Peptide Driven Identification of TCRs (PDI-TCR) reveals dynamics and phenotypes of CD4 T cells in tuberculosis

Accurately assigning antigen specificity to T cell receptor (TCR) sequences is challenging due to the complexity of the TCR-antigen recognition process in humans. We developed the Peptide-Driven Identification of TCRs (PDI-TCR) assay, a novel method combining in vitro expansion with peptide pools, bulk TCR sequencing, and statistical analysis to identify antigen-specific TCRs from human blood. A key feature of PDI-TCR is its ability to account for bystander T cell expansion using both antigen-specific and non-specific peptide pools, distinguishing true antigen-specific TCRs from the unspecific ones. We applied PDI-TCR to Tuberculosis (TB) patients, sampling blood at diagnosis and throughout therapy, and Mycobacterium tuberculosis (Mtb)-sensitized healthy individuals (IGRA+). We identified hundreds of Mtb-specific and unspecific TCRs, and in combination with single-cell mRNA and TCR sequencing of CD4 T cells, we characterized the transcriptome of Mtb-specific and unspecific T cells in each cohort. Mtb-specific T cells were highly diverse, with the presence of short-lived effector phenotypes only in TB at diagnosis and memory phenotypes maintained through treatment. In contrast, unspecific T cells were more clonally restricted and expanded, cytotoxic, and maintained through treatment. Thus, PDI-TCR is a powerful tool for identifying antigen-specific TCRs. Combined with single-cell sequencing of T cell populations, it enables direct ex vivo monitoring of antigen-specific T cells.