CD8+肿瘤浸润淋巴细胞（TILs）经常在非小细胞肺癌（NSCLC）中观察到。然而，CD8+ TILs的特征，尤其是针对肿瘤抗原的T细胞人群，尚不明确。本研究对来自三例手术切除的肺癌标本中的CD8+ TILs进行了高通量单细胞RNA测序和单细胞T细胞受体（TCR）测序。通过统一流形近似和投影对聚类进行了维度缩减。研究了特异性靶向肿瘤抗原KK-LC-1和预测新抗原的CD8+ TIL TCR。进行了差异表达基因分析、基因集富集分析（GSEA）和单样本GSEA以对特异性抗原T细胞进行表征。共分析了6998个CD8+ T细胞，根据其基因表达谱将其分为10个聚类。发现了通过ENTPD1（CD39）、TOX、PDCD1（PD1）、HAVCR2（TIM3）等基因的表达以及T细胞寡克隆性来表征的枯竭T细胞（exhausted T (Tex)）聚类。Tex TCR库（Tex-TCRs）包括9个不同的TCR克隆型，可以识别5个肿瘤抗原，包括KK-LC-1抗原和4个新抗原。通过对肿瘤抗原特异性T细胞（n=140）进行重新聚类，可以看到即使在同一Tex聚类内，个体T细胞克隆型也存在在不同分化阶段和功能状态的细胞上。用预测的亲和肽刺激这些T细胞，表明TCR信号强度及随后的T细胞增殖和细胞因子产生是可变的，但相对于KK-LC-1，新抗原总是更高。我们的方法将重点放在CD8+ TILs中表现出枯竭表型的T细胞上，这有助于鉴定肿瘤抗原并阐明特定肿瘤抗原特异性T细胞的性质，以确定非小细胞肺癌患者中有前景的免疫治疗靶点。© 作者（或其雇主）2023。在CC BY-NC下允许重新使用。不得进行商业再利用。详见权限和版权。由BMJ出版。

CD8+tumor infiltrating lymphocytes (TILs) are often observed in non-small cell lung cancers (NSCLC). However, the characteristics of CD8+ TILs, especially T-cell populations specific for tumor antigens, remain poorly understood.High throughput single-cell RNA sequencing and single-cell T-cell receptor (TCR) sequencing were performed on CD8+ TILs from three surgically-resected lung cancer specimens. Dimensional reduction for clustering was performed using Uniform Manifold Approximation and Projection. CD8+ TIL TCR specific for the cancer/testis antigen KK-LC-1 and for predicted neoantigens were investigated. Differentially-expressed gene analysis, Gene Set Enrichment Analysis (GSEA) and single sample GSEA was performed to characterize antigen-specific T cells.A total of 6998 CD8+ T cells was analyzed, divided into 10 clusters according to their gene expression profile. An exhausted T-cell (exhausted T (Tex)) cluster characterized by the expression of ENTPD1 (CD39), TOX, PDCD1 (PD1), HAVCR2 (TIM3) and other genes, and by T-cell oligoclonality, was identified. The Tex TCR repertoire (Tex-TCRs) contained nine different TCR clonotypes recognizing five tumor antigens including a KK-LC-1 antigen and four neoantigens. By re-clustering the tumor antigen-specific T cells (n=140), it could be seen that the individual T-cell clonotypes were present on cells at different stages of differentiation and functional states even within the same Tex cluster. Stimulating these T cells with predicted cognate peptide indicated that TCR signal strength and subsequent T-cell proliferation and cytokine production was variable but always higher for neoantigens than KK-LC-1.Our approach focusing on T cells with an exhausted phenotype among CD8+ TILs may facilitate the identification of tumor antigens and clarify the nature of the antigen-specific T cells to specify the promising immunotherapeutic targets in patients with NSCLC.© 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.