Identification of Antigen-Specific T Cell Receptors with combinatorial peptide pooling

T cell receptor (TCR) repertoire diversity enables the antigen-specific immune responses against the vast space of possible pathogens. Identifying TCR-antigen binding pairs from the large TCR repertoire and antigen space is crucial for biomedical research. Here, we introduce copepodTCR, an open-access tool to design and interpret high-throughput experimental TCR specificity assays. copepodTCR implements a combinatorial peptide pooling scheme for efficient experimental testing of T cell responses against large overlapping peptide libraries, that can be used to identify the specificity of (or "deorphanize") TCRs. The scheme detects experimental errors and, coupled with a hierarchical Bayesian model for unbiased interpretation, identifies the response-eliciting peptide sequence for a TCR of interest out of hundreds of peptides tested using a simple experimental set-up. Using in silico simulations, we demonstrate the varied experimental settings in which copepodTCR yields efficient and interpretable TCR specificity results. We validated our approach on a library of 253 overlapping peptides covering the SARS-CoV-2 spike protein, split across 12 pools. A single stimulation with combinatorial pools identified the correct epitope of two TCRs with known specificity and then deorphanized two SARS-CoV-2 associated TCRs shared among a large cohort of COVID-19 patients. We provide experimental guides to efficiently design larger screens covering thousands of peptides which will be crucial to identify antigen-specific T cells and their targets from limited clinical material.