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Deficiency of AMPK in CD8+ T cells suppresses their anti-tumor function by inducing protein phosphatase-mediated cell death.

Rao E, Zhang Y, Zhu G, Hao J, Persson XM, Egilmez NK, Suttles J, Li B - Oncotarget (2015)

Bottom Line: A number of studies have linked AMPK, a major metabolic sensor coordinating of multiple cellular functions, to tumor development and progression.Here we report that activation of AMPK promotes survival and anti-tumor function of T cells, in particular CD8+ T cells, resulting in superior tumor suppression in vivo.Moreover, we demonstrate that protein phosphatases are the key mediators of AMPK-dependent effects on T cell death, and inhibition of phosphatase activity by okadaic acid successfully restores T cell survival and function.

View Article: PubMed Central - PubMed

Affiliation: The Hormel Institute, University of Minnesota, Austin, MN, USA.

ABSTRACT
A number of studies have linked AMPK, a major metabolic sensor coordinating of multiple cellular functions, to tumor development and progression. However, the exact role of AMPK in tumor development is still controversial. Here we report that activation of AMPK promotes survival and anti-tumor function of T cells, in particular CD8+ T cells, resulting in superior tumor suppression in vivo. While AMPK expression is dispensable for T cell development, genetic deletion of AMPK promotes T cell death during in vitro activation and in vivo tumor development. Moreover, we demonstrate that protein phosphatases are the key mediators of AMPK-dependent effects on T cell death, and inhibition of phosphatase activity by okadaic acid successfully restores T cell survival and function. Altogether, our data suggest a novel mechanism by which AMPK regulates protein phosphatase activity in control of survival and function of CD8+ T cells, thereby enhancing their role in tumor immunosurveillance.

No MeSH data available.


Related in: MedlinePlus

AMPK deficiency promotes CD8 T cell death during activationA, cells from LNs of WT and AMPK KO mice were stimulated with PMA (10ng/ml) plus indicated concentrations of ionomycin for 6 hours. Flow cytometric staining for 7-AAD and Annexin V in CD8+ T cells. B, cells from LNs of WT and KO mice were stimulated with PMA (10ng/ml)/ionomycin (500ng/ml) for indicated time periods. CD8+ T cell death was analyzed by flow cytometric staining of 7-AAD (*, p<0.05). C, cells from LNs of WT and KO were stimulated with anti-CD3/CD28 for 48h and CD8+ T cell death was analyzed by flow cytometric staining. Average cell death was shown in the right panel (*, p<0.05). D, cells from LNs were stimulated with PMA/ionomycin in the presence or absence of indicated concentrations of Z-VAD-FMK or BHA for 6h. Cell death in CD8+ T cells was analyzed by flow staining for 7-AAD and Annexin V. E, western blot analysis of LC-3A/3B expression in PMA/ion-stimulated CD8+ T cells as indicated time points. F, intracellular staining for LC-3A/3B expression in CD8+ T cells after stimulation with PMA/ionomycin for 6h. Data shown are representative of 3 experiments.
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Figure 5: AMPK deficiency promotes CD8 T cell death during activationA, cells from LNs of WT and AMPK KO mice were stimulated with PMA (10ng/ml) plus indicated concentrations of ionomycin for 6 hours. Flow cytometric staining for 7-AAD and Annexin V in CD8+ T cells. B, cells from LNs of WT and KO mice were stimulated with PMA (10ng/ml)/ionomycin (500ng/ml) for indicated time periods. CD8+ T cell death was analyzed by flow cytometric staining of 7-AAD (*, p<0.05). C, cells from LNs of WT and KO were stimulated with anti-CD3/CD28 for 48h and CD8+ T cell death was analyzed by flow cytometric staining. Average cell death was shown in the right panel (*, p<0.05). D, cells from LNs were stimulated with PMA/ionomycin in the presence or absence of indicated concentrations of Z-VAD-FMK or BHA for 6h. Cell death in CD8+ T cells was analyzed by flow staining for 7-AAD and Annexin V. E, western blot analysis of LC-3A/3B expression in PMA/ion-stimulated CD8+ T cells as indicated time points. F, intracellular staining for LC-3A/3B expression in CD8+ T cells after stimulation with PMA/ionomycin for 6h. Data shown are representative of 3 experiments.

