Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Aug 17;9(1):3296.
doi: 10.1038/s41467-018-05854-6.

The Tumor Suppressor Menin Prevents Effector CD8 T-cell Dysfunction by Targeting mTORC1-dependent Metabolic Activation

Affiliations
Free PMC article

The Tumor Suppressor Menin Prevents Effector CD8 T-cell Dysfunction by Targeting mTORC1-dependent Metabolic Activation

Junpei Suzuki et al. Nat Commun. .
Free PMC article

Abstract

While menin plays an important role in preventing T-cell dysfunction, such as senescence and exhaustion, the regulatory mechanisms remain unclear. We found that menin prevents the induction of dysfunction in activated CD8 T cells by restricting the cellular metabolism. mTOR complex 1 (mTORC1) signaling, glycolysis, and glutaminolysis are augmented by menin deficiency. Rapamycin treatment prevents CD8 T-cell dysfunction in menin-deficient CD8 T cells. Limited glutamine availability also prevents CD8 T-cell dysfunction induced by menin deficiency, and its inhibitory effect is antagonized by α-ketoglutarate (α-KG), an intermediate metabolite of glutaminolysis. α-KG-dependent histone H3K27 demethylation seems to be involved in the dysfunction in menin-deficient CD8 T cells. We also found that α-KG activates mTORC1-dependent central carbon metabolism. These findings suggest that menin maintains the T-cell functions by limiting mTORC 1 activity and subsequent cellular metabolism.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Menin deficiency induces dysfunction of CD8 T cells. a A representative staining profile of CD62L/CD27 on the cell surface of the WT and menin KO effector CD8 T cells. Naive CD8 T cells were stimulated with anti-TCR-β mAb plus anti-CD28 mAb with IL-2 for 2 days, and then the cells were further expanded with IL-2 for an additional 5 days. An analysis was performed on day 7 after the initial stimulation. b A representative staining profile of PD-1 on the cell surface of the WT and menin KO CD8 T cells on day 7. c Representative results of the intracellular FACS analysis of IFN-γ/OPN in the WT and menin KO effector CD8 T cells on day 7. The percentages of cells are indicated in each quadrant. d The results of ELISA for IL-6, IL-10, and OPN in the supernatants of the cells in c restimulated with immobilized anti-TCR-β for 16 h are shown with the standard deviation (n = 3: biological replicates). e The results of the quantitative RT-PCR analysis of mRNAs encoding pro-inflammatory enzymes in the WT and menin KO effector CD8 T cells on day 7. The results are presented relative to the mRNA expression of Cd3ε with the standard deviations (n = 3: technical replicates). f The percentages of SA β-galactosidase (SA β-Gal)-positive cells on day 12 are shown with the standard deviation (n = 3: biological replicates). g A representative staining profile of CD62L/CD27 on the cell surface of the WT and menin KO OT1 Tg splenic CD8 T cells on day 7 after Lm-OVA infection. h A representative staining profile of PD-1 on the cell surface of the cells in g. i Representative results of the intracellular FACS analysis of IFN-γ/OPN in the cells in g stimulated with an OVA-peptide (SIINFEKL) for 6 h. **p < 0.01 (Student’s t-test)
Fig. 2
Fig. 2
Menin KO CD8 T cells rapidly proliferate and acquire effector functions. a The results of the immunoblot analysis of the phospho-Akt (Ser473 or Thr308) and total Akt protein in the WT or menin KO activated CD8 T cells. WT or menin KO naive CD8 T cells were stimulated with anti-TCR-β mAb plus anti-CD28 mAb for 36 h and subjected to immunoblotting. b The results of the immunoblot analysis of the phospho-mTOR (Ser2448/2481), total mTOR protein and α-tubulin (control) of the cells in a. c The result of the flow cytometry analysis of phospho-ribosomal S6 (Ser235/236 and Ser240/244) and total ribosomal S6 protein in the cells in a. d The cell division between eFluor670-labeled WT or menin KO naive CD8 T cell was compared upon the indicated concentration of anti-TCR-β mAb plus anti-CD28 mAb in the presence of IL-2 for 48 h. e Naive CD8 T cells from the spleen of the WT and menin KO mice were stimulated with anti-TCR-β mAb plus anti-CD28 mAb in the presence of IL-2 for 2 days. The cells were then further expanded with IL-2 for the indicated days. Representative results of the intracellular FACS analysis of IL-2/IFN-γ in the WT and menin KO CD8 T cells on the indicated days. The percentages of cells are indicated in each quadrant. f The results of the intracellular FACS analysis of GzmB in the WT and menin KO CD8 T cells on day 3
Fig. 3
Fig. 3
