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

Publication ,  Journal Article
Siska, PJ; Kim, B; Ji, X; Hoeksema, MD; Massion, PP; Beckermann, KE; Wu, J; Chi, J-T; Hong, J; Rathmell, JC
Published in: J Immunol Methods
November 2016

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.

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Published In

J Immunol Methods

DOI

EISSN

1872-7905

Publication Date

November 2016

Volume

438

Start / End Page

51 / 58

Location

Netherlands

Related Subject Headings

  • Up-Regulation
  • T-Lymphocytes
  • Signal Transduction
  • Receptors, Antigen, T-Cell
  • Reactive Oxygen Species
  • Microscopy, Fluorescence
  • Lymphocyte Activation
  • Immunology
  • Humans
  • Glutathione
 

Citation

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Siska, P. J., Kim, B., Ji, X., Hoeksema, M. D., Massion, P. P., Beckermann, K. E., … Rathmell, J. C. (2016). Fluorescence-based measurement of cystine uptake through xCT shows requirement for ROS detoxification in activated lymphocytes. J Immunol Methods, 438, 51–58. https://doi.org/10.1016/j.jim.2016.08.013
Siska, Peter J., Bumki Kim, Xiangming Ji, Megan D. Hoeksema, Pierre P. Massion, Kathryn E. Beckermann, Jianli Wu, Jen-Tsan Chi, Jiyong Hong, and Jeffrey C. Rathmell. “Fluorescence-based measurement of cystine uptake through xCT shows requirement for ROS detoxification in activated lymphocytes.J Immunol Methods 438 (November 2016): 51–58. https://doi.org/10.1016/j.jim.2016.08.013.
Siska PJ, Kim B, Ji X, Hoeksema MD, Massion PP, Beckermann KE, et al. Fluorescence-based measurement of cystine uptake through xCT shows requirement for ROS detoxification in activated lymphocytes. J Immunol Methods. 2016 Nov;438:51–8.
Siska, Peter J., et al. “Fluorescence-based measurement of cystine uptake through xCT shows requirement for ROS detoxification in activated lymphocytes.J Immunol Methods, vol. 438, Nov. 2016, pp. 51–58. Pubmed, doi:10.1016/j.jim.2016.08.013.
Siska PJ, Kim B, Ji X, Hoeksema MD, Massion PP, Beckermann KE, Wu J, Chi J-T, Hong J, Rathmell JC. Fluorescence-based measurement of cystine uptake through xCT shows requirement for ROS detoxification in activated lymphocytes. J Immunol Methods. 2016 Nov;438:51–58.
Journal cover image

Published In

J Immunol Methods

DOI

EISSN

1872-7905

Publication Date

November 2016

Volume

438

Start / End Page

51 / 58

Location

Netherlands

Related Subject Headings

  • Up-Regulation
  • T-Lymphocytes
  • Signal Transduction
  • Receptors, Antigen, T-Cell
  • Reactive Oxygen Species
  • Microscopy, Fluorescence
  • Lymphocyte Activation
  • Immunology
  • Humans
  • Glutathione