biostudies-literatureNagaoka KJSPS KAKENHIJapan Agency for Medical Research and Development (AMED)1789https://www.ebi.ac.uk/biostudies/studies/S-EPMC12650950biostudies-literatureUnknown14(22)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-TCR Pairing Assay using reporter T cells), a functional assay based on co-culture of TCR-T reporter cells and autologous antigen-presenting cells (APCs) to screen neoantigen-TCR 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 tCellsNeoPAIR-T: Functional Mapping of Neoantigen-TCR Pairs Using a CRISPR-Engineered Jurkat Reporter System.PMC1265095024ama221321h000223H02762Nagaoka KKobayashi YKakimi KfalseNeoPAIR-T: Functional Mapping of Neoantigen-TCR Pairs Using a CRISPR-Engineered Jurkat Reporter System.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-TCR Pairing Assay using reporter T cells), a functional assay based on co-culture of TCR-T reporter cells and autologous antigen-presenting cells (APCs) to screen neoantigen-TCR 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 t2025-01-01T00:00:00Z2025 Nov2026-05-20T03:20:19.081Z2026-05-20T03:13:48.265ZS-EPMC126509504129484210.3390/cells14221789