Proteomics of immune cells from liver tumors reveals immunotherapy targets [tumor]

Elucidating the mechanisms by which immune cells become dysfunctional in tumors is critical to developing next-generation immunotherapies. We profiled proteomes of cancer cells, as well as monocyte/macrophages, and CD4+ and CD8+ T cells isolated from tumors, livers, and blood of 48 patients with hepatocellular carcinoma. We found that tumor macrophages induce the sphingosine-1-phospate-degrading enzyme SGPL1, which dampened their inflammatory phenotype and anti-tumor function in vivo. We further discovered that the signaling scaffold protein AFAP1L2 is upregulated in chronically activated CD8+ T cells in tumors but is not present during the canonical T cell activation program. Ablation of AFAP1L2 in CD8+ T cells increased their viability upon repeated stimulation and enhanced their antitumor activity synergistically with PD-L1 blockade in mouse models. Our data revealed new targets for immunotherapy and provide a resource on immune cell proteomes in liver cancer (www.immunomics.ch/liver). Overall design: Fresh tumor tissue samples were collected. Tumors were enzymatically digested. Cell suspensions were filtered through, and mononuclear immune cells were separated from tumor, stromal cells and red blood cells by Ficoll gradient centrifugation. Mononuclear immune cells were stained with antibodies to CD45 and CD14, plus antibodies to CD8, CD4, CD56 to exclude other cell types and sorted. Cell pellets were washed and snap frozen in liquid nitrogen.

阐明免疫细胞在肿瘤内功能失调的机制，对于开发下一代免疫治疗方案至关重要。我们对48例肝细胞癌患者的肿瘤、肝脏及血液中分离得到的癌细胞、单核细胞/巨噬细胞，以及CD4+和CD8+ T细胞的蛋白质组（proteome）进行了谱分析。研究发现，肿瘤巨噬细胞可诱导鞘氨醇-1-磷酸降解酶SGPL1的表达，该酶会在体内削弱巨噬细胞的炎症表型与抗肿瘤功能。我们进一步揭示，信号转导支架蛋白AFAP1L2在肿瘤内慢性活化的CD8+ T细胞中表达上调，但在经典T细胞活化程序中并无该蛋白的表达。在小鼠模型中，敲除CD8+ T细胞内的AFAP1L2可提升其在反复刺激下的存活能力，并与PD-L1阻断疗法协同增强其抗肿瘤活性。本研究不仅揭示了免疫治疗的全新靶点，还为肝癌免疫细胞蛋白质组提供了研究资源（www.immunomics.ch/liver）。整体实验设计：收集新鲜肿瘤组织样本，通过酶解法消化肿瘤组织，将细胞悬液过滤后，采用Ficoll密度梯度离心法从肿瘤细胞、基质细胞及红细胞中分离出单核免疫细胞。随后使用靶向CD45、CD14的抗体，以及靶向CD8、CD4、CD56的抗体对单核免疫细胞进行染色，以排除其他细胞类型并完成细胞分选。收集细胞沉淀并洗涤后，将其置于液氮中快速冷冻保存。