Tippalagama, R.; Trevizani, R.; Chihab, L.; Chawla, A.; Fung, K.; Greenbaum, J.; Kearns, K.; De Silva, A.; Gunasinghe, W.; Perera, J.; Gunasekera, H.; Senevirathne, D.; Scriba, T.; Sette, A.; Arlehamn, C. L.; Burel, J.; Peters, B.

Assigning antigen specificity to T cell receptor (TCR) sequences is challenging due to the TCR repertoire\'s diversity and the complexity of TCR:antigen recognition. We developed the Peptide-Driven Identification of TCRs (PDI-TCR) assay that combines in vitro expansion of cells with peptide pools, bulk TCR sequencing, and statistical analysis to identify antigen-specific TCRs from human blood. A key feature of PDI-TCR is the ability to distinguish true antigen-specific TCR clonotypes from TCRs associated with unspecific bystander activation by comparing responses to non-overlapping peptide pools. We applied PDI-TCR to Tuberculosis (TB) patients, sampling blood at diagnosis and throughout treatment, and Mycobacterium tuberculosis (Mtb)-sensitized healthy individuals (IGRA+). We identified hundreds of Mtb-specific TCRs, as well as unspecific TCRs, and characterized their phenotype in each cohort by single-cell RNA sequencing ex vivo. Mtb-specific T cells were highly diverse, with short-lived effector phenotypes only present in TB at diagnosis, while memory phenotypes were maintained through treatment. In contrast, unspecific expanded T cells were more clonally restricted, had a cytotoxic phenotype, and were maintained throughout treatment. This showcases PDI-TCR as a powerful tool for identifying antigen-specific TCRs, which enables direct ex vivo identification and monitoring of antigen-specific T cells.