Mentions: As a major metabolic sensor, AMPK can be activated to promote cell survival (12). To dissect how AMPK deficiency impairs CD8+ T cell function, we reasoned that AMPK deficiency in T cells may promote cell death due to metabolic demands during activation. Since AMPK can be activated by signals either from Ca2+ or from TCR in T lymphocytes [29], we first used ionomycin triggering Ca2+ signals in cells from LNs and measured T cell death. We found that ionomycin induced CD8+ T cell death in a dose-dependent manner. Particularly, deletion of AMPK increased CD8+ T cell death under conditions when ionomycin was present at the high concentrations (500 and 1000ng/ml) (Figure 5A). Similar observations were observed when we analyzed CD4+ T cells from WT and KO mice (Supplementary Figure 4A). Of note, there was no difference in cell death between non-T cell populations in LNs from WT and KO mice (Supplementary Figure 4B). To substantiate these observations, we dynamically measured T cell death with the same levels of external stimulation. Again, more CD8+ and CD4+ T cells died in the absence of AMPK in a time-dependent manner (Figure 5B, Supplementary Figure 4C). In contrast, cell death of non-T cell populations was the same between the two strains (Supplementary Figure 4D). Moreover, when we used TCR triggering of cells from LNs in vitro, we observed AMPK deficiency induced more cell death in both CD8+ and CD4+ populations as compared to AMPK sufficient T cells (Figure 5C, Supplementary Figure 4E). However, cell death exhibited no difference in non-T cell populations where AMPK was not ablated in both WT and KO mice (Supplementary Figure 4F). Altogether, these data clearly demonstrated that AMPK deficiency promotes T cell death in situations where it is activated by different signals.


Deficiency of AMPK in CD8+ T cells suppresses their anti-tumor function by inducing protein phosphatase-mediated cell death.

Rao E, Zhang Y, Zhu G, Hao J, Persson XM, Egilmez NK, Suttles J, Li B - Oncotarget (2015)

