背景：免疫疗法已证明其具有改善肝细胞癌（HCC）患者预后的潜力；然而，患者对免疫治疗的反应差异很大。对有反应者和无反应者的肿瘤微环境（TME）进行比较分析有望揭示导致免疫治疗耐药的机制并提供潜在的治疗靶点。方法：我们使用 10x Genomics 技术对 6 名对抗 PD-1 治疗有反应的 HCC 患者和 1 名无反应的 HCC 患者进行了测序分析。此外，我们从公共数据库中获得了未经治疗、有反应和无反应 HCC 患者的单细胞数据，并使用部分数据集作为验证队列。这些数据使用 Harmony 等算法进行整合。建立了一个独立的验证队列。此外，我们使用 10x Genomics 空间转录组技术对三名 HCC 反应性患者的肿瘤邻近组织进行空间转录组测序。此外，我们还分析了从公共数据库获得的三名 HCC 患者的数据。最后，我们使用免疫荧光、流式细胞术和体内实验验证了我们的结论。结果：我们的研究结果证实了“免疫屏障”的存在，部分解释了免疫疗法疗效有限的原因。我们的分析显示，在表达多种免疫抑制信号的无反应患者中，TREM2 巨噬细胞显着增加。抗 Csf1r 单克隆抗体有效消除了这些巨噬细胞，增强了抗 PD-1 疗法的治疗效果。 TCR巨噬细胞具有直接杀伤肿瘤的能力。 IL1B cDC2 是 cDC2 细胞的主要功能亚型。 THEMIShi CD8 T 亚型的缺失可能会降低免疫治疗效果。此外，CD8 T 细胞在抗 PD-1 治疗后进入应激状态，这可能与 CD8 T 细胞耗竭和衰老有关。结论：免疫 TME 谱显示有反应、无反应和未治疗的 HCC 患者存在差异。这些差异可能解释了免疫疗法在某些 HCC 患者中疗效降低的原因。我们发现这些细胞和分子具有独特的功能，可用于增强 HCC 患者的免疫治疗效果。© 作者。

Background: Immunotherapy has demonstrated its potential to improve the prognosis of patients with hepatocellular carcinoma (HCC); however, patients' responses to immunotherapy vary a lot. A comparative analysis of the tumor microenvironment (TME) in responders and non-responders is expected to unveil the mechanisms responsible for the immunotherapy resistance and provide potential treatment targets. Methods: We performed sequencing analyses using 10x Genomics technology on six HCC patients who responded to anti-PD-1 therapy and one HCC patient who did not respond. Additionally, we obtained single cell data from untreated, responsive, and nonresponsive HCC patients from public databases, and used part of the datasets as a validation cohort. These data were integrated using algorithms such as Harmony. An independent validation cohort was established. Furthermore, we performed spatial transcriptomic sequencing on the tumor adjacent tissues of three HCC responsive patients using 10x Genomics spatial transcriptomic technology. Additionally, we analyzed data about three HCC patients obtained from public databases. Finally, we validated our conclusions using immunofluorescence, flow cytometry, and in vivo experiments. Results: Our findings confirmed the presence of "immune barrier" partially accounting for the limited efficacy of immunotherapy. Our analysis revealed a significant increase in TREM2+ Macrophages among non-responsive patients expressing multiple immunosuppressive signals. anti-Csf1r monoclonal antibodies effectively eliminated these macrophages and augmented the therapeutic effects of anti-PD-1 therapy. TCR+ Macrophages possessed direct tumor-killing capabilities. IL1B+ cDC2 was the primary functional subtype of cDC2 cells. Absence of THEMIShi CD8+ T subtypes might diminish immunotherapeutic effects. Furthermore, CD8+ T cells entered a state of stress after anti-PD-1 treatment, which might be associated with CD8+ T cell exhaustion and senescence. Conclusions: The profiles of immune TMEs showed differences in HCC patients responsive, non-responsive and untreated. These differences might explain the discounted efficacy of immunotherapy in some HCC patients. The cells and molecules, which we found to carry unique capabilities, may be targeted to enhance immunotherapeutic outcomes in patients with HCC.© The author(s).