P-glycoprotein expression skews mitochondrial dye measurements in T cells

Assays to monitor metabolic parameters of immune cells at a single cell level provide efficient means to study immunometabolism. We show here that staining intensity of mitochondria targeted probes in T cells is drastically influenced by P-glycoprotein/P-gp expression, a xenobiotic efflux pump that extrudes these fluorescent dyes. Discrepancies between MTG signals and multiple dye-independent measurements are seen in CD4 T and CD8 T cell subsets and are corrected by P-gp inhibition (PSC833) during MTG staining. We further investigate invariant Natural Killer T (iNKT) cells, which express the highest level of P-glycoprotein among T cells. Using mtDNA quantification, mitochondria targeted reporter, respiration and proteomics, we establish that iNKT cells have higher mitochondrial content and activity than CD4 T cells, opposite to what MTG signals reveal. A similar phenomenon is also seen in human PBMCs, and with TMRE, a dye indicator of mitochondrial membrane potential. Collectively, these data argue that P-glycoprotein expression is a significant confounding factor when analyzing T cells using mitochondrial specific dyes. Complementary methods are necessary to reliably assess mitochondrial features in T cells. The proteomics data are raw files acquired on Orbitrap Exploris 480 mass spectrometer using data-independent analysis at the University of Arkansas Medical Sciences Proteomics Core.