针对适应性免疫系统的免疫检查点封锁彻底改变了癌症的治疗。尽管观察到了令人印象深刻的临床益处，但患者亚组仍然没有反应，这凸显了利用额外免疫细胞的联合疗法的必要性。自然杀伤 (NK) 细胞是癌症治疗的新兴工具。然而，只有受抑制性受体差异控制的 NK 细胞亚群才会对特定癌症类型产生反应。如何充分利用所有 NK 细胞亚群的完整抗肿瘤潜力，同时又不利于 NK 细胞耐药肿瘤细胞的出现，这一问题仍然悬而未决。我们在与人类原代细胞共培养的黑色素瘤细胞中进行了全基因组 CRISPR/Cas9 敲除耐药性筛选NK 细胞。我们通过关注同种异体环境中的 NK 细胞亚群，全面评估了调节肿瘤耐药性和易感性的因素。此外，我们还测试了目前在临床试验中使用的治疗性阻断抗体。在剩余的 NK 细胞共培养的黑色素瘤细胞中，缺乏抗原呈递或 IFNγ 信号通路的黑色素瘤细胞被耗尽，并表现出对 NK 细胞的敏感性增强。用 IFNγ 治疗可诱导黑色素瘤细胞对静息、IL-2 培养和 ADCC 激活的 NK 细胞产生强大的抵抗力，这些抵抗力依赖于表达经典和非经典 MHC-I 所需的 B2M。 IFNγ诱导的HLA-E表达介导黑色素瘤细胞对NKG2A KIR-和部分对NKG2A KIR NK细胞亚群的耐药性。经典MHC-I的表达本身足以抑制NKG2A-KIR，但不能抑制NKG2A KIR NK细胞亚群。用 monalizumab（一种 NKG2A 阻断单克隆抗体）处理 NK 细胞，增强了相应 NK 细胞亚群的反应性。 monalizumab 与 lirilumab（阻断 KIR2 受体）与 DX9（阻断 KIR3DL1）的组合需要恢复所有 NK 细胞亚群对黑色素瘤和不同来源肿瘤中 IFNγ 诱导的耐药肿瘤细胞的细胞毒性。我们的数据表明，在这种情况下在 NK 细胞中，IFNγ 通过上调经典和非经典 MHC-I 诱导肿瘤细胞产生耐药性。此外，我们揭示了对 NK 细胞亚群反应性的见解，并提出了一种涉及 monalizumab/lirilumab/DX9 联合治疗的治疗策略，以完全恢复 NK 细胞亚群的抗肿瘤反应。© 作者（或其雇主）2024。Re - CC BY-NC 允许使用。禁止商业再利用。请参阅权利和权限。英国医学杂志出版。

Immune checkpoint blockade targeting the adaptive immune system has revolutionized the treatment of cancer. Despite impressive clinical benefits observed, patient subgroups remain non-responsive underscoring the necessity for combinational therapies harnessing additional immune cells. Natural killer (NK) cells are emerging tools for cancer therapy. However, only subpopulations of NK cells that are differentially controlled by inhibitory receptors exert reactivity against particular cancer types. How to leverage the complete anti-tumor potential of all NK cell subsets without favoring the emergence of NK cell-resistant tumor cells remains unresolved.We performed a genome-wide CRISPR/Cas9 knockout resistance screen in melanoma cells in co-cultures with human primary NK cells. We comprehensively evaluated factors regulating tumor resistance and susceptibility by focusing on NK cell subsets in an allogenic setting. Moreover, we tested therapeutic blocking antibodies currently used in clinical trials.Melanoma cells deficient in antigen-presenting or the IFNγ-signaling pathways were depleted in remaining NK cell-co-cultured melanoma cells and displayed enhanced sensitivity to NK cells. Treatment with IFNγ induced potent resistance of melanoma cells to resting, IL-2-cultured and ADCC-activated NK cells that depended on B2M required for the expression of both classical and non-classical MHC-I. IFNγ-induced expression of HLA-E mediated the resistance of melanoma cells to the NKG2A+ KIR- and partially to the NKG2A+ KIR+ NK cell subset. The expression of classical MHC-I by itself was sufficient for the inhibition of the NKG2A- KIR+, but not the NKG2A+ KIR+ NK cell subset. Treatment of NK cells with monalizumab, an NKG2A blocking mAb, enhanced the reactivity of a corresponding subset of NK cells. The combination of monalizumab with lirilumab, blocking KIR2 receptors, together with DX9, blocking KIR3DL1, was required to restore cytotoxicity of all NK cell subsets against IFNγ-induced resistant tumor cells in melanoma and tumors of different origins.Our data reveal that in the context of NK cells, IFNγ induces the resistance of tumor cells by the upregulation of classical and non-classical MHC-I. Moreover, we reveal insights into NK cell subset reactivity and propose a therapeutic strategy involving combinational monalizumab/lirilumab/DX9 treatment to fully restore the antitumor response across NK cell subsets.© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.