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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 impairs CD8 T cell activationA, sorted naïve CD8+ T cells were cultured in anti-CD3/CD28-coated wells with indicated concentrations of IL-12 for 72 hours. Production of IFNγ in these cells was analyzed by intracellular staining. The mean fluorescent intensity of IFNγ in these differentiated cells is shown in panel B. Total splenic CD8+ T cells were stimulated with anti-CD3/CD28 for 24h. Relative mRNA levels of IFNγ were measured by real-time RT-PCR C, Protein levels of IFNγ in the supernatants were measured by ELISA D,. Expression of IFNγ in CD8+ T cells stimulated with anti-CD3/CD28 for 48h was measured by intracellular flow staining. The average percentage was shown in the right panel (E) (*, p<0.05; **, p<0.01). Data shown are representative of at least 3 experiments.
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Figure 4: AMPK deficiency impairs CD8 T cell activationA, sorted naïve CD8+ T cells were cultured in anti-CD3/CD28-coated wells with indicated concentrations of IL-12 for 72 hours. Production of IFNγ in these cells was analyzed by intracellular staining. The mean fluorescent intensity of IFNγ in these differentiated cells is shown in panel B. Total splenic CD8+ T cells were stimulated with anti-CD3/CD28 for 24h. Relative mRNA levels of IFNγ were measured by real-time RT-PCR C, Protein levels of IFNγ in the supernatants were measured by ELISA D,. Expression of IFNγ in CD8+ T cells stimulated with anti-CD3/CD28 for 48h was measured by intracellular flow staining. The average percentage was shown in the right panel (E) (*, p<0.05; **, p<0.01). Data shown are representative of at least 3 experiments.

Mentions: Given the impaired anti-tumor function of AMPK deficient CD8+ T cells in tumors, we next investigated the role of AMPK in regulation of anti-tumor functions of CD8+ T cells in vitro. Naïve CD8+ T cells were purified and differentiated into cytotoxic T cells with the stimulation of anti-CD3/CD28 and IL-12. The expression of IFNγ was determined by intracellular staining. Strikingly, both the percentage and intensity of IFNγ expression in AMPK deficient CD8+ T cells were significantly lower than those in AMPK sufficient population (Figure 4A, 4B). When total CD8+ T cells isolated from the spleen of WT and KO mice were stimulated with anti-CD3/CD28 antibodies, we demonstrated that production of IFNγ in CD8+ T cells was also significantly reduced in the absence of AMPK as determined by both mRNA levels and protein levels (Figure 4C-4E). Besides TCR activation of CD8+ T cells, we also activated T cells from the spleen and LNs with PMA/Ionomycin and confirmed that the percentage of IFNγ positive population was significantly decreased in CD8+ T cells from KO mice as compared to WT mice (Supplementary Figure 3A, 3B). IFNγ levels in the cultural supernatants were also reduced in the absence of AMPK (Supplementary Figure 3C, 3D). In agreement with above in vivo observation, these in vitro data substantiated that AMPK activation is indispensable in promoting the anti-tumor functions of CD8+ 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)

AMPK deficiency impairs CD8 T cell activationA, sorted naïve CD8+ T cells were cultured in anti-CD3/CD28-coated wells with indicated concentrations of IL-12 for 72 hours. Production of IFNγ in these cells was analyzed by intracellular staining. The mean fluorescent intensity of IFNγ in these differentiated cells is shown in panel B. Total splenic CD8+ T cells were stimulated with anti-CD3/CD28 for 24h. Relative mRNA levels of IFNγ were measured by real-time RT-PCR C, Protein levels of IFNγ in the supernatants were measured by ELISA D,. Expression of IFNγ in CD8+ T cells stimulated with anti-CD3/CD28 for 48h was measured by intracellular flow staining. The average percentage was shown in the right panel (E) (*, p<0.05; **, p<0.01). Data shown are representative of at least 3 experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4480727&req=5

Figure 4: AMPK deficiency impairs CD8 T cell activationA, sorted naïve CD8+ T cells were cultured in anti-CD3/CD28-coated wells with indicated concentrations of IL-12 for 72 hours. Production of IFNγ in these cells was analyzed by intracellular staining. The mean fluorescent intensity of IFNγ in these differentiated cells is shown in panel B. Total splenic CD8+ T cells were stimulated with anti-CD3/CD28 for 24h. Relative mRNA levels of IFNγ were measured by real-time RT-PCR C, Protein levels of IFNγ in the supernatants were measured by ELISA D,. Expression of IFNγ in CD8+ T cells stimulated with anti-CD3/CD28 for 48h was measured by intracellular flow staining. The average percentage was shown in the right panel (E) (*, p<0.05; **, p<0.01). Data shown are representative of at least 3 experiments.
Mentions: Given the impaired anti-tumor function of AMPK deficient CD8+ T cells in tumors, we next investigated the role of AMPK in regulation of anti-tumor functions of CD8+ T cells in vitro. Naïve CD8+ T cells were purified and differentiated into cytotoxic T cells with the stimulation of anti-CD3/CD28 and IL-12. The expression of IFNγ was determined by intracellular staining. Strikingly, both the percentage and intensity of IFNγ expression in AMPK deficient CD8+ T cells were significantly lower than those in AMPK sufficient population (Figure 4A, 4B). When total CD8+ T cells isolated from the spleen of WT and KO mice were stimulated with anti-CD3/CD28 antibodies, we demonstrated that production of IFNγ in CD8+ T cells was also significantly reduced in the absence of AMPK as determined by both mRNA levels and protein levels (Figure 4C-4E). Besides TCR activation of CD8+ T cells, we also activated T cells from the spleen and LNs with PMA/Ionomycin and confirmed that the percentage of IFNγ positive population was significantly decreased in CD8+ T cells from KO mice as compared to WT mice (Supplementary Figure 3A, 3B). IFNγ levels in the cultural supernatants were also reduced in the absence of AMPK (Supplementary Figure 3C, 3D). In agreement with above in vivo observation, these in vitro data substantiated that AMPK activation is indispensable in promoting the anti-tumor functions of CD8+ 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