{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T06:49:55Z","timestamp":1791355795118,"version":"4.3.1"},"reference-count":46,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T00:00:00Z","timestamp":1791331200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100020595","name":"National Science and Technology Council","doi-asserted-by":"publisher","award":["NSTC113-2314-B-038-137, NSTC14-2314-B-038-013"],"award-info":[{"award-number":["NSTC113-2314-B-038-137, NSTC14-2314-B-038-013"]}],"id":[{"id":"10.13039\/100020595","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"\n Background<\/jats:title>\n \n The tumor microenvironment exerts intense survival pressure on CD8\n +<\/jats:sup>\n tumor-infiltrating lymphocytes (TILs), yet the cellular basis for an earlier, less-exhausted state associated with response to immune checkpoint blockade (ICB) in non-small-cell lung cancer (NSCLC) remains unclear. We previously identified\n BCL2A1<\/jats:italic>\n as a CD8\n +<\/jats:sup>\n TIL-derived marker predictive of a response to PD-1 blockade. Here we combine deep single-cell discovery with experimental validation in primary human T cells to characterise the BCL2A1-associated CD8\n +<\/jats:sup>\n TIL state and its candidate upstream signal.\n <\/jats:p>\n <\/jats:sec>\n \n Methods<\/jats:title>\n \n We integrated four transcriptomic cohorts\u2014pre-treatment bulk RNA-seq with CD8\n +<\/jats:sup>\n TIL deconvolution (n=60), on-treatment scRNA-seq\/TCR-seq (n=11), longitudinal scRNA-seq\/TCR-seq (n=18), and an independent post-treatment cohort (n=59)\u2014with in silico perturbation, pathway, and metabolic-flux analyses, and experimentally tested a key prediction of this state by flow cytometry in primary human CD8\n +<\/jats:sup>\n T cells.\n <\/jats:p>\n <\/jats:sec>\n \n Results<\/jats:title>\n \n BCL2A1<\/jats:italic>\n high<\/jats:sup>\n CD8\n +<\/jats:sup>\n TILs showed elevated BCL2A1 expression without coordinated upregulation of anti-apoptotic BCL2-family genes, maintained minimal exhaustion, and occupied an early,\n BCL6<\/jats:italic>\n -,\n TCF7<\/jats:italic>\n -, and\n NFKB1<\/jats:italic>\n -regulated pseudotime state. Across longitudinal and post-treatment cohorts, they formed diverse, low-expansion repertoires rather than dominant expanded clones, with reduced biosynthetic flux (branched-chain amino acid and purine metabolism) consistent with a non-proliferative state. NF-\u03baB signaling was elevated, and TNF-\u03b1 was nominated as the leading candidate upstream ligand of\n BCL2A1<\/jats:italic>\n . We then experimentally tested this prediction: in primary human CD8\n +<\/jats:sup>\n T cells, TNF-\u03b1 induced\n BCL2A1<\/jats:italic>\n protein \u224813-fold (44.5% vs. 3.4% BCL2A1\n +<\/jats:sup>\n ; MFI 2.93-fold; P &lt; 0.0001), whereas CD3\/CD28 activation alone did not, establishing a TNF-\u03b1-specific, protein-level response.\n <\/jats:p>\n <\/jats:sec>\n \n Conclusions<\/jats:title>\n \n Combining integrative single-cell analysis with experimental validation in primary human T cells, we define\n BCL2A1<\/jats:italic>\n as a marker of an early, less-exhausted CD8\n +<\/jats:sup>\n TIL state associated with improved ICB outcomes in NSCLC; a survival-related role is supported by these associations but was not functionally tested.\n <\/jats:p>\n <\/jats:sec>","DOI":"10.3389\/fimmu.2026.1908216","type":"journal-article","created":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T05:53:25Z","timestamp":1791352405000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["BCL2A1 marks an early, less-exhausted CD8+ tumor-infiltrating lymphocyte state associated with PD-1 blockade response in NSCLC"],"prefix":"10.3389","volume":"17","author":[{"given":"Hoang Minh Quan","family":"Pham","sequence":"first","affiliation":[{"name":"International Ph.D. Program in Medicine, College of Medicine, Taipei Medical University","place":["Taipei, Taiwan"]},{"name":"Department of Oncology, Faculty of Medicine, Can Tho University of Medicine and Pharmacy","place":["Can Tho, Vietnam"]},{"name":"Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University","place":["Taipei, Taiwan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chia-Ling","family":"Chen","sequence":"additional","affiliation":[{"name":"School of Respiratory Therapy, College of Medicine, Taipei Medical University","place":["Taipei, Taiwan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Po-Hao","family":"Feng","sequence":"additional","affiliation":[{"name":"Division of Pulmonary Medicine, Department of Internal Medicine, Shuang Ho Hospital, Taipei Medical University","place":["New Taipei, Taiwan"]},{"name":"Division of Pulmonary Medicine, Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University","place":["Taipei, Taiwan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kang-Yun","family":"Lee","sequence":"additional","affiliation":[{"name":"School of Respiratory Therapy, College of Medicine, Taipei Medical University","place":["Taipei, Taiwan"]},{"name":"Division of Pulmonary Medicine, Department of Internal Medicine, Shuang Ho Hospital, Taipei Medical University","place":["New Taipei, Taiwan"]},{"name":"Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University","place":["Taipei, Taiwan"]},{"name":"TMU Research Center of Thoracic Medicine, Taipei Medical University","place":["Taipei, Taiwan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu-Ping","family":"Hung","sequence":"additional","affiliation":[{"name":"Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University","place":["Taipei, Taiwan"]},{"name":"Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University","place":["Taipei, Taiwan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chiou-Feng","family":"Lin","sequence":"additional","affiliation":[{"name":"International Ph.D. Program in Medicine, College of Medicine, Taipei Medical University","place":["Taipei, Taiwan"]},{"name":"Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University","place":["Taipei, Taiwan"]},{"name":"TMU Research Center of Thoracic Medicine, Taipei Medical University","place":["Taipei, Taiwan"]},{"name":"Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University","place":["Taipei, Taiwan"]},{"name":"Core Laboratory of Immune Monitoring, Office of Research & Development, Taipei Medical University","place":["Taipei, Taiwan"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,10,7]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1056\/nejmoa1917346","article-title":"Atezolizumab for first-line treatment of PD-L1-selected patients with NSCLC","volume":"383","author":"Herbst","year":"2020","journal-title":"N Engl J Med"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1200\/jco.21.00174","article-title":"Five-year 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