Abstract 4678: High-throughput single-cell linking of antigen specificities with T cell receptor sequences using de novo generated DNA-linked MHC tetramers

Abstract

Abstract Identification of correlated antigens recognized by T cell and its T cell receptor (TCR) sequences at single cell level is extremely useful to the understanding and treating immune-related diseases. Although mass cytometry, CyTOF, has pushed the number of antigenic peptides that can be interrogated at once to around 100, the destructive nature of CyTOF prohibits linking pMHCs recognized by a T cell with its TCR receptor sequences. In addition, the high cost associated with peptide synthesis process prevents the quick generation of a peptide library that can be tailored to any pathogens or diseases. Here we describe a new technology, TetATCR-Seq for Tetramer Associated TCR sequencing, that is capable of de novo generating a large amount of peptides and mapping antigen recognition with single cell TCR sequences by DNA barcoded tetramers. It evolves three major steps: 1) de novo peptide generation, 2) DNA barcoded tetramer generation, and 3) single cell sequencing of tetramer DNA barcode and TCR sequences. By spiking in 5 altered peptide ligands (APL) that are known to bind to a HCV T cell clone with other 90 irrelevant peptides, we showed that the binding of all these 5 APLs can be faithfully detected by sequencing while non-specific binding is minimum. Using a published set of cancer neo-antigen sequences, we showed that these peptides and their wild type counterparts can be quickly generated. Resulted tetramers can be used to clearly identify a population of neo-antigen only or wild type antigen only or double positive T cells. Subsequent single cell TCR sequencing showed that TCR paired α and β chain sequences can be obtained in more than 80% of single sorted T cells. Simultaneous tetramer DNA barcode sequencing showed that many T cells are found to cross-reactive to both neo-antigen and its wild type counterpart, suggesting the necessity of strict screening for neo-antigen only TCR in adoptive cell transfer therapy. In summary, the TetaTCR-seq tool we developed here enables one to quickly survey antigen repertoire again TCR repertoire at single cell level for a large number of peptides. This is an extremely useful tool in profiling T cell repertoire reactivity in infection and vaccination. It is also a useful tool to screen for T cell cross-reactivity and select neo-antigen specific TCR for cancer immunotherapy. Citation Format: Ning Jiang, Shu-qi Zhang, Chenfeng He, Eric Sun, Mingliang Zhang, Weiping Jia. High-throughput single-cell linking of antigen specificities with T cell receptor sequences using de novo generated DNA-linked MHC tetramers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4678.