Protocol

Regulatory T Cells

Volume 707 of the series Methods in Molecular Biology pp 243-261

Date:

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

  • Milica Vukmanovic-StejicAffiliated withDepartment of Immunology, Infection, and Immunity, University College London
  • , Yan ZhangAffiliated withCentre for Infection, Cellular, and Molecular Medicine, St George’s, University of London
  • , Arne N. AkbarAffiliated withDepartment of Immunology, Infection, and Immunity, University College London
  • , Derek C. MacallanAffiliated withDepartment of Immunology, Infection, and Immunity, University College London

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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.

Key words

Lymphocyte Regulatory T cell Treg Kinetics Isotope Tracer Proliferation Death