Scalable TCR synthesis and screening enable antigen reactivity mapping in vitiligo.

T cells initiate targeted immune responses using T cell receptors (TCRs) to recognize specific antigens. Mapping TCRs to antigens at scale remains a major challenge. Here, we developed an approach to synthesize and functionally screen tens of thousands of TCRs simultaneously. TCR rapid assembly for functional testing (TCRAFT) uses a modular strategy to rapidly and inexpensively construct large pools of TCRs from sequences while maintaining TCRα/β pairing. We applied TCRAFT to reconstruct over 3,800 TCRs from vitiligo blister fluid and mapped these TCRs to specific peptide-major histocompatibility complexes using RAPTR, an activation-based library-on-library screening approach. Vitiligo antigen-specific T cells displayed pronounced clonal expansion and transcriptomic signatures similar to antigen-specific T cells in melanoma, pointing to shared features of disease-relevant T cells in autoimmunity and cancer. Demonstrating scalability, we synthesized and screened over 30,800 TCRs from donors with pancreatic ductal adenocarcinoma to capture antigen-reactive TCRs. Our approach expands the scale and accessibility of TCR-antigen screening, which is critical to understanding immunity and developing new immunotherapies.