AbstractAntigen discovery technologies have largely focused on major histocompatibility complex (MHC) class I-restricted human T cell receptors (TCRs), leaving methods for MHC class II-restricted and mouse TCR reactivities relatively undeveloped. Here we present TCR mapping of antigenic peptides (TCR-MAP), an antigen discovery method that uses a synthetic TCR-stimulated circuit in immortalized T cells to activate sortase-mediated tagging of engineered antigen-presenting cells (APCs) expressing processed peptides on MHCs. Live, tagged APCs can be directly purified for deconvolution by sequencing, enabling TCRs with unknown specificity to be queried against barcoded peptide libraries in a pooled screening context. TCR-MAP accurately captures self-reactivities or viral reactivities with high throughput and sensitivity for both MHC class I-restricted and class II-restricted TCRs. We elucidate problematic cross-reactivities of clinical TCRs targeting the cancer/testis melanoma-associated antigen A3 and discover targets of myocarditis-inciting autoreactive T cells in mice. TCR-MAP has the potential to accelerate T cell antigen discovery efforts in the context of cancer, infectious disease and autoimmunity.