Abstract

Natural killer (NK) cells are key innate effector cells in cancer surveillance and in the defence against virus infections. However, much is still unknown about how NK cells distinguish between malignant and healthy cells. To learn more about what structures that determine the outcome of interactions between NK cells and target cells, we performed a genome-wide CRISPR screen with the leukemic K562 cells using the Brunello gRNA library and NK cell susceptibility as selection. The screen identified genes that provide protection or susceptibility to NK cell cytotoxicity. We could confirm the major role of B7-H6, the ligand for the NK cell activating receptor NKp30, for NK cell recognition, along with CD58 that ligates the co-stimulatory receptor CD2. Knock-out (KO) cell lines with depleted B7-H6 and/or CD58 expression displayed altered interactions with NK cells with regards to degranulation, cytotoxicity and cytokine secretion. Deletion of the gene TMEM30A provided protection against NK cell cytotoxicity. The encoded protein is a subunit of P4 ATPase flippase, which is involved in transfer of phosphatidylserine (PS) to the inner leaflet of the cell membrane. Accordingly, TMEM30A KO K562 cells displayed increased levels of PS on the surface as well as lower susceptibility to NK cell cytotoxicity. Blockade of PS by Annexin-V or blocking the inhibitory receptor IRp60/CD300a on NK cells restored NK cell killing of TMEM30A-deficient cells. Notably, TMEM30A is commonly mutated in diffuse large B cell lymphoma and this may serve as an escape mechanism for NK cell immunosurveillance. Our study highlights the potential role for agents targeting the interaction between PS and IRp60 in cancer immunotherapy.