Barcoding intracellular reverse transcription enables high-throughput phenotype-coupled T cell receptor analyses

Sahana Jayaraman; Janelle M. Montagne; Thomas R. Nirschl; Emily Marcisak; Jeanette Johnson; Amanda Huff; Meng-Hsuan Hsiao; Julie Nauroth; Thatcher Heumann; Jelani C. Zarif; Elizabeth M. Jaffee; Nilo Azad; Elana J. Fertig; Neeha Zaidi; H. Benjamin Larman

Cell Reports: Methods (Oct 2023)

Barcoding intracellular reverse transcription enables high-throughput phenotype-coupled T cell receptor analyses

Sahana Jayaraman,
Janelle M. Montagne,
Thomas R. Nirschl,
Emily Marcisak,
Jeanette Johnson,
Amanda Huff,
Meng-Hsuan Hsiao,
Julie Nauroth,
Thatcher Heumann,
Jelani C. Zarif,
Elizabeth M. Jaffee,
Nilo Azad,
Elana J. Fertig,
Neeha Zaidi,
H. Benjamin Larman

Affiliations

Sahana Jayaraman: Institute for Cell Engineering, Division of Immunology, Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
Janelle M. Montagne: Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Convergence Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Bloomberg Kimmel Immunology Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Division of Quantitative Sciences, Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
Thomas R. Nirschl: Pathobiology Graduate Program, Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Bloomberg∼Kimmel Institute for Cancer Immunotherapy, Johns Hopkins Medicine Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD 21205, USA
Emily Marcisak: Convergence Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
Jeanette Johnson: Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Immunology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
Amanda Huff: Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Convergence Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Bloomberg Kimmel Immunology Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
Meng-Hsuan Hsiao: Institute for Cell Engineering, Division of Immunology, Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
Julie Nauroth: Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
Thatcher Heumann: Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Division of Hematology Oncology, Vanderbilt-Ingram Comprehensive Cancer Center, Vanderbilt University Medical Center, Nashville, TN, USA
Jelani C. Zarif: Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Bloomberg∼Kimmel Institute for Cancer Immunotherapy, Johns Hopkins Medicine Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD 21205, USA
Elizabeth M. Jaffee: Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Convergence Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Bloomberg Kimmel Immunology Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
Nilo Azad: Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Convergence Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Bloomberg Kimmel Immunology Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
Elana J. Fertig: Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Convergence Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Division of Quantitative Sciences, Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Applied Mathematics and Statistics, Johns Hopkins University, Baltimore, MD 21218, USA; Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
Neeha Zaidi: Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Convergence Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Bloomberg Kimmel Immunology Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
H. Benjamin Larman: Institute for Cell Engineering, Division of Immunology, Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; Corresponding author

Journal volume & issue: Vol. 3, no. 10
p. 100600

Abstract

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Summary: Assays linking cellular phenotypes with T cell or B cell antigen receptor sequences are crucial for characterizing adaptive immune responses. Existing methodologies are limited by low sample throughput and high cost. Here, we present INtraCEllular Reverse Transcription with Sorting and sequencing (INCERTS), an approach that combines molecular indexing of receptor repertoires within intact cells and fluorescence-activated cell sorting (FACS). We demonstrate that INCERTS enables efficient processing of millions of cells from pooled human peripheral blood mononuclear cell (PBMC) samples while retaining robust association between T cell receptor (TCR) sequences and cellular phenotypes. We used INCERTS to discover antigen-specific TCRs from patients with cancer immunized with a novel mutant KRAS peptide vaccine. After ex vivo stimulation, 28 uniquely barcoded samples were pooled prior to FACS into peptide-reactive and non-reactive CD4+ and CD8+ populations. Combining complementary patient-matched single-cell RNA sequencing (scRNA-seq) data enabled retrieval of full-length, paired TCR alpha and beta chain sequences for future validation of therapeutic utility. Motivation: Existing methodologies that integrate T or B cell phenotypes with their antigen receptor sequences are limited by low sample throughput and high cost. Such approaches include single-cell RNA sequencing (scRNA-seq) and flow cytometric sorting of cells into subpopulations for bulk repertoire sequencing, both of which are difficult to scale across many samples and millions of cells. Therefore, we developed INtraCEllular Reverse Transcription with Sorting and sequencing (INCERTS). INCERTS successfully links cellular phenotype with antigen receptor sequence via barcoding during reverse transcription within intact cells. Subsequent cell sorting, sequencing, and demultiplexing enables high-throughput characterization of phenotype-coupled antigen receptor sequences across millions of cells.

Published in Cell Reports: Methods

ISSN: 2667-2375 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Technology: Chemical technology: Biotechnology; Science: Chemistry: Organic chemistry: Biochemistry
Website: https://www.cell.com/cell-reports-methods

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