Discovery of rare antigen-specific TCRs via replicate profiling

Abstract Development of effective vaccines and targeted immunotherapies for cancer, autoimmunity, allergy, and infectious diseases requires comprehensive understanding of functionality and antigenic specificity of involved T cell clones. A major technical challenge remains the high-throughput identification of antigen-specific T cells. Here, we present a rapid cost-efficient replicate-based TCR discovery assay starting from PBMC that enables sensitive identification of clonal alpha–beta paired TCRs responding to individual or pooled peptides. In a small-scale experiment with a single donor, assay identified over 90 CD4 + and CD8 + TCRβ clonotypes selectively and reproducibly expanding in presence of SARS-CoV-2 peptide pools. Their specificity was validated by time tracking and comparison against known SARS-CoV-2-specific TCRs. Further experiments on a cohort of five donors identified nearly 1,000 CD4 + and CD8 + TCR clonotypes responding to viral and fungal peptide antigens. The assay demonstrated high sensitivity in capturing low-frequency clones—represented by 2–3 T cells per million, and allowed frequency correlation-based TCRα/TCRβ pairing, validated using single-cell transcriptomics and reporter TCR cell lines. TCR positioning within the scRNA-Seq map revealed distinct helper T cell subsets involved in primary and secondary response to SARS-CoV-2, establishing an integrated pipeline that links antigen-responsive clones to their precise functional phenotypes.