Mdr1 expression skews mitochondrial dye staining in T cells 4309

Abstract Description Mitochondria actively adapt to the cellular metabolic demands, and change their quantity, quality and activity to modulate T cells. Many mitochondria specific dyes have been used to profile mitochondrial properties in T cells using flow cytometry. As a prominent example, MitoTrackerGreen/MTG is widely used to report mitochondrial mass. However, we found MTG fluorescence intensity is skewed by Multidrug resistance-1 (Mdr1; encoded by Abcb1a and Abcb1b in mice and ABCB1 in humans) expression in T cells, a xenobiotic transporter that mediates dye efflux leading to artificially low signals. iNKT cells, a group of innate like T cells, express highest level of Mdr1, and show significantly lower MTG stain compared to other T cells. Pharmacological blockade of Mdr1 restores higher MTG signals in iNKT cells in mouse and human. Using mtDNA quantification, PhAMfloxed mitochondrial reporter, and proteomic analysis, we unequivocally show that iNKT cells have higher mitochondrial content. Consistently, iNKT cells show higher steady state and maximal respiratory activities than other T cells, indicating that mitochondria play important roles in these cells. Overall, we found that mitochondrial dye efflux mediated by Mdr1 leads to erroneous assessment of mitochondrial parameters in T cells. Mdr1 blockade may be incorporated in the standard staining procedure for dye dependent metabolic assays in immune cells. Funding Sources NIH/NIGMS R35GM147713 Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)