Fluorescence-based measurement of cystine uptake through xCT shows requirement for ROS detoxification in activated lymphocytes.
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.
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.
Peter J
PJ
Siska
Department of Chemistry, Duke University, Durham, NC 27708, United States.
Bumki
B
Kim
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.
Xiangming
X
Ji
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.
Megan D
MD
Hoeksema
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.
Pierre P
PP
Massion
Division of Hematology/Oncology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37323, United States.
Kathryn E
KE
Beckermann
Molecular Genetics and Microbiology, Duke University Medical Center Durham, NC 27710, United States.
Jianli
J
Wu
Molecular Genetics and Microbiology, Duke University Medical Center Durham, NC 27710, United States.
Jen-Tsan
JT
Chi
Department of Chemistry, Duke University, Durham, NC 27708, United States.
Jiyong
J
Hong
Department of Pathology, Microbiology, and Immunology, Vanderbilt Center for Immunobiology, Vanderbilt University Medical Center, Nashville, TN 37323, United States.
Jeffrey C
JC
Rathmell
D027182:Amino Acid Transport System y+ / Q000378:metabolism; D001402:B-Lymphocytes / Q000378:metabolism*; D045744:Cell Line, Tumor; D065150:Cellular Reprogramming; D003553:Cystine / Q000378:metabolism*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D008213:Lymphocyte Activation; D008856:Microscopy, Fluorescence; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism*; D015854:Up-Regulation
D027182:Amino Acid Transport System y+; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D011948:Receptors, Antigen, T-Cell; C118895:SLC7A11 protein, human; D003553:Cystine; D005978:Glutathione; D016650:Fluorescein-5-isothiocyanate
Cystine; CystineFITC; Metabolism; Reactive oxygen species (ROS); T cells; xCT