Abstract: Objective To target at the NKG2A-HLA-E inhibitory axis, a pluripotent stem cell(iPSC)-derived genetically engineered natural killer cells(NK cells) with NKG2A knockout (NKG2A KO-iNK) were prepared and then their tumor-killing efficacy was evaluated in vitro. Methods NKG2A was knocked out in iPSCs using gene-editing technology. These cells were then differentiated into NKG2A KO-iNK cells. Surface markers at each differentiation stage were analyzed by flow cytometry. Western blot confirmed NKG2A knockout, and flow cytometry assessed expression of activating receptors (NKG2D) and natural cytotoxicity receptors (NKp30, NKp44, NKp46) in NKG2A KO-iNK cells. Cytotoxic activity against tumor cell lines with varying human leukocyte antigen E (HLA-E) expression level was evaluated via lactate dehydrogenase (LDH) release assay. Results Co-transfection of iPSCs with Cas9 protein and three small-guide RNAs (sgRNAs) targeting at exons 1 and 2 of the KLRC1 gene (encoding NKG2A) successfully generated monoclonal NKG2A-knockout iPSCs (NKG2A KO-iPSCs) with a single T-base insertion in exon 1. During iPSC differentiation into NK cells, CD34 expression reached 30%-50% at the embryoid body (EB) stage (day 8), while CD56 and CD16 expression exceeded 80% by day 28. Western blot confirmed complete NKG2A knockout in NKG2A KO-iNK cells. Flow cytometry revealed comparable expression level of activating receptor NKG2D and cytotoxicity receptors (NKp30, NKp44, NKp46) between NKG2A KO-iNK and wild-type iNK (WT-iNK) cells. The LDH assay results indicated that the cytotoxic activity of NKG2A KO-iNK cells against the HLA-E highly-expressed B-cell precursor leukemia cell line Nalm6 cells was significantly higher than that of WT-iNK cells, while there was no significant difference between them and human myeloma cell line H929 cells with low HLA-E expression and human hepatocellular carcinoma cell line HepG2 cells with almost no HLA-E expression. Interferon-γ (IFN-γ) pretreatment up regulated HLA-E expression in Nalm6 cells, further amplifying NKG2A KO-iNK-mediated cytotoxicity. Conclusions By disrupting the NKG2A-HLA-E inhibitory axis, NKG2A KO-iNK cells exhibit markedly enhanced in vitro cytotoxicity against HLA-E-high tumor cells. This result highlights their potential function as a novel adoptive cell therapy strategy for cancers reliant on HLA-E-mediated immune evasion.

Key words: induced pluripotent stem cells (iPSCs), NKG2A, natural killer (NK) cell, immunotherapy