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Single-Cell–Based High-Throughput Ig and TCR Repertoire Sequencing Analysis in Rhesus Macaques

Evan S. Walsh, Tammy S. Tollison, Hayden N. Brochu, Brian I. Shaw, Kayleigh R. Diveley, Hsuan Chou, Lynn Law, Allan D. Kirk, Michael Gale Jr. and Xinxia Peng
J Immunol February 1, 2022, 208 (3) 762-771; DOI: https://doi.org/10.4049/jimmunol.2100824
Evan S. Walsh
*Department of Molecular Biomedical Sciences, North Carolina State University College of Veterinary Medicine, Raleigh, NC;
†Bioinformatics Graduate Program, North Carolina State University, Raleigh, NC;
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Tammy S. Tollison
*Department of Molecular Biomedical Sciences, North Carolina State University College of Veterinary Medicine, Raleigh, NC;
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Hayden N. Brochu
*Department of Molecular Biomedical Sciences, North Carolina State University College of Veterinary Medicine, Raleigh, NC;
†Bioinformatics Graduate Program, North Carolina State University, Raleigh, NC;
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Brian I. Shaw
‡Department of Surgery, Duke University, Durham, NC;
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Kayleigh R. Diveley
*Department of Molecular Biomedical Sciences, North Carolina State University College of Veterinary Medicine, Raleigh, NC;
§Genetics Graduate Program, North Carolina State University, Raleigh, NC;
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Hsuan Chou
*Department of Molecular Biomedical Sciences, North Carolina State University College of Veterinary Medicine, Raleigh, NC;
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Lynn Law
¶Department of Immunology, University of Washington, Seattle, WA;
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Allan D. Kirk
‡Department of Surgery, Duke University, Durham, NC;
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Michael Gale Jr.
¶Department of Immunology, University of Washington, Seattle, WA;
‖Center for Innate Immunity and Immune Diseases, University of Washington, Seattle, WA;
#Washington National Primate Research Center, University of Washington, Seattle, WA; and
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Xinxia Peng
*Department of Molecular Biomedical Sciences, North Carolina State University College of Veterinary Medicine, Raleigh, NC;
†Bioinformatics Graduate Program, North Carolina State University, Raleigh, NC;
**Bioinformatics Research Center, North Carolina State University, Raleigh, NC
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Key Points

  • Single-cell–based rhesus-specific Ig and TCR repertoire sequencing is established.

  • All rhesus Ig and TCR isotypes and chain types are recovered.

Abstract

Recent advancements in microfluidics and high-throughput sequencing technologies have enabled recovery of paired H and L chains of Igs and VDJ and VJ chains of TCRs from thousands of single cells simultaneously in humans and mice. Despite rhesus macaques being one of the most well-studied model organisms for the human adaptive immune response, high-throughput single-cell immune repertoire sequencing assays are not yet available due to the complexity of these polyclonal receptors. We used custom primers that capture all known rhesus macaque Ig and TCR isotypes and chains that are fully compatible with a commercial solution for single-cell immune repertoire profiling. Using these rhesus-specific assays, we sequenced Ig and TCR repertoires in >60,000 cells from cryopreserved rhesus PBMCs, splenocytes, and FACS-sorted B and T cells. We were able to recover every Ig isotype and TCR chain, measure clonal expansion in proliferating T cells, and pair Ig and TCR repertoires with gene expression profiles of the same single cells. Our results establish the ability to perform high-throughput immune repertoire analysis in rhesus macaques at the single-cell level.

This article is featured in Top Reads, p.

Footnotes

  • This work was supported by the U.S. Department of Health and Human Services (HHS), National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID) (Contract HHSN272201800008C) and by the Washington National Primate Research Center Core (Grant P51 OD010425 to M.G. and Grant R21AI120713 to X.P. from the HHS, NIH, NIAID, Office of the Director). The nonhuman primate work has also been funded by multiple awards from the HHS, NIH, NIAID, including Grants R38AI40297 (to B.I.S.) and U19AI131471 (to A.D.K.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article

    DC
    dendritic cell
    IRS
    immune repertoire sequencing
    NP
    nonproliferating
    P
    proliferating
    QC
    quality-control
    scIRS
    single-cell immune repertoire sequencing
    UMAP, uniform manifold approximation and projection; UMI
    unique molecular identifier
    UTR
    untranslated region

  • Received August 19, 2021.
  • Accepted November 17, 2021.
  • Copyright © 2022 by The American Association of Immunologists, Inc.
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The Journal of Immunology: 208 (3)
The Journal of Immunology
Vol. 208, Issue 3
1 Feb 2022
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Single-Cell–Based High-Throughput Ig and TCR Repertoire Sequencing Analysis in Rhesus Macaques
Evan S. Walsh, Tammy S. Tollison, Hayden N. Brochu, Brian I. Shaw, Kayleigh R. Diveley, Hsuan Chou, Lynn Law, Allan D. Kirk, Michael Gale, Xinxia Peng
The Journal of Immunology February 1, 2022, 208 (3) 762-771; DOI: 10.4049/jimmunol.2100824

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Single-Cell–Based High-Throughput Ig and TCR Repertoire Sequencing Analysis in Rhesus Macaques
Evan S. Walsh, Tammy S. Tollison, Hayden N. Brochu, Brian I. Shaw, Kayleigh R. Diveley, Hsuan Chou, Lynn Law, Allan D. Kirk, Michael Gale, Xinxia Peng
The Journal of Immunology February 1, 2022, 208 (3) 762-771; DOI: 10.4049/jimmunol.2100824
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