目的·分析半乳糖凝集素9阳性肿瘤相关巨噬细胞（galectin-9⁺ tumor-associated macrophages， galectin-9⁺TAMs）在肌层浸润性膀胱癌（muscle-invasive bladder cancer，MIBC）组织中的表型特征，探究MIBC微环境对galectin-9⁺TAMs的调控作用，galectin-9⁺TAMs抑制CD8⁺T细胞反应的机制及其临床治疗意义。方法·通过流式细胞术检测MIBC与癌旁组织中galectin-9⁺TAMs的表型特征。利用TCGA（The Cancer Genome Atlas）数据库筛选与LGALS9和LGALS9巨噬细胞基因集显著相关的细胞因子。用人重组细胞因子体外刺激巨噬细胞，分为人重组巨噬细胞集落刺激因子（recombinant human macrophage-stimulating factor，rhM-CSF）刺激组、人重组白介素-16（recombinant human interleukin-16，rhIL-16）刺激组和人重组干扰素γ（recombinant human interferon-γ，rhIFN-γ）刺激组，并通过流式细胞术检测3组galectin-9表达情况。流式细胞术检测加入rhM-CSF中和性抗体后巨噬细胞表达galectin-9的情况。Anti-galectin-9抗体处理MIBC单细胞悬液后，通过流式细胞术检测TAMs的效应功能变化。分选癌与癌旁组织中galectin-9⁺TAMs和人外周血CD8⁺T细胞并进行体外共培养，通过流式细胞术检测CD8⁺T细胞效应功能变化。采用anti-galectin-9抗体和程序性死亡受体1（programmed cell death protein 1，PD-1）抗体单独及协同处理体外培养的肿瘤组织，通过流式细胞术检测肿瘤细胞凋亡及CD8⁺T细胞效应功能变化。结果·Galectin-9⁺TAMs高表达人类白细胞DR抗原（human leukocyte antigen DR，HLA-DR）、CD86、CD206和细胞程序性死亡-配体1（programmed cell death-ligand 1，PD-L1），分泌IL-10和转化生长因子-β（transforming growth factor-β，TGF-β）增加，分泌肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）减少。TCGA数据库筛选结果显示，M-CSF、IL-16和IFN-γ与LGALS9和LGALS9巨噬细胞基因集的相关性最显著。用rhM-CSF、rhIL-16和rhIFN-γ体外刺激巨噬细胞，rhM-CSF刺激组中galectin-9的表达显著升高，加入中和性抗体后表达显著下调。阻断galectin-9后，TAMs表型从高表达抑制性分子向促炎症分子转换，其表面表达PD-L1显著下降。体外共培养galectin-9⁺TAMs和CD8⁺T细胞后，galectin-9⁺TAMs能够抑制CD8⁺T细胞的效应功能，该作用部分依赖于galectin-9。联合阻断galectin-9和PD-1后，肿瘤细胞凋亡比例、CD8⁺T细胞的增殖能力和效应分子的分泌与单独阻断PD-1相比，均显著增多或增强。结论·Galectin-9⁺TAMs具有免疫抑制表型和功能。肿瘤来源M-CSF诱导TAMs高表达galectin-9。Galectin-9⁺TAMs抑制CD8⁺T细胞功能从而促进MIBC免疫逃逸。联合阻断galectin-9和PD-1能够更有效地重激活CD8⁺T细胞功能。

Objective ·To explore the phenotype of galectin-9⁺ tumor-associated macrophages (galectin-9⁺TAMs) in muscle-invasive bladder cancer (MIBC). To clarify the regulation of galectin-9⁺TAMs in MIBC microenvironment, and elucidate the mechanism of galectin-9⁺TAMs inhibiting the effector function of CD8⁺T cells and its clinical therapeutic significance in MIBC. Methods ·Phenotype of galectin-9⁺TAMs from MIBC peritumor and tumor tissues was detected by flow cytometry. TCGA (The Cancer Genome Atlas) database was used to sort out the cytokines most relevant to LGALS9 and LGALS9 macrophage gene set. Macrophages were stimulated by recombinant human cytokines in vitro, divided into recombinant human macrophage stimulating factor (rhM-CSF) stimulation group, recombinant human interleukin-16 (rhIL-16) stimulation group and recombinant human interferon-γ (rhIFN-γ) stimulation group. Expression level of galectin-9 among the three groups was verified by flow cytometry. Expression level of galectin-9 on macrophages was detected by flow cytometry after adding M-CSF neutralizing antibody. Effector functions of TAMs were detected by flow cytometry after treating MIBC single cell suspension with anti-galectin-9 antibody. Galectin-9⁺TAMs from peritumor and tumor tissues, and human peripheral blood CD8⁺T cells were sorted and co-cultured in vitro. Effector functions of CD8⁺T cells were detected by flow cytometry. Tumor tissues cultured in vitro were treated with anti-galectin-9 antibody and programmed cell death protein 1 (PD-1) antibody alone or in combination. Tumor cell apoptosis and effector function of CD8⁺T cells were detected by flow cytometry. Results ·Galectin-9⁺TAMs exhibited the phenotype with high expression of human leukocyte antigen DR (HLA-DR), CD86, CD206 and programmed cell death-ligand 1 (PD-L1). Increased IL-10 and transforming growth factor-β (TGF-β) and decreased tumor necrosis factor-α (TNF-α) were secreted by itself. M-CSF, IL-16 and IFN-γ showed the significant difference with LGALS9 and LGALS9 macrophage gene set in TCGA database. Galectin-9 on macrophages increased significantly in rhM-CSF stimulated group and its expression decreased by adding neutralizing antibody. Galectin-9 inhibition switched the activation of TAMs from an immunosuppressive phenotype to a more inflammatory state and PD-L1 on its surface significantly decreased. After cultured in vitro, galectin-9⁺TAMs inhibited the effect of CD8⁺T cells in a partly galectin-9-dependent manner. Compared with applying PD-1 inhibitor alone, percentage of tumor cell apoptosis, the proliferation of CD8⁺T cells and its effector molecules were significantly enhanced or increased in both galectin-9 and PD-1 blockade. Conclusion ·Galectin-9⁺TAMs exhibit an immunosuppressive phenotype and function. Tumor-derived M-CSF induced TAMs to express galectin-9. Galectin-9⁺TAMs inhibit the function of CD8⁺T cells to promote the immune escape of MIBC. Galectin-9 and PD-1 blockade can reactivate the function of CD8⁺T cells more effectively and synergistically.