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Measurement of Proliferation and Disappearance of Regulatory T Cells in Human Studies Using Deuterium-Labeled Glucose

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Regulatory T Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 707))

Abstract

The in vivo proliferation and disappearance kinetics of lymphocytes may be estimated in humans from rates of deuterium-labeled glucose (2H2-glucose) incorporation into DNA. This protocol describes its application to regulatory T cells (Treg). Because Treg divide frequently, 2H2-glucose is a suitable precursor, achieving high levels of enrichment over a short period. Being nonradioactive and readily administered, it is appropriate for human studies.

There are four phases to the method: labeling, sampling, analysis and modeling. Labeling consists of administration of 2H2-glucose, either intravenously or orally; during this phase, small blood samples are taken to monitor plasma glucose enrichment. Sampling occurs over the ensuing ∼3 weeks; PBMC are collected and sorted according to surface marker expression. Cell separation can be achieved by fluorescence-activated cell sorting (FACS) using CD4, CD45RA and CD25 to define memory Treg (CD4+CD25hi), or by a combination of magnetic bead separation and FACS. Analysis consists of DNA extraction, hydrolysis, derivatization to the pentafluoro tri-acetate (PFTA) derivative, and quantitation of deuterium content by gas-chromatography mass-spectrometry (GC/MS). The ratio of deuterium enrichment in cellular DNA relative to plasma glucose is used to derive the fraction of new cells in the sorted population, and this is modeled as a function of time to derive proliferation and disappearance kinetics.

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Acknowledgments

We acknowledge financial support from the Medical Research Council (UK), BBSRC (UK), the Wellcome Trust, Merck Serono and the Charitable Trustees of St George’s Hospital, London during the execution of studies included in this report.

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Authors and Affiliations

  1. Department of Immunology, Infection, and Immunity, University College London, London, UK

    Milica Vukmanovic-Stejic, Arne N. Akbar & Derek C. Macallan

  2. Centre for Infection, Cellular, and Molecular Medicine, St George’s, University of London, London, UK

    Yan Zhang

Authors
  1. Milica Vukmanovic-Stejic
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  2. Yan Zhang
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  3. Arne N. Akbar
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  4. Derek C. Macallan
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Editor information

Editors and Affiliations

  1. , Division of Immunoregulation, MRC National Institute for Medical Resea, The Ridgeway, Mill Hill, London, NW7 1AA, United Kingdom

    George Kassiotis

  2. , VIB Autoimmune Genetics Laboratory, Katholieke Universiteit Leuven, Herestraat 49, Leuven, 3000, Belgium

    Adrian Liston

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Vukmanovic-Stejic, M., Zhang, Y., Akbar, A.N., Macallan, D.C. (2011). Measurement of Proliferation and Disappearance of Regulatory T Cells in Human Studies Using Deuterium-Labeled Glucose. In: Kassiotis, G., Liston, A. (eds) Regulatory T Cells. Methods in Molecular Biology, vol 707. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61737-979-6_16

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  • DOI: https://doi.org/10.1007/978-1-61737-979-6_16

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61737-978-9

  • Online ISBN: 978-1-61737-979-6

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