We present tetramer-associated T-cell receptor sequencing (TetTCR-seq) to link T cell receptor (TCR) sequences to their cognate antigens in single cells at high throughput. Binding is determined using a library of DNA-barcoded antigen tetramers that is rapidly generated by in vitro transcription and translation. We applied TetTCR-seq to identify patterns in TCR cross-reactivity with cancer neoantigens and to rapidly isolate neoantigen-specific TCRs with no cross-reactivity to the wild-type antigen.
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We thank B. Wendel for discussions and for producing recombinant HLA-A2; M.M. Davis and H. Huang at Stanford University for discussion of the lentiviral transduction protocol and providing a template TCR construct and HLA-A2 construct; W. Uckert at the Max Delbruck Center for Molecular Medicine for sharing the Jurkat 76 cell line; A. Brock at University of Texas Austin for sharing the HEK 293T cell line; J. Lou at the Institute of Biophysics, Chinese Academy of Sciences, for helping with HCV APL prediction; P. Parker, K. Patel and H. Pan for assistance with initial prototyping; and the NIH tetramer center for additional pMHC tetramer reagents. We also thank anonymous blood donors and staff members at We Are Blood for sample collection. This work was supported by NIH grants R00AG040149 (N.J.), S10OD020072 (N.J.) and R33CA225539 (N.J.), by NSF CAREER Award 1653866 (N.J.), by Welch Foundation grant F1785 (N.J.), by the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation (N.J.) and by National Natural Science Foundation of China major international (regional) joint research project 81220108006 (W.J.) and NSFC-NHMRC joint research grant 81561128016 (W.J.). N.J. is a Cancer Prevention and Research Institute of Texas (CPRIT) Scholar and a Damon Runyon-Rachleff Innovator. S.-Q.Z. is a recipient of Thrust 2000 – Archie W. Straiton Endowed Graduate Fellowship in Engineering No. 1. A.A.S. is a recipient of the Cockrell School of Engineering fellowship and the Thrust 2000 – Mario E. Ramirez Endowed Graduate Fellowship in Engineering.
Peptide library name, sequence, and fluorescent encoding for each of the six main experiments
TetTCR-seq summary for experiment 1
TetTCR-Seq summary for experiment 2
Description of neoantigen and wild-type peptides used for experiments 3 and 4
TetTCR-seq summary for experiment 3
TetTCR-seq summary for experiment 4
Description of neoantigen and wild-type peptides used for experiments 5 and 6
TetTCR-seq summary for experiment 5
TetTCR-seq summary for experiment 6
Primer and DNA template sequences for generating DNA-BC pMHC tetramers and amplification of the DNA-BC