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 targeting probes in T cells is dramatically influenced by P-glycoprotein/P-gp expression, a xenobiotic efflux pump tha...
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Frontiers Media S.A.
2025-06-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2025.1560104/full |
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| author | Sophia P. M. Sok Jianan Cheng Klaudia Strucinska Narcis I. Popescu Lihua Wu Qiuqing Ke William B. Kiosses David Stanford Willard M. Freeman Satoshi Matsuzaki Tommy L. Lewis Tommy L. Lewis Meng Zhao Meng Zhao Meng Zhao |
| author_facet | Sophia P. M. Sok Jianan Cheng Klaudia Strucinska Narcis I. Popescu Lihua Wu Qiuqing Ke William B. Kiosses David Stanford Willard M. Freeman Satoshi Matsuzaki Tommy L. Lewis Tommy L. Lewis Meng Zhao Meng Zhao Meng Zhao |
| author_sort | Sophia P. M. Sok |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-b160efbe18264c8f88b4e7db1c99b58d |
| institution | OA Journals |
| issn | 1664-3224 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj-art-b160efbe18264c8f88b4e7db1c99b58d2025-08-20T02:07:30ZengFrontiers Media S.A.Frontiers in Immunology1664-32242025-06-011610.3389/fimmu.2025.15601041560104P-glycoprotein expression skews mitochondrial dye measurements in T cellsSophia P. M. Sok0Jianan Cheng1Klaudia Strucinska2Narcis I. Popescu3Lihua Wu4Qiuqing Ke5William B. Kiosses6David Stanford7Willard M. Freeman8Satoshi Matsuzaki9Tommy L. Lewis10Tommy L. Lewis11Meng Zhao12Meng Zhao13Meng Zhao14Arthritis and Clinical Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma, OK, United StatesArthritis and Clinical Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma, OK, United StatesAging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma, OK, United StatesArthritis and Clinical Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma, OK, United StatesArthritis and Clinical Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma, OK, United StatesArthritis and Clinical Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma, OK, United StatesDivision of Inflammation Biology, La Jolla Institute for Immunology, La Jolla, CA, United StatesCenter for Biomedical Data Science, Oklahoma Medical Research Foundation, Oklahoma, OK, United StatesGenes and Human Disease Research Program, Oklahoma Medical Research Foundation, Oklahoma, OK, United StatesAging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma, OK, United StatesAging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma, OK, United StatesDepartments of Biochemistry & Molecular Biology, Neuroscience and Physiology, University of Oklahoma Health Sciences Center, Oklahoma, OK, United StatesArthritis and Clinical Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma, OK, United StatesDepartment of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma, OK, United StatesStephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma, OK, United StatesAssays 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.https://www.frontiersin.org/articles/10.3389/fimmu.2025.1560104/fullT cellsmitochondriaoxidative phosphorilationmitotrackerTMREP-glycolprotein |
| spellingShingle | Sophia P. M. Sok Jianan Cheng Klaudia Strucinska Narcis I. Popescu Lihua Wu Qiuqing Ke William B. Kiosses David Stanford Willard M. Freeman Satoshi Matsuzaki Tommy L. Lewis Tommy L. Lewis Meng Zhao Meng Zhao Meng Zhao P-glycoprotein expression skews mitochondrial dye measurements in T cells Frontiers in Immunology T cells mitochondria oxidative phosphorilation mitotracker TMRE P-glycolprotein |
| title | P-glycoprotein expression skews mitochondrial dye measurements in T cells |
| title_full | P-glycoprotein expression skews mitochondrial dye measurements in T cells |
| title_fullStr | P-glycoprotein expression skews mitochondrial dye measurements in T cells |
| title_full_unstemmed | P-glycoprotein expression skews mitochondrial dye measurements in T cells |
| title_short | P-glycoprotein expression skews mitochondrial dye measurements in T cells |
| title_sort | p glycoprotein expression skews mitochondrial dye measurements in t cells |
| topic | T cells mitochondria oxidative phosphorilation mitotracker TMRE P-glycolprotein |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2025.1560104/full |
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