TIRTL-seq: Deep, quantitative, and affordable paired TCR repertoire sequencing 3040

Abstract Description α/β T cells are key players in adaptive immunity, with their specificity determined by T cell receptor (TCR) α and β chain sequences. While bulk TCR sequencing enables cost-effective repertoire profiling without chain pairing information, single-cell approaches provide paired data but are limited in throughput and costly. Here, we present TIRTL-seq (Throughput-Intensive Rapid TCR Library sequencing), a novel approach generating ready-to-sequence TCR libraries from live cells in less than 7 hours. The automated protocol uses non-contact liquid handlers in 384-well plates, with miniaturized reactions costing <$0.50/well. TIRTL-seq enables precise inference of clone frequencies and TCR chain pairings from well-occurrence patterns, scaling to 1 million unique paired αβTCR clonotypes at <$2000/sample. We benchmarked TIRTL-seq against state-of-the-art 5’RACE bulk TCR-seq and 10x Genomics Chromium technologies on longitudinal samples. We show that TIRTL-seq is able to quantitatively identify expanding and contracting clonotypes between timepoints while providing accurate TCR chain pairings, including distinct temporal dynamics of SARS-CoV-2-specific and EBV-specific CD8+ T cell responses after infection. While clonal expansion was followed by sharp contraction for SARS-CoV-2 specific TCRs, EBV-specific TCRs remained stable once established. We anticipate that our paired TCR-seq method will be instrumental for extracting therapeutically relevant information from the TCR repertoire. Funding Sources Supported by grants AI136514, AI144616, and AI165077, the St. Jude Center for Influenza Research and Response (SJCEIRR) contract 75N93021C00016, the Center for Influenza Vaccine Research in High Risk Populations (CIVR-HRP) contract 75N93019C00052, the TIRTL Bluesky Initiative, and ALSAC at St. Jude. Topic Categories Technological Innovations in Immunology (TECH)