High-throughput tools for decoding T cell receptor specificity

Abstract

T cells play a central role in adaptive immunity by recognizing specific antigens through their T cell receptors (TCRs). These receptors bind to peptides presented by major histocompatibility complex (pMHC) proteins, driving immune responses in cancer, infection, and autoimmunity. Understanding how TCRs recognize antigens is crucial for developing cancer immunotherapies and identifying therapeutic targets in autoimmunity, infectious disease, and allergy. However, large-scale mapping of TCR-antigen interactions remains a challenge due to the vast diversity of both TCRs and antigens, as well as the limitations in current screening technologies in cost, throughput, and accessibility. This work presents two advances in large-scale TCR-antigen screening. The first aim introduces a scalable and cost-effective platform for synthesizing tens of thousands of TCRs from sequence data to create synthetic TCR libraries. We integrate this approach with a high-throughput antigen discovery platform that leverages pMHC-pseudotyped viruses to identify TCR-pMHC pairs. Using this system, we screen 3,808 vitiligo patient-derived TCRs against 101 antigens, and synthesize 30,810 TCRs from patients with pancreatic ductal adenocarcinoma (PDAC). By streamlining TCR assembly and antigen screening, this pipeline has the potential to advance immunotherapy, accelerate vaccine design, and deepen our understanding of TCR recognition. The second aim presents a new method that couples pMHC-displaying virus-like particles with yeast display, enabling efficient screening of millions of TCR variants against ~100 pMHCs at once. Yeast display is a powerful tool for studying TCR-antigen interactions but is constrained by its reliance on recombinant protein production. Our approach overcomes this limitation by replacing recombinant protein with barcoded lentiviral particles, allowing large-scale, multiplexed screening of TCR libraries. By overcoming key technical barriers, these tools significantly expand our ability to study TCR specificity and engineer new antigen-specific therapeutics.