Rapamycin inhibits dysfunction of menin KO CD8 T cells. a A representative staining profile of CD62L/CD27 on the cell surface of the WT and menin KO effector CD8 T cells on day 7. The percentages of cells are indicated in each quadrant. Naive CD8 T cells were stimulated with anti-TCR-β mAb plus anti-CD28 mAb with IL-2 in the presence or absence of rapamycin for 2 days, and then the cells were further expanded with IL-2 in the absence of rapamycin for an additional 5 days. b Representative staining profiles of CD226 and PD-1 on the surface of the cells in a. c The ELISA for IL-6, IL-10, and OPN in the supernatants of the cells in a restimulated with immobilized anti-TCR-β for 16 h are shown with the standard deviation (n = 3: biological replicates). d The results of the quantitative RT-PCR analysis of the pro-inflammatory enzymes in the cells in a. The results are presented relative to the expression of Cd3εmRNA with the standard deviations (n = 3: technical replicates). e The percentages of SA β-galactosidase (SA β-Gal)-positive cells on day 12 are shown with the standard deviation (n = 3: biological replicates). f A 1:1 mixture of WT OT-1 Tg effector CD8 T (Thy1.1+)/menin KO OT-1 Tg effector CD8 T cells (Thy1.2+) or WT (Thy1.1+)/rapamycin-treated menin KO (Thy1.2+) was adoptively transferred into WT congenic (Thy1.1+ Thy1.2+) mice. Twenty days after the transfer, the mice were infected with Lm-OVA to activate the donor cells. The donor cells were collected from the spleen on day 5 after Lm-OVA infection and analyzed by FACS. The absolute number of donor cells in the spleen was indicated (mean ± SD, n = 4 per group: biological replicates). g WT (Thy1.1+ or Thy1.2+), menin KO or rapamycin-treated menin KO OT-1 Tg memory CD8 T cells (Thy1.2+) were mixed and transferred into WT congenic mice (Thy1.1+ Thy1.2+) as in f. The mice were infected with Lm-OVA the next day and analyzed as in f. The absolute number of donor cells in the spleen is shown (mean ± SD, n = 4 per group: biological replicates). *p < 0.05, **p < 0.01 (Student’s t-test)
Fig. 4
Fig. 4
Metabolic profiling of menin KO activated CD8 T cells. a WT or menin KO naive CD8 T cells were stimulated with anti-TCR-β and anti-CD28 mAbs in the presence of IL-2 for 24 h. 2-NBDG was then added to the cultures for 30 min, and its incorporation was determined by FACS. A representative FACS profile is shown. b The intracellular level of glutamine in naive and activated CD8 T cells with WT or menin KO background are indicated (n = 3: biological replicates). The results are presented with the standard deviation. c The intracellular amount of glucose 6-phosphate (G6P), fructose 1,6-diphosphate (F1, 6P), pyruvate, and lactate in the WT and menin KO activated CD8 T cells are presented with the standard deviation (n = 3: biological replicates). Naive CD8 T cells were stimulated with anti-TCR-β and anti-CD28 mAbs in the presence of IL-2 for 36 h. d The intracellular amount of glutamine and glutamate of the cells in c. The results are presented with the standard deviation (n = 3: biological replicates). e The intracellular amounts of TCA cycle intermediates of the cells in c. The results are presented with the standard deviation (n = 3: biological replicates). f, g Naive CD8 T cells were stimulated with anti-TCR-β and anti-CD28 mAbs in the presence of IL-2 for 36 h, and then glycolysis (f) and the OCR (g) were determined before or 20 min after glucose (10 mM) injection. b, f, g **p < 0.01 (Student’s t-test), c, d, e *p < 0.05, **p < 0.01, ***p < 0.001 (Welch’s t-test)
Fig. 5
Fig. 5
The glutamine-α-KG axis is involved in the dysfunction in menin KO CD8 T cells. a A representative staining profile of CD62L/CD27 on the cell surface of the WT and menin KO effector CD8 T cells on day 7. The percentages of cells are indicated in each quadrant. Naive CD8 T cells were activated and cultured for 3 days under normal (Ctrl), glutamine-deprived (dGln), or glutamine-deprived supplemented with DM-α-KG (dGln/α-KG) conditions for 3 days, and then the cells were further expanded with IL-2 under normal conditions for an additional 4 days. An analysis was performed on day 7 after the initial anti-TCR-β/CD28 stimulation. b Representative staining profiles of CD226 and PD-1 on the cell surface of the cells in a. c The ELISA results for IL-6, IL-10, and OPN in the supernatants of the cells in a restimulated with immobilized anti-TCR-β for 16 h are shown with standard deviation (n = 3: biological replicates). d The results of the quantitative RT-PCR analysis of the pro-inflammatory enzymes in the cells in a. The results are presented relative to the expression of Cd3ε mRNA with the standard deviations (n = 3: technical replicates). e The percentages of SA β-galactosidase (SA β-Gal)-positive cells on day 12 after the initial anti-TCR-β/CD28 mAb stimulation are shown with the standard deviation (n = 3: biological replicates). f A 1:1 mixture of WT OT-1 Tg effector CD8 T (Thy1.1+)/menin KO OT-1 Tg effector CD8 T cells under normal conditions (Thy1.2+) or WT (Thy1.1+)/menin KO under glutamine-deprived conditions (Thy1.2+) was adoptively transferred into WT congenic (Thy1.1+ Thy1.2+) mice. Twenty days after the transfer, the mice were infected with Lm-OVA to activate the donor cells. The donor cells were collected from the spleen on day 5 after Lm-OVA infection and analyzed by FACS. The absolute number of donor cells in the spleen is shown (mean ± SD, n = 4 per group: biological replicates). *p < 0.05, **p < 0.01 (Student’s t-test)
