BackgroundProgrammed cell death protein 1 (PD-1) receptor has two ligands,programmed death-ligand 1 (PD-L1) and PD-L2. When compared with PD-L1, PD-L2 has not received much attention, and its role remains unclear.MethodsThe expression profiles of pdcd1lg2 (PD-L2-encoding gene) mRNA and PD-L2 protein were analyzed using TCGA, ICGC, and HPA databases. Kaplan-Meier and Cox regression analyses were used to assess the prognostic significance of PD-L2. We used GSEA, Spearman’s correlation analysis and PPI network to explore the biological functions of PD-L2. PD-L2-associated immune cell infiltration was evaluated using the ESTIMATE algorithm and TIMER 2.0. The expressions of PD-L2 in tumor-associated macrophages (TAMs) in human colon cancer samples, and in mice in an immunocompetent syngeneic setting were verified using scRNA-seq datasets, multiplex immunofluorescence staining, and flow cytometry. After fluorescence-activated cell sorting, flow cytometry and qRT-PCR and transwell and colony formation assays were used to evaluate the phenotype and functions of PD-L2+TAMs. Immune checkpoint inhibitors (ICIs) therapy prediction analysis was performed using TIDE and TISMO. Last, a series of targeted small-molecule drugs with promising therapeutic effects were predicted using the GSCA platform.ResultsPD-L2 was expressed in all the common human cancer types and deteriorated outcomes in multiple cancers. PPI network and Spearman’s correlation analysis revealed that PD-L2 was closely associated with many immune molecules. Moreover, both GSEA results of KEGG pathways and GSEA results for Reactome analysis indicated that PD-L2 expression played an important role in cancer immune response. Further analysis showed that PD-L2 expression was strongly associated with the infiltration of immune cells in tumor tissue in almost all cancer types, among which macrophages were the most positively associated with PD-L2 in colon cancer. According to the results mentioned above, we verified the expression of PD-L2 in TAMs in colon cancer and found that PD-L2+TAMs population was not static. Additionally, PD-L2+TAMs exhibited protumor M2 phenotype and increased the migration, invasion, and proliferative capacity of colon cancer cells. Furthermore, PD-L2 had a substantial predictive value for ICIs therapy cohorts.ConclusionPD-L2 in the TME, especially expressed on TAMs, could be applied as a potential therapeutic target.

中文翻译：

---

泛癌分析确定 PD-L2 是肿瘤微环境中的肿瘤启动子

研究背景程序性细胞死亡蛋白1（PD-1）受体有两种配体，程序性死亡配体1（PD-L1）和PD-L2。与PD-L1相比，PD-L2并未受到太多关注，其作用仍不清楚。方法PDCD1LG2使用 TCGA、ICGC 和 HPA 数据库分析（PD-L2 编码基因）mRNA 和 PD-L2 蛋白。Kaplan-Meier 和 Cox 回归分析用于评估 PD-L2 的预后意义。我们利用GSEA、Spearman相关分析和PPI网络来探索PD-L2的生物学功能。使用 ESTIMATE 算法和 TIMER 2.0 评估 PD-L2 相关免疫细胞浸润。使用 scRNA-seq 数据集、多重免疫荧光染色和流式细胞术验证人类结肠癌样本中的肿瘤相关巨噬细胞 (TAM) 以及免疫活性同基因环境中的小鼠中 PD-L2 的表达。荧光激活细胞分选后，使用流式细胞术、qRT-PCR、Transwell 和集落形成测定来评估 PD-L2 的表型和功能+TAM。使用 TIDE 和 TISMO 进行免疫检查点抑制剂 (ICIs) 治疗预测分析。最后，利用GSCA平台预测了一系列具有良好治疗效果的靶向小分子药物。结果PD-L2在所有常见的人类癌症类型中表达，并且在多种癌症中恶化结果。PPI网络和Spearman相关分析显示PD-L2与许多免疫分子密切相关。此外，KEGG通路的GSEA结果和Reactome分析的GSEA结果均表明PD-L2表达在癌症免疫反应中发挥重要作用。进一步分析表明PD-L2在几乎所有癌症类型中，PD-L2的表达与肿瘤组织中免疫细胞的浸润密切相关，其中巨噬细胞与结肠癌中PD-L2的正相关最为显着。根据上述结果，我们验证了结肠癌TAMs中PD-L2的表达情况，发现PD-L2+TAM 的数量并不是静态的。此外，PD-L2+TAM 表现出促肿瘤 M2 表型，并增加结肠癌细胞的迁移、侵袭和增殖能力。此外，PD-L2对ICIs治疗队列具有显着的预测价值。结论TME中的PD-L2，特别是在TAM上表达的PD-L2可以作为潜在的治疗靶点。