13.46%, n=3) relative to GalKO (21.68%, n=3) and 10GE (16.8%, n=2) targets. Furthermore, blockade of the CD94 receptor on NK cells reversed the inhibitory effect of HLA-E, restoring cytotoxicity and degranulation to levels comparable with HLA-E–negative targets. This effect was reproduced in two independent experiments, supporting the reliability.
The proportion of PI-positive cells was markedly lower in co-cultures with HLA-E-expressing PAECs (36%, n=5) versus GalKO (66%, n=5) and 10GE cells (54%, n=4), corroborating the LDH and CD107a findings.
Conclusion: These results provide direct mechanistic evidence that HLA-E expression inhibits hNK cell effector functions via the CD94/NKG2A inhibitory pathway. The observed reduction in cytotoxicity and degranulation underscores the relevance of this checkpoint in the xenogeneic context. The significant difference between 11GE and 10GE PAECs—differing only by HLA-E—highlights its specific immunomodulatory role and supports the inclusion of HLA-E in future genetically engineered pigs to enhance innate immune compatibility in pig-to-human xenotransplantation. Additional studies using baboon NK cells are ongoing to validate translational relevance in preclinical models.