Fig. 6
Fig. 6
Demethylation of histone H3K27 is involved in CD8 T-cell dysfunction. a The result of the immunoblot analysis of the di- or tri-methylated histone H3K27, tri-methylated histone H3K4 and total histone H3 in the WT CD8 T cells cultured under the indicated conditions for 48 h. The protein amount of histone H3 was used as a loading control. b The results of the immunoblot analysis of the di- or tri-methylated histone H3K27, tri-methylated histone H3K4 and total histone H3 in the WT or menin KO CD8 T cells cultured under normal conditions for 3 days. c A representative staining profile of CD62L/CD27 on the cell surface of the WT, utx KO menin KO or menin/utx-double KO effector CD8 T cells on day 7. The percentages of cells are indicated in each quadrant. d Representative staining profile of PD-1 on the cell surface of the cells in c. e The ELISA results for IL-6, IL-10, OPN, and IFN-γ in the supernatants of the cells in c restimulated with immobilized anti-TCR-β for 16 h are shown with the standard deviation (n = 3: biological replicates). f The results of the quantitative RT-PCR analysis of the pro-inflammatory enzymes of the cells in c. The results are presented relative to the expression of Cd3ε mRNA with the standard deviations (n = 3: technical replicates). g The percentages of SA β-galactosidase (SA β-Gal)-positive cells on day 12 after the initial anti-TCR-β/CD28 stimulation are shown with the standard deviation (n = 3: biological replicates). *p < 0.05, **p < 0.01 (Student’s t-test)
Fig. 7
Fig. 7
α-KG induces the activation of the central carbon metabolism. a The results of the immunoblot analysis of phospho-mTOR (Ser2448), mTOR and histone H3 in CD8 T cells cultured under the indicated conditions for 48 h. The protein amount of histone H3 was used as a loading control. b DM-α-KG-dependent induction of the phosphorylation (Ser240/244) of ribosomal S6 protein in CD8 T cells stimulated for 24 h. c, d Naive CD8 T cells were stimulated with anti-TCR-β, anti-CD28 mAbs plus IL-2 without glutamine in the presence or absence of DM-α-KG for 36 h and then ECAR (c) and the OCR (d) were determined. e The results of the quantitative RT-PCR analysis of the metabolic enzymes in the CD8 T cells cultured under as in the indicated conditions for 36 h. The results are presented relative to the expression of Cd3ε mRNA with the standard deviation (n = 3: technical replicates). f Naive CD8 T cells were stimulated with anti-TCR-β, anti-CD28 mAbs plus IL-2, and DM-α-KG without glutamine in the presence or absence of rapamycin (10 nM) for 36 h, and then the ECAR (left) and the OCR (right) were determined. Glycolysis was calculated by subtracting the ECAR before glucose injection from the ECAR 20 min after injection. Basal mitochondria respiration was calculated by subtracting the OCR in the presence of rotenone/antimycin A from the basal OCR at 15 min after the start of measurement. g Menin prevents effector CD8 T-cell dysfunction by targeting mTORC1-dependent metabolic activation. e, f *p < 0.05, **p < 0.01 (Student’s t-test)

Similar articles

See all similar articles

Cited by 3 articles

References

    1. Schietinger A, Greenberg PD. Tolerance and exhaustion: defining mechanisms of T cell dysfunction. Trends Immunol. 2014;35:51–60. doi: 10.1016/j.it.2013.10.001. - DOI - PMC - PubMed
    1. Finkel T, Serrano M, Blasco MA. The common biology of cancer and ageing. Nature. 2007;448:767–774. doi: 10.1038/nature05985. - DOI - PubMed
    1. Gavazzi G, Krause KH. Ageing and infection. Lancet Infect. Dis. 2002;2:659–666. doi: 10.1016/S1473-3099(02)00437-1. - DOI - PubMed
    1. Appay V, Almeida JR, Sauce D, Autran B, Papagno L. Accelerated immune senescence and HIV-1 infection. Exp. Gerontol. 2007;42:432–437. doi: 10.1016/j.exger.2006.12.003. - DOI - PubMed
    1. Tu W, Rao S. Mechanisms underlying T cell immunosenescence: Aging and cytomegalovirus infection. Front. Microbiol. 2016;7:2111. doi: 10.3389/fmicb.2016.02111. - DOI - PMC - PubMed

Publication types

MeSH terms

Feedback