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J Immunol Methods. 2016 Nov;438:51-58. doi: 10.1016/j.jim.2016.08.013. Epub 2016 Sep 2.

Fluorescence-based measurement of cystine uptake through xCT shows requirement for ROS detoxification in activated lymphocytes.

Author information

1
Department of Pathology, Microbiology, and Immunology, Vanderbilt Center for Immunobiology, Vanderbilt University Medical Center, Nashville, TN 37323, United States. Electronic address: peter.siska@vanderbilt.edu.
2
Department of Chemistry, Duke University, Durham, NC 27708, United States.
3
Division of Allergy, Pulmonary and Critical Care Medicine, Thoracic Oncology Center, Vanderbilt Ingram Comprehensive Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37323, United States.
4
Division of Hematology/Oncology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37323, United States.
5
Molecular Genetics and Microbiology, Duke University Medical Center Durham, NC 27710, United States.
6
Department of Pathology, Microbiology, and Immunology, Vanderbilt Center for Immunobiology, Vanderbilt University Medical Center, Nashville, TN 37323, United States.

Abstract

T and B lymphocytes undergo metabolic re-programming upon activation that is essential to allow bioenergetics, cell survival, and intermediates for cell proliferation and function. To support changes in the activity of signaling pathways and to provide sufficient and necessary intracellular metabolites, uptake of extracellular nutrients increases sharply with metabolic re-programming. One result of increased metabolic activity can be reactive oxygen species (ROS), which can be toxic when accumulated in excess. Uptake of cystine allows accumulation of cysteine that is necessary for glutathione synthesis and ROS detoxification. Cystine uptake is required for T cell activation and function but measurements based on radioactive labeling do not allow analysis on single cell level. Here we show the critical role for cystine uptake in T cells using a method for measurement of cystine uptake using a novel CystineFITC probe. T cell receptor stimulation lead to upregulation of the cystine transporter xCT (SLC7a11) and increased cystine uptake in CD4+ and CD8+ human T cells. Similarly, lipopolysaccharide stimulation increased cystine uptake in human B cells. The CystineFITC probe was not toxic and could be metabolized to prevent cystine starvation induced cell death. Furthermore, blockade of xCT or competition with natural cystine decreased uptake of CystineFITC. CystineFITC is thus a versatile tool that allows measurement of cystine uptake on single cell level and shows the critical role for cystine uptake for T cell ROS regulation and activation.

KEYWORDS:

Cystine; CystineFITC; Metabolism; Reactive oxygen species (ROS); T cells; xCT

PMID:
27594594
PMCID:
PMC5065394
DOI:
10.1016/j.jim.2016.08.013
[Indexed for MEDLINE]
Free PMC Article

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