{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,18]],"date-time":"2025-11-18T05:15:13Z","timestamp":1763442913071,"version":"3.45.0"},"reference-count":43,"publisher":"MDPI AG","issue":"22","license":[{"start":{"date-parts":[[2025,11,14]],"date-time":"2025-11-14T00:00:00Z","timestamp":1763078400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"crossref","award":["24ama221321h0002"],"award-info":[{"award-number":["24ama221321h0002"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"crossref"}]},{"name":"JSPS KAKENHI","award":["23H02762"],"award-info":[{"award-number":["23H02762"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Targeting mutation-derived neoantigens is a promising strategy for personalized immunotherapies. However, identifying true neoantigens and cognate T cell receptors (TCRs) remains challenging because computational prediction of neoantigen peptides is uncertain and most tumor-infiltrating lymphocytes are bystanders rather than tumor-reactive, necessitating functional validation. Here, we developed NeoPAIR-T (Neoantigen\u2013TCR Pairing Assay using reporter T cells), a functional assay based on co-culture of TCR\u2013T reporter cells and autologous antigen-presenting cells (APCs) to screen neoantigen\u2013TCR pairs. Reporter T cells are Jurkat-derived cells engineered to express a luciferase\/eGFP dual reporter, providing quantitative readouts of TCR activation, while APCs are immortalized autologous cells transfected with tandem minigenes (TMGs) encoding predicted neoantigens, bypassing peptide synthesis. NeoPAIR-T also includes TCR\u03b1-knockout with targeted knock-in of candidate TCRs at the TCR\u03b2 locus to prevent mispairing and enables parallel testing of multiple reporter T cell clones co-cultured with the same APCs for efficient identification of functional pairs. Using lung cancer samples, whole-exome and RNA sequencing predicted 63 candidate peptides assembled into three TMGs. Single-cell RNA\/TCR sequencing identified eight TCR clonotypes, introduced into reporter T cells and tested in parallel. Co-culture with TMG-expressing APCs revealed two functional neoantigen\u2013TCR pairs validated by peptide assays (EC50: 10\u22129.2\u201310\u22126.7 M). Collectively, NeoPAIR-T streamlines neoantigen\u2013TCR identification for vaccine and TCR-T applications.<\/jats:p>","DOI":"10.3390\/cells14221789","type":"journal-article","created":{"date-parts":[[2025,11,14]],"date-time":"2025-11-14T14:37:52Z","timestamp":1763131072000},"page":"1789","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["NeoPAIR-T: Functional Mapping of Neoantigen\u2013TCR Pairs Using a CRISPR-Engineered Jurkat Reporter System"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8202-031X","authenticated-orcid":false,"given":"Koji","family":"Nagaoka","sequence":"first","affiliation":[{"name":"Department of Immunology, Kindai University Faculty of Medicine, Sakai 590-0197, Osaka, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2600-2181","authenticated-orcid":false,"given":"Yukari","family":"Kobayashi","sequence":"additional","affiliation":[{"name":"Department of Immunology, Kindai University Faculty of Medicine, Sakai 590-0197, Osaka, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2631-3040","authenticated-orcid":false,"given":"Kazuhiro","family":"Kakimi","sequence":"additional","affiliation":[{"name":"Department of Immunology, Kindai University Faculty of Medicine, Sakai 590-0197, Osaka, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1126\/science.aaa4971","article-title":"Neoantigens in cancer immunotherapy","volume":"348","author":"Schumacher","year":"2015","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"474","DOI":"10.1038\/s41586-024-08507-5","article-title":"A neoantigen vaccine generates antitumour immunity in renal cell carcinoma","volume":"639","author":"Braun","year":"2025","journal-title":"Nature"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1038\/s41591-024-03334-7","article-title":"Autogene cevumeran with or without atezolizumab in advanced solid tumors: A phase 1 trial","volume":"31","author":"Lopez","year":"2025","journal-title":"Nat. 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