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Single-Cell Multiparametric Analysis of Rare HIV-Infected Cells Identified by Duplexed RNAflow-FISH

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HIV Reservoirs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2407))

Abstract

HIV-infected cells are difficult to characterize in vivo because of their great paucity and their diversity. This chapter describes a duplexed flow cytometry method that enables detection, quantification and phenotyping of these rare cells at single-cell resolution. Primary CD4+ T cells are enriched from PBMCs, stained for surface and intracellular proteins and then subjected to fluorescent in situ hybridization to label viral RNA before acquisition on a flow cytometer. Technical and analytical advices are provided to improve the quality of the data. This flow cytometric RNA fluorescent in situ hybridization (RNAflow-FISH) procedure can be applied to the characterization of both HIV-infected cells from viremic people living with HIV and reactivated viral reservoirs from virally suppressed individuals on therapy.

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Acknowledgments

We thank Amy Baxter for the raw data used to exemplify the RNAflow-FISH gating strategy displayed in Fig. 1. We also thank Gérémy Sannier for his insight on the manuscript.

DEK is supported by a FRQS Merit Award (# 268471), the Canadian Institutes of Health Research (CIHR # 152977, CIHR # 164062, CIHR # 168901), and the US National Institutes of Health (NIH UM1-AI-144462 (CHAVD), NIH R01-AI-143411).

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Authors and Affiliations

  1. Research Centre of the Centre Hospitalier de l’Université de Montréal (CRCHUM), Montréal, QC, Canada

    Mathieu Dubé & Daniel E. Kaufmann

  2. Université de Montréal, Montreal, QC, Canada

    Daniel E. Kaufmann

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  1. Mathieu Dubé
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  2. Daniel E. Kaufmann
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Correspondence to Daniel E. Kaufmann .

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Editors and Affiliations

  1. School of Medicine and Human Immuno-Virology (H.I.V.) Group, San Raffaele University and Scientific Institute, Milano, Italy

    Guido Poli

  2. Viral Pathogenesis and Biosafety Group, San Raffaele Scientific Institute, Milano, Italy

    Elisa Vicenzi

  3. Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Fabio Romerio

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Dubé, M., Kaufmann, D.E. (2022). Single-Cell Multiparametric Analysis of Rare HIV-Infected Cells Identified by Duplexed RNAflow-FISH. In: Poli, G., Vicenzi, E., Romerio, F. (eds) HIV Reservoirs. Methods in Molecular Biology, vol 2407. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1871-4_20

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  • DOI: https://doi.org/10.1007/978-1-0716-1871-4_20

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1870-7

  • Online ISBN: 978-1-0716-1871-4

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