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

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.

Mitochondrial dysregulation and glycolytic insufficiency functionally impair CD8 T cells infiltrating human renal cell carcinoma

“…Glucose and cystine uptake was measured using the fluorescent glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) and CystineFITC (44). Cells were cultured in glucose-free media and incubated in 50 μM 2-NBDG for 45 minutes or in 1 μM CystineFITC for 20 minutes.…”

Distinct Regulation of Th17 and Th1 Cell Differentiation by Glutaminase-Dependent Metabolism

“…Activated T cells upregulate amino acid transporters (Sinclair et al, 2013) and enzymes that metabolize glutamine (Nakaya et al, 2014; Wang et al, 2011). Glutamine is initially hydrolyzed via the enzyme glutaminase (GLS) (Wang et al, 2010) to produce glutamate, which is used in protein synthesis and generation of glutathione to regulate reactive oxygen species (ROS) and exchanged to promote cystine uptake (Siska et al, 2016). Glutamate is further metabolized to α-ketoglutarate (α-KG), which provides anaplerotic support of the tricarboxylic acid cycle (TCA) cycle in growing cells (Yuan et al, 2014) and is a substrate for histone and DNA demethylases that regulate chromatin accessibility (Nakajima and Kunimoto, 2014).…”

Cell-surface marker discovery for lung cancer

“…Five of these markers, CXorf61, DSG3, FAT2, GPR87, and LYPD3, were selected based primarily on their high and broad expression among the lung cancer samples relative to normal lung. Additional markers were selected based on their profile and that there are currently available molecular imaging probes targeting these markers (CA9, CA12, KISS1R and SLC7A11) [ 25 , 35 – 70 ]. KISS1R and TMPRSS4 have known high affinity ligands and inhibitors, respectively, for potential use in targeting [ 71 – 80 ].…”

“…Two 18 F-glutamate derivative PET agents and an 18 F-aminosuberic acid derivative PET agent have also been developed that target the x C - transporter (SLC7A11) [ 62 – 69 ]. Recently, a fluorescent cystine derivative has also been developed for imaging the x C - transporter (SLC7A11) [ 70 ]. High-affinity agonist (including metastin analogs and fluorobenzoyl pentapeptides) [ 71 – 75 , 79 ] and antagonist (including 2-acylamino-4,6-diphenyl-pyridine derivatives) [ 76 – 78 ] ligands are known for the KiSS-1 receptor (KISS1R).…”