免疫逃逸是肝细胞癌（HCC）的一个重要特征。免疫检查点抑制剂（ICIs）在 HCC 中的总体缓解率仍然有限。揭示免疫调节机制、寻找新的免疫靶点有望进一步提高免疫治疗的疗效。我们的研究旨在利用 CRISPR 筛选小鼠模型来识别在 HCC 免疫逃避中起关键作用的潜在靶点，并进一步探讨其在改善免疫治疗中的价值。我们在两种不同免疫背景的小鼠模型（C57BL/6 和 NPG 小鼠）中进行了 CRISPR 筛选）并确定免疫抑制基因 Gsk3a 作为进一步研究的候选基因。采用流式细胞术分析Gsk3a对免疫细胞浸润和T细胞功能的影响。 RNA测序用于鉴定Gsk3a诱导的中性粒细胞基因表达的变化以及下游分子的改变。还探讨了Gsk3a抑制剂与抗程序性细胞死亡蛋白1（PD-1）抗体组合的治疗价值。Gsk3a作为免疫抑制靶点，显着促进免疫功能正常小鼠而非免疫缺陷小鼠的肿瘤生长。 Gsk3a 通过诱导中性粒细胞趋化性来抑制细胞毒性 T 淋巴细胞 (CTL) 功能。 Gsk3a 促进中性粒细胞表达谱的自我趋化性和中性粒细胞胞外陷阱 (NET) 的形成，通过富含亮氨酸的 α-2-糖蛋白 1 (LRG1) 阻断 T 细胞活性。当Gsk3a抑制剂与抗PD-1抗体联合使用时，观察到显着的协同效应。我们通过CRISPR筛选鉴定了潜在的HCC免疫逃避靶点Gsk3a。 Gsk3a 通过中间分子 LRG1 诱导中性粒细胞募集和 NET 形成，从而抑制 CTL 功能。以 Gsk3a 为目标可以增强 CTL 功能并提高 ICI 的功效。© 作者（或其雇主）2024。根据 CC BY-NC 允许重复使用。禁止商业再利用。请参阅权利和权限。英国医学杂志出版。

Immune escape is an important feature of hepatocellular carcinoma (HCC). The overall response rate of immune checkpoint inhibitors (ICIs) in HCC is still limited. Revealing the immune regulation mechanisms and finding new immune targets are expected to further improve the efficacy of immunotherapy. Our study aims to use CRISPR screening mice models to identify potential targets that play a critical role in HCC immune evasion and further explore their value in improving immunotherapy.We performed CRISPR screening in two mice models with different immune backgrounds (C57BL/6 and NPG mice) and identified the immunosuppressive gene Gsk3a as a candidate for further investigation. Flow cytometry was used to analyze the impact of Gsk3a on immune cell infiltration and T-cell function. RNA sequencing was used to identify the changes in neutrophil gene expression induced by Gsk3a and alterations in downstream molecules. The therapeutic value of the combination of Gsk3a inhibitors and anti-programmed cell death protein-1 (PD-1) antibody was also explored.Gsk3a, as an immune inhibitory target, significantly promoted tumor growth in immunocompetent mice rather than immune-deficient mice. Gsk3a inhibited cytotoxic T lymphocytes (CTLs) function by inducing neutrophil chemotaxis. Gsk3a promoted self-chemotaxis of neutrophil expression profiles and neutrophil extracellular traps (NETs) formation to block T-cell activity through leucine-rich α-2-glycoprotein 1 (LRG1). A significant synergistic effect was observed when Gsk3a inhibitor was in combination with anti-PD-1 antibody.We identified a potential HCC immune evasion target, Gsk3a, through CRISPR screening. Gsk3a induces neutrophil recruitment and NETs formation through the intermediate molecule LRG1, leading to the inhibition of CTLs function. Targeting Gsk3a can enhance CTLs function and improve the efficacy of ICIs.© 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.