AMPK deficiency promotes CD8 T cell death during activationA, cells from LNs of WT and AMPK KO mice were stimulated with PMA (10ng/ml) plus indicated concentrations of ionomycin for 6 hours. Flow cytometric staining for 7-AAD and Annexin V in CD8+ T cells. B, cells from LNs of WT and KO mice were stimulated with PMA (10ng/ml)/ionomycin (500ng/ml) for indicated time periods. CD8+ T cell death was analyzed by flow cytometric staining of 7-AAD (*, p<0.05). C, cells from LNs of WT and KO were stimulated with anti-CD3/CD28 for 48h and CD8+ T cell death was analyzed by flow cytometric staining. Average cell death was shown in the right panel (*, p<0.05). D, cells from LNs were stimulated with PMA/ionomycin in the presence or absence of indicated concentrations of Z-VAD-FMK or BHA for 6h. Cell death in CD8+ T cells was analyzed by flow staining for 7-AAD and Annexin V. E, western blot analysis of LC-3A/3B expression in PMA/ion-stimulated CD8+ T cells as indicated time points. F, intracellular staining for LC-3A/3B expression in CD8+ T cells after stimulation with PMA/ionomycin for 6h. Data shown are representative of 3 experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: AMPK deficiency promotes CD8 T cell death during activationA, cells from LNs of WT and AMPK KO mice were stimulated with PMA (10ng/ml) plus indicated concentrations of ionomycin for 6 hours. Flow cytometric staining for 7-AAD and Annexin V in CD8+ T cells. B, cells from LNs of WT and KO mice were stimulated with PMA (10ng/ml)/ionomycin (500ng/ml) for indicated time periods. CD8+ T cell death was analyzed by flow cytometric staining of 7-AAD (*, p<0.05). C, cells from LNs of WT and KO were stimulated with anti-CD3/CD28 for 48h and CD8+ T cell death was analyzed by flow cytometric staining. Average cell death was shown in the right panel (*, p<0.05). D, cells from LNs were stimulated with PMA/ionomycin in the presence or absence of indicated concentrations of Z-VAD-FMK or BHA for 6h. Cell death in CD8+ T cells was analyzed by flow staining for 7-AAD and Annexin V. E, western blot analysis of LC-3A/3B expression in PMA/ion-stimulated CD8+ T cells as indicated time points. F, intracellular staining for LC-3A/3B expression in CD8+ T cells after stimulation with PMA/ionomycin for 6h. Data shown are representative of 3 experiments.
Mentions: As a major metabolic sensor, AMPK can be activated to promote cell survival (12). To dissect how AMPK deficiency impairs CD8+ T cell function, we reasoned that AMPK deficiency in T cells may promote cell death due to metabolic demands during activation. Since AMPK can be activated by signals either from Ca2+ or from TCR in T lymphocytes [29], we first used ionomycin triggering Ca2+ signals in cells from LNs and measured T cell death. We found that ionomycin induced CD8+ T cell death in a dose-dependent manner. Particularly, deletion of AMPK increased CD8+ T cell death under conditions when ionomycin was present at the high concentrations (500 and 1000ng/ml) (Figure 5A). Similar observations were observed when we analyzed CD4+ T cells from WT and KO mice (Supplementary Figure 4A). Of note, there was no difference in cell death between non-T cell populations in LNs from WT and KO mice (Supplementary Figure 4B). To substantiate these observations, we dynamically measured T cell death with the same levels of external stimulation. Again, more CD8+ and CD4+ T cells died in the absence of AMPK in a time-dependent manner (Figure 5B, Supplementary Figure 4C). In contrast, cell death of non-T cell populations was the same between the two strains (Supplementary Figure 4D). Moreover, when we used TCR triggering of cells from LNs in vitro, we observed AMPK deficiency induced more cell death in both CD8+ and CD4+ populations as compared to AMPK sufficient T cells (Figure 5C, Supplementary Figure 4E). However, cell death exhibited no difference in non-T cell populations where AMPK was not ablated in both WT and KO mice (Supplementary Figure 4F). Altogether, these data clearly demonstrated that AMPK deficiency promotes T cell death in situations where it is activated by different signals.

Bottom Line: A number of studies have linked AMPK, a major metabolic sensor coordinating of multiple cellular functions, to tumor development and progression.Here we report that activation of AMPK promotes survival and anti-tumor function of T cells, in particular CD8+ T cells, resulting in superior tumor suppression in vivo.Moreover, we demonstrate that protein phosphatases are the key mediators of AMPK-dependent effects on T cell death, and inhibition of phosphatase activity by okadaic acid successfully restores T cell survival and function.

View Article: PubMed Central - PubMed

Affiliation: The Hormel Institute, University of Minnesota, Austin, MN, USA.

ABSTRACT
A number of studies have linked AMPK, a major metabolic sensor coordinating of multiple cellular functions, to tumor development and progression. However, the exact role of AMPK in tumor development is still controversial. Here we report that activation of AMPK promotes survival and anti-tumor function of T cells, in particular CD8+ T cells, resulting in superior tumor suppression in vivo. While AMPK expression is dispensable for T cell development, genetic deletion of AMPK promotes T cell death during in vitro activation and in vivo tumor development. Moreover, we demonstrate that protein phosphatases are the key mediators of AMPK-dependent effects on T cell death, and inhibition of phosphatase activity by okadaic acid successfully restores T cell survival and function. Altogether, our data suggest a novel mechanism by which AMPK regulates protein phosphatase activity in control of survival and function of CD8+ T cells, thereby enhancing their role in tumor immunosurveillance.

No MeSH data available.


Related in: MedlinePlus