AUTHOR=Sok Sophia P. M. , Cheng Jianan , Strucinska Klaudia , Popescu Narcis I. , Wu Lihua , Ke Qiuqing , Kiosses William B. , Stanford David , Freeman Willard M. , Matsuzaki Satoshi , Lewis Tommy L. , Zhao Meng 

TITLE=P-glycoprotein expression skews mitochondrial dye measurements in T cells

JOURNAL=Frontiers in Immunology

VOLUME=Volume 16 - 2025

YEAR=2025

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2025.1560104

DOI=10.3389/fimmu.2025.1560104

ISSN=1664-3224

ABSTRACT=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 targeting probes in T cells is dramatically influenced by P-glycoprotein/P-gp expression, a xenobiotic efflux pump that extrudes these fluorescent dyes. Discrepancies between MitoTracker Green FM/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 abundance, mitochondrial volume, 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.