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

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.