In the myeloid compartment of the tumor microenvironment, CD244 signaling has been implicated in immunosuppressive phenotype of monocytes. However, the precise molecular mechanism and contribution of CD244 to tumor immunity in monocytes/macrophages remains elusive due to the co-existing lymphoid cells expressing CD244. To directly assess the role of CD244 in tumor-associated macrophages, monocyte-lineage-specific CD244-deficient mice were generated using cre-lox recombination and challenged with B16F10 melanoma. The phenotype and function of tumor-infiltrating macrophages along with antigen-specific CD8 T cells were analyzed by flow cytometry and single cell RNA sequencing data analysis, and the molecular mechanism underlying anti-tumorigenic macrophage differentiation, antigen presentation, phagocytosis was investigated ex vivo. Finally, the clinical feasibility of CD244-negative monocytes as a therapeutic modality in melanoma was confirmed by adoptive transfer experiments. CD244fl/flLysMcre mice demonstrated a significant reduction in tumor volume (61% relative to that of the CD244fl/fl control group) 14 days after tumor implantation. Within tumor mass, CD244fl/flLysMcre mice also showed higher percentages of Ly6Clow macrophages, along with elevated gp100+IFN-γ+ CD8 T cells. Flow cytometry and RNA sequencing data demonstrated that ER stress resulted in increased CD244 expression on monocytes. This, in turn, impeded the generation of anti-tumorigenic Ly6Clow macrophages, phagocytosis and MHC-I antigen presentation by suppressing autophagy pathways. Combining anti-PD-L1 antibody with CD244−/− bone marrow-derived macrophages markedly improved tumor rejection compared to the anti-PD-L1 antibody alone or in combination with wild-type macrophages. Consistent with the murine data, transcriptome analysis of human melanoma tissue single-cell RNA-sequencing dataset revealed close association between CD244 and the inhibition of macrophage maturation and function. Furthermore, the presence of CD244-negative monocytes/macrophages significantly increased patient survival in primary and metastatic tumors. Our study highlights the novel role of CD244 on monocytes/macrophages in restraining anti-tumorigenic macrophage generation and tumor antigen-specific T cell response in melanoma. Importantly, our findings suggest that CD244-deficient macrophages could potentially be used as a therapeutic agent in combination with immune checkpoint inhibitors. Furthermore, CD244 expression in monocyte-lineage cells serve as a prognostic marker in cancer patients.

中文翻译：

---

单核细胞上 CD244 的靶向删除促进分化为抗肿瘤巨噬细胞并增强黑色素瘤中的 PD-L1 阻断

在肿瘤微环境的骨髓区室中，CD244 信号传导与单核细胞的免疫抑制表型有关。然而，由于表达 CD244 的淋巴细胞共存，CD244 对单核细胞/巨噬细胞肿瘤免疫的精确分子机制和贡献仍然难以捉摸。为了直接评估 CD244 在肿瘤相关巨噬细胞中的作用，使用 cre-lox 重组产生单核细胞谱系特异性 CD244 缺陷小鼠，并用 B16F10 黑色素瘤进行攻击。通过流式细胞术和单细胞RNA测序数据分析，分析肿瘤浸润巨噬细胞和抗原特异性CD8 T细胞的表型和功能，并体外研究抗肿瘤巨噬细胞分化、抗原呈递、吞噬作用的分子机制。最后，过继转移实验证实了 CD244 阴性单核细胞作为黑色素瘤治疗方式的临床可行性。CD244fl/flLysMcre小鼠在肿瘤植入后14天表现出肿瘤体积显着减小（相对于CD244fl/fl对照组减少61%）。在肿瘤块内，CD244fl/flLysMcre 小鼠还显示出较高百分比的 Ly6Clow 巨噬细胞，以及升高的 gp100+IFN-γ+ CD8 T 细胞。流式细胞术和 RNA 测序数据表明 ER 应激导致单核细胞上 CD244 表达增加。这反过来又通过抑制自噬途径阻碍了抗肿瘤 Ly6Clow 巨噬细胞的产生、吞噬作用和 MHC-I 抗原呈递。与单独的抗PD-L1抗体或与野生型巨噬细胞组合相比，抗PD-L1抗体与CD244−/−骨髓源性巨噬细胞的组合显着改善了肿瘤排斥。与小鼠数据一致，人类黑色素瘤组织单细胞 RNA 测序数据集的转录组分析揭示了 CD244 与巨噬细胞成熟和功能的抑制之间的密切关联。此外，CD244阴性单核细胞/巨噬细胞的存在显着增加了原发性和转移性肿瘤患者的生存率。我们的研究强调了 CD244 对单核细胞/巨噬细胞在抑制黑色素瘤中抗肿瘤巨噬细胞生成和肿瘤抗原特异性 T 细胞反应方面的新作用。重要的是，我们的研究结果表明，CD244 缺陷型巨噬细胞有可能与免疫检查点抑制剂联合用作治疗剂。此外，单核细胞谱系细胞中的 CD244 表达可作为癌症患者的预后标志物。