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However, the characteristics of CD8<jats:sup>+<\/jats:sup> TILs, especially T-cell populations specific for tumor antigens, remain poorly understood.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>High throughput single-cell RNA sequencing and single-cell T-cell receptor (TCR) sequencing were performed on CD8<jats:sup>+<\/jats:sup> TILs from three surgically-resected lung cancer specimens. Dimensional reduction for clustering was performed using Uniform Manifold Approximation and Projection. CD8<jats:sup>+<\/jats:sup> 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.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>A total of 6998\u2009CD8<jats:sup>+<\/jats:sup> 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 <jats:italic>ENTPD1<\/jats:italic> (CD39), <jats:italic>TOX<\/jats:italic>, <jats:italic>PDCD1<\/jats:italic> (PD1), <jats:italic>HAVCR2<\/jats:italic> (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.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Conclusions<\/jats:title>\n                  <jats:p>Our approach focusing on T cells with an exhausted phenotype among CD8<jats:sup>+<\/jats:sup> 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.<\/jats:p>\n               <\/jats:sec>","DOI":"10.1136\/jitc-2023-007180","type":"journal-article","created":{"date-parts":[[2023,8,6]],"date-time":"2023-08-06T20:25:12Z","timestamp":1691353512000},"page":"e007180","update-policy":"https:\/\/doi.org\/10.1136\/crossmarkpolicy","source":"Crossref","is-referenced-by-count":42,"title":["Single-cell sequencing on CD8<sup>+<\/sup> TILs revealed the nature of exhausted T cells recognizing neoantigen and cancer\/testis antigen in non-small cell lung cancer"],"prefix":"10.1136","volume":"11","author":[{"given":"Hiroyasu","family":"Komuro","sequence":"first","affiliation":[{"name":"Division of Translational Oncoimmunology, Aichi Cancer Center Research Institute, Nagoya, Japan"},{"name":"Department of General Thoracic Surgery, Gifu University School of Medicine Graduate School of Medicine, Gifu, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuichi","family":"Shinohara","sequence":"additional","affiliation":[{"name":"Division of Translational Oncoimmunology, Aichi Cancer Center Research Institute, Nagoya, Japan"},{"name":"Department of Thoracic Surgery, Aichi Cancer Center Hospital, Nagoya, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yasunori","family":"Fukushima","sequence":"additional","affiliation":[{"name":"Division of 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