Abstract 5387: Novel targeted single-cell TCR sequencing method used for analysis of a T cell infiltrate from a glioblastoma patient
Authors: Li Shuqiang , Sun Jing , Anandappa Annabelle , Spiro Oliver , Leshchiner Ignaty , Datta Krishnalekha , Bao Yun , Hu Zhuting , Hacohen Nir , Mathewson Nathan D. , Tirosh Itay , Wucherpfennig Kai , Regev Aviv , Suva Mario , Keskin Derin B. , Wu Catherine J. , Livak Kenneth J. 
Published: August 2018

Abstract
Obtaining paired alpha- and beta-chain information of the T cell receptor (TCR) enables identification of which TCR binds to a particular antigen, providing critical knowledge for understanding how a tumor interacts with its immune microenvironment and for monitoring responses to immunotherapy. We have developed a rapid, cost-effective single-cell TCR sequencing protocol (scTCRseq) that processes single T cells flow sorted into 384-well plates. Exploiting the added specificity provided by RNase H-dependent PCR (rhPCR; Dobosy et al., BMC Biotechnology 11:80, 2011), our scTCRseq protocol mixes primers for all known productive alpha and beta TCR alleles and obtains specific amplification of only the alleles present in a single-cell lysate (typically one alpha allele and one beta allele) without the use of nested PCR. Applying this protocol to T cell samples from normal donors, we detect TCRs arising from all productive TRAV and TRBV genes, and observe the expected highly diverse repertoires. Our method also includes targeted sequencing of 96 (or more) marker transcripts in order to obtain cell phenotype information. Our scTCRseq protocol has been adapted to analyze low-input samples (100 pg-10 ng) of PBMC RNA for characterization of the TCR repertoire and to improve the sensitivity of paired TCR detection in Smart-seq2 libraries prepared from single-cell RNA.
We recently applied this approach to analyze T cell clonality in the T cell infiltrate from a glioblastoma patient receiving neoantigen-specific peptide vaccination. Fresh tumor obtained post-vaccination at the time of recurrence was dissociated and single CD3+ T cells were isolated by flow cytometry. Smart-seq2 analysis suggested the presence of clones, but this was based largely on inference from one-chain data. Using our scTCRseq protocol to re-amplify the TCR segments, paired alpha- and beta-chain information was obtained from 277 of 363 (76%) single cells that passed quality control. Out of 231 distinct clonotypes detected, 25 were identified as clones, where a clone is defined as two or more cells with identical alpha- and beta-CDR3 segments. GLIPH analysis (Glanville et al., Nature 547:94-98, 2017) was used to cluster infiltrate TCRs by specificity. A representative TCR from each specificity cluster was cloned in a TCR-negative Jurkat cell line. Cellular assays were used to determine which specificity groups react with epitopes from peptides used in the vaccination. TCRs specific for neoantigens included in the vaccine were detected.
We have demonstrated a robust workflow that combines sensitive detection of paired alpha- and beta-chain TCR sequences with TCR cloning and expression to determine which TCRs in a tumor infiltrate have neoantigen specificity.
Citation Format: Shuqiang Li, Jing Sun, Annabelle Anandappa, Oliver Spiro, Ignaty Leshchiner, Krishnalekha Datta, Yun Bao, Zhuting Hu, Nir Hacohen, Nathan D. Mathewson, Itay Tirosh, Kai Wucherpfennig, Aviv Regev, Mario Suva, Derin B. Keskin, Catherine J. Wu, Kenneth J. Livak. Novel targeted single-cell TCR sequencing method used for analysis of a T cell infiltrate from a glioblastoma patient [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 5387.