T cell antigenic recognition sequencing (TAR-seq) enables precise characterization of vaccine-elicited cellular immunity ex vivo and

Introduction: The functional responses of antigen (Ag) specific T cells are complex, clonotype-specific, context-dependent, and incompletely captured by dominant analytic approaches. Here we present T Cell Antigenic Recognition Sequencing (TAR-seq), a two-part process that uses single-cell RNA + T cell receptor sequencing (scRNA/TCR-seq) to provide a precise and comprehensive characterization of the differentiation state and functional response of T cells specific to a given antigen, Methods: Many approaches use stimulation and activation-induced markers to identify Ag-specific T cells. We extend this by adding an scRNA/TCR-seq readout to identify the responding TCRs for each subject. We performed rigorous validation using tetramer-sorted T cells and developed a reusable probabilistic model to differentiate TCR-stimulated cells from bystander activation. These TCRs provide molecular barcodes to identify Ag-specific cells from unsorted scRNA/TCR-seq data, irrespective of stimulation, enabling a comprehensive view of their activity. Results: We applied this method to a cohort of SIV-vaccinated rhesus macaques (RMs). We performed SIV-infected cell recognition assays to identify the SIV-specific TCRs for each RM. We used these TCRs to subset SIV-specific cells and perform precise contrasts. Despite identical vaccination, we identified clone- and subject-level variability in their cytotoxic differentiation and cytokine production after incubation with SIV-infected targets. Per clonotype, the percentage of cells that responded after antigen exposure varied widely. Finally, we used TCRs as barcodes to precisely map the Discussion: Together, our data validate a powerful method to comprehensively understand T cell activity, with relevance to infectious disease, cancer, and autoimmunity, across species.