深入了解非小细胞肺癌（NSCLC）中免疫抑制通路的机制非常重要，以开发新的诊断和治疗方法。在这里，我们调查了CD39和CD73在NSCLC中的预后意义。利用多重免疫荧光技术对162例早期未经治疗的NSCLC患者的CD39、CD73和CD103的表达和定位进行了数字化定量分析，并与患者预后进行了相关研究。通过流式细胞术评估了不同细胞群体中的表达情况。对消化的NSCLC肿瘤组织中的CD4+和CD8+T细胞进行了靶向RNA-Seq，分析了单细胞RNA-Seq数据以探究CD39+ T细胞群体的功能意义。 我们证明了早期未经治疗的NSCLC患者的流式细胞仪显示瘤组织中天然杀伤细胞（NK细胞）、成纤维细胞和T细胞中CD39表达上调。CD73表达主要在肿瘤组织中的成纤维细胞和Epcam+细胞中发现。对162例早期未经治疗的NSCLC患者进行的多重免疫荧光分析表明，CD39表达主要定位于肿瘤间质，而CD73表达在肿瘤巢和间质之间均匀分布，在肿瘤间质中CD39和CD73的高表达与5年内无复发生存（RFS）不良有关。另外，我们发现定位于肿瘤巢中的CD8+T细胞表达CD103，并且肿瘤巢中CD39+CD103+CD8+ T细胞的密度预测了5年内改善的RFS。靶向RNA-Seq显示NSCLC的肿瘤微环境在CD4+ T细胞中上调调节性途径，在CD8+ T细胞中引起衰竭，并且通过单细胞RNA测序数据分析表明，CD39+CD4+细胞富集了调节性T细胞特异基因集，而CD39+CD103+细胞毒性T淋巴细胞表现出衰竭的毒性表型特征，CXCL13的表达上调。要了解CD39+ T细胞群体在NSCLC中的预后影响，需要了解其分布和定位的模式。本研究提供了对CD39+ T细胞的空间和功能特征及其与患者预后的重要性的深入了解。 ©作者（或其雇主）2023。遵循CC BY-NC许可进行再使用。无商业再利用。参见权利和权限。由BMJ出版。

An improved mechanistic understanding of immunosuppressive pathways in non-small cell lung cancer (NSCLC) is important to develop novel diagnostic and therapeutic approaches. Here, we investigate the prognostic significance of the ectonucleotidases CD39 and CD73 in NSCLC.The expression and localization of CD39, CD73 and CD103 was digitally quantified in a cohort of 162 early treatment naïve NSCLC patients using multiplex-immunofluorescence and related to patient outcome. Expression among different cell-populations was assessed via flow cytometry. Targeted RNA-Seq was performed on CD4+ and CD8+ T cells from digested NSCLC tumor tissue and single-cell RNA-Seq data was analyzed to investigate the functional significance of CD39+ T cell populations.We demonstrate that flow cytometry of early untreated NSCLC patients shows an upregulation of CD39 expression in the tumor tissue among natural killer (NK) cells, fibroblasts and T cells. CD73 expression is mainly found among fibroblasts and Epcam+cells in the tumor tissue. Multiplex Immunofluorescence in a cohort of 162 early untreated NSCLC patients demonstrates that CD39 expression is mainly localized in the tumor stroma while CD73 expression is equally distributed between tumor nest and stroma, and high expression of CD39 and CD73 in the tumor stroma is associated with poor recurrence-free survival (RFS) at 5 years. Additionally, we find that CD8+T cells located in the tumor nest express CD103 and the density of CD39+CD103+CD8+ T cells in the tumor nest predicts improved RFS at 5 years. Targeted RNA-Seq shows that the tumor microenvironment of NSCLC upregulates regulatory pathways in CD4+ T cells and exhaustion in CD8+ T cells, and analysis of a single cell RNA sequencing dataset shows that CD39+CD4+ cells are enriched in Treg signature gene-sets, and CD39+CD103+ cytotoxic T lymphocyte show gene signatures indicative of an exhausted cytotoxic phenotype with upregulated expression of CXCL13.Knowledge of patterns of distribution and location are required to understand the prognostic impact of CD39+ T cell populations in NSCLC. This study provides an improved understanding of spatial and functional characteristics of CD39+ T cells and their significance to patient outcome.© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.