IFN? mediates the resistance of tumor cells to distinct NK cell subsets.

Tumor resistance to immune cells remains a major obstacle for successful treatment. NK cells are emerging tools for cancer therapy. However, due to differential and subset-specific expression of inhibitory NK cell receptors, often only subsets of NK cells exert reactivity against particular cancer types. Using a genome-wide CRISPR/Cas9 knockout (KO) screen in melanoma, we revealed resistance factors against NK cell attack and evaluated their differential impact on NK cell subpopulations. We show that melanoma cells deficient in antigen-presenting machinery or the IFN? signaling pathway displayed enhanced sensitivity to NK cell killing. Treatment with IFN? induced melanoma cell resistance that depended on B2M required for both classical and non-classical MHC-I expression. HLA-E mediated melanoma cell resistance to NKG2A+ KIR-, and partially to NKG2A+ KIR+ NK cells. Treatment of NK cells with the NKG2A blocking monalizumab resulted in enhanced NK cell reactivity to similar extent as deletion of HLA-E in melanoma cells. The combination of monalizumab with lirilumab, blocking KIR2 receptors, together with DX9, an anti-KIR3DL1 mAb, was required to restore the response of all NK cells against IFN?-pretreated tumor cells of different origins. Our data reveal insights into NK cell subset reactivity and strategies how to leverage their full anti-tumor potential. Overall design: To investigate genetic changes crucial to the NK cells' effector function during encounters with melanoma cells, a CRISPR mediated genetic screen was carried out on A375 melanoma cell line using a genome-wide CRISPR sgRNA library. The pool of edited A375 cells were treated with different amounts of NK cells to reach medium or high killing efficiency during 24 hours. Important modulators in the NK cell- melanoma interaction can be inferred from the abundance of each of the remaining sgRNA in the pool of the 3 samples. The medium-killing and high-killing samples were collapsed as replicates in the analysis.