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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

Enhanced phosphatase activity in AMPK deficient CD8 T cells promotes cell death during activationA, purified CD8+ T cells from LNs were stimulated with PMA/ionomycin for indicated time periods. Cells were lyzed and analyzed for activation of AMPK, ACC, AKT, AKT2, PI3K, S6P, ERK, p38 by western blot. β-actin was used as a loading control. B, intracellular staining for phosphorylation of S6P in CD8+ T cells with PMA/ionomycin stimulation for indicated time periods. C, intracellular analysis of phosphorylation of S6P in PMA/ionomycin-activated CD8+ T cells in the presence or absence of okadaic acid (OA, 0.5μM). D, flow cytometric analysis of PMA/ionomycin-activated CD8+ T cell death in the presence or absence of OA (1μM). Average percentage of dead CD8+ T cells was shown in panel E. Intracellular staining for IFNγ expression in PMA/ionomycin-activated CD8+ T cells in the presence or absence of OA (1μM). Average percentage of IFNγ+ cells was shown panel G. Data shown are representative of 3 experiments.
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Figure 6: Enhanced phosphatase activity in AMPK deficient CD8 T cells promotes cell death during activationA, purified CD8+ T cells from LNs were stimulated with PMA/ionomycin for indicated time periods. Cells were lyzed and analyzed for activation of AMPK, ACC, AKT, AKT2, PI3K, S6P, ERK, p38 by western blot. β-actin was used as a loading control. B, intracellular staining for phosphorylation of S6P in CD8+ T cells with PMA/ionomycin stimulation for indicated time periods. C, intracellular analysis of phosphorylation of S6P in PMA/ionomycin-activated CD8+ T cells in the presence or absence of okadaic acid (OA, 0.5μM). D, flow cytometric analysis of PMA/ionomycin-activated CD8+ T cell death in the presence or absence of OA (1μM). Average percentage of dead CD8+ T cells was shown in panel E. Intracellular staining for IFNγ expression in PMA/ionomycin-activated CD8+ T cells in the presence or absence of OA (1μM). Average percentage of IFNγ+ cells was shown panel G. Data shown are representative of 3 experiments.

Mentions: To dissect the molecular mechanisms by which AMPK deficiency promotes T cell death during activation, we analyzed major AMPK-mediated signaling pathways in CD8+ T cells from AMPK KO and WT mice. As shown in Figure 6A, ablation of AMPK in CD8+ T cells abrogated phosphorylation of AMPK and its substrate acetyl CoA carboxylase (ACC). AMPK deficiency seemed to have no discernible impact on the phosphorylation of AKT1, AKT2, PI3K, P38, ERK in activated T cells. However, we noticed that phosphorylation of S6 ribosomal protein (S6P) (Ser235/236) was not sustained in activated CD8+ T cells when AMPK was absent. To confirm this observation, we performed intracellular staining by flow cytometry to dynamically track the phosphorylation levels of S6P in specific lymphocyte populations. Compared to unstimulated lymphocytes from LNs, ionomycin stimulation induced rapid phosphorylation of S6P (Ser 235/236) in CD8+ T cells from both WT and KO mice (10 min), suggesting that protein kinase activity of S6P was not affected by AMPK deficiency. On the contrary, dephosphorylation of S6P was obviously enhanced in AMPK-deficient CD8+ or CD4+ T cells at 60min and 120min after ionomycin stimulation (Figure 6B, Supplementary Figure 5A). Of note, under the same condition dephosphorylation levels of S6P remained the same for non-T cell populations (Supplementary Figure 5B). These data suggest that, instead of affecting protein kinase activity, AMPK deficiency may enhance protein phosphatase activity in T cells.


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)

Enhanced phosphatase activity in AMPK deficient CD8 T cells promotes cell death during activationA, purified CD8+ T cells from LNs were stimulated with PMA/ionomycin for indicated time periods. Cells were lyzed and analyzed for activation of AMPK, ACC, AKT, AKT2, PI3K, S6P, ERK, p38 by western blot. β-actin was used as a loading control. B, intracellular staining for phosphorylation of S6P in CD8+ T cells with PMA/ionomycin stimulation for indicated time periods. C, intracellular analysis of phosphorylation of S6P in PMA/ionomycin-activated CD8+ T cells in the presence or absence of okadaic acid (OA, 0.5μM). D, flow cytometric analysis of PMA/ionomycin-activated CD8+ T cell death in the presence or absence of OA (1μM). Average percentage of dead CD8+ T cells was shown in panel E. Intracellular staining for IFNγ expression in PMA/ionomycin-activated CD8+ T cells in the presence or absence of OA (1μM). Average percentage of IFNγ+ cells was shown panel G. Data shown are representative of 3 experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Enhanced phosphatase activity in AMPK deficient CD8 T cells promotes cell death during activationA, purified CD8+ T cells from LNs were stimulated with PMA/ionomycin for indicated time periods. Cells were lyzed and analyzed for activation of AMPK, ACC, AKT, AKT2, PI3K, S6P, ERK, p38 by western blot. β-actin was used as a loading control. B, intracellular staining for phosphorylation of S6P in CD8+ T cells with PMA/ionomycin stimulation for indicated time periods. C, intracellular analysis of phosphorylation of S6P in PMA/ionomycin-activated CD8+ T cells in the presence or absence of okadaic acid (OA, 0.5μM). D, flow cytometric analysis of PMA/ionomycin-activated CD8+ T cell death in the presence or absence of OA (1μM). Average percentage of dead CD8+ T cells was shown in panel E. Intracellular staining for IFNγ expression in PMA/ionomycin-activated CD8+ T cells in the presence or absence of OA (1μM). Average percentage of IFNγ+ cells was shown panel G. Data shown are representative of 3 experiments.
Mentions: To dissect the molecular mechanisms by which AMPK deficiency promotes T cell death during activation, we analyzed major AMPK-mediated signaling pathways in CD8+ T cells from AMPK KO and WT mice. As shown in Figure 6A, ablation of AMPK in CD8+ T cells abrogated phosphorylation of AMPK and its substrate acetyl CoA carboxylase (ACC). AMPK deficiency seemed to have no discernible impact on the phosphorylation of AKT1, AKT2, PI3K, P38, ERK in activated T cells. However, we noticed that phosphorylation of S6 ribosomal protein (S6P) (Ser235/236) was not sustained in activated CD8+ T cells when AMPK was absent. To confirm this observation, we performed intracellular staining by flow cytometry to dynamically track the phosphorylation levels of S6P in specific lymphocyte populations. Compared to unstimulated lymphocytes from LNs, ionomycin stimulation induced rapid phosphorylation of S6P (Ser 235/236) in CD8+ T cells from both WT and KO mice (10 min), suggesting that protein kinase activity of S6P was not affected by AMPK deficiency. On the contrary, dephosphorylation of S6P was obviously enhanced in AMPK-deficient CD8+ or CD4+ T cells at 60min and 120min after ionomycin stimulation (Figure 6B, Supplementary Figure 5A). Of note, under the same condition dephosphorylation levels of S6P remained the same for non-T cell populations (Supplementary Figure 5B). These data suggest that, instead of affecting protein kinase activity, AMPK deficiency may enhance protein phosphatase activity in T cells.

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