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. 2013 Dec 12;39(6):1043-56.
doi: 10.1016/j.immuni.2013.09.015. Epub 2013 Dec 5.

T cell exit from quiescence and differentiation into Th2 cells depend on Raptor-mTORC1-mediated metabolic reprogramming

Kai Yang  1 , Sharad Shrestha  1 , Hu Zeng  1 , Peer W F Karmaus  1 , Geoffrey Neale  2 , Peter Vogel  3 , David A Guertin  4 , Richard F Lamb  5 , Hongbo Chi  6
Affiliations

T cell exit from quiescence and differentiation into Th2 cells depend on Raptor-mTORC1-mediated metabolic reprogramming

Kai Yang et al. Immunity. .

Abstract

Naive T cells respond to antigen stimulation by exiting from quiescence and initiating clonal expansion and functional differentiation, but the control mechanism is elusive. Here we describe that Raptor-mTORC1-dependent metabolic reprogramming is a central determinant of this transitional process. Loss of Raptor abrogated T cell priming and T helper 2 (Th2) cell differentiation, although Raptor function is less important for continuous proliferation of actively cycling cells. mTORC1 coordinated multiple metabolic programs in T cells including glycolysis, lipid synthesis, and oxidative phosphorylation to mediate antigen-triggered exit from quiescence. mTORC1 further linked glucose metabolism to the initiation of Th2 cell differentiation by orchestrating cytokine receptor expression and cytokine responsiveness. Activation of Raptor-mTORC1 integrated T cell receptor and CD28 costimulatory signals in antigen-stimulated T cells. Our studies identify a Raptor-mTORC1-dependent pathway linking signal-dependent metabolic reprogramming to quiescence exit, and this in turn coordinates lymphocyte activation and fate decisions in adaptive immunity.

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Figures

Figure 1
Figure 1. Raptor is required for T cell activation and proliferation in vitro and in vivo
(A) Flow cytometry of CD4+ and CD8+ T cells in the spleens of WT and Rptor−/− mice. Right panels, proportions and numbers of CD4+ and CD8+ T cells (n=3 mice per group). (B) Expression of CD62L and CD44 on CD4+ and CD8+ T cells. (C,D) IL-2 secretion (C) and mRNA expression (D) in CD4+ T cells stimulated with α -CD3-CD28. (E) [3H]Thymidine incorporation of CD4+ T cells stimulated with α-CD3 or α-CD3-CD28 for 64 h and pulsed with [3H]thymidine for an additional 8 h. (F) Flow cytometry of CFSE-labeled CD4+ T cells stimulated with α-CD3-CD28 for 72 h. (G,H) CFSE-labeled WT OT-II or Rptor−/− OT-II T cells (CD45.2+) were transferred into CD45.1+ recipients and immunized s.c. with OVA323–339 in CFA 24 h later, followed by analysis after an additional 3 d. G, Proportion and number of donor OT-II T cells in the draining lymph nodes of recipients (n=3 mice per group). H, Flow cytometry of CFSE (left) and cell size (right) of donor OT-II T cells. (I,J) CFSE-labeled CD4+ T cells were transferred into sublethally irradiated CD45.1+ (I) or non-irradiated Rag1−/− mice (J), followed by analysis at day 7 after transfer. Data are representative of 2 (A-D,G-J) or 3 (E,F) independent experiments, and error bars represent the SEM. See also Figure S1.
Figure 2
Figure 2. Deficiency of Raptor but not Rictor markedly impairs antigen-specific immune responses in vivo and in vitro
(A,B) Flow cytometry of OVA-reactive IFN- γ+ CD4+ T cells from mice infected with OVA-expressing L. monocytogenes, detected after LLO189–201 stimulation and intracellular cytokine staining (A). B, proportion and number of LLO-reactive IFN-γ+ CD4+ T cells. (C,D) Flow cytometry of OVA-reactive IFN-γ+ CD8+ T cells from infected mice, detected after OVA257–264 stimulation and intracellular cytokine staining (C). D, proportion and number of OVA-reactive IFN-γ+ CD8+ T cells. (E) [3H]Thymidine incorporation of CD4+ T cells stimulated with α-CD3 or α-CD3-CD28 for 64 h, and pulsed with [3H]thymidine for an additional 8 h. Data are combined results from 3 (A–D) independent or representative of 2 (E) independent experiments, and error bars represent the SEM. See also Figure S2.
Figure 3
Figure 3. Raptor-mTORC1 signaling is mainly required for cell cycle entry from quiescence instead of continuous proliferation
(A) BrdU staining of CD4+ T cells stimulated with α -CD3-CD28 for 22 h, followed by pulsing with BrdU for 2 h. (B) Expression of CD71 and CD98 on CD4+ T cells stimulated with α-CD3-CD28 for 18 h. (C) Scatter plot comparing global gene expression profiles between WT and Rptor−/− CD4+ T cells stimulated with α-CD3-CD28 for 24 h (n=4 mice per group). Probe sets containing the cell cycle genes with twofold or greater difference between WT and Rptor−/−T cells are shown in red, with select genes annotated. mRNA expression levels are on a log2 scale. (D) Expression of Raptor, cyclin D2, cyclin E, CDK2, CDK4, and CDK6 in CD4+ T cells stimulated with α-CD3-CD28. (E) Expression and phosphorylation of Rb in CD4+ T cells stimulated with α-CD3-CD28. (F) CFSE-labeled CD4+ T cells were stimulated with α-CD3-CD28 for 70 h, pulsed with BrdU for 2 h, and subjected to 7-AAD and BrdU staining. Presented is BrdU staining of the gated CFSE-diluted population. (G) WT and Rptorfl/fl CD4+ T cells were infected with EGFP-Cre retrovirus at 24 h after α-CD3-CD28 stimulation, and 4 d later, GFP+ cells were sorted and stimulated with α-CD3-CD28 for 24 h for BrdU incorporation assay. (H) WT CD4+ T cells were stimulated with α-CD3-CD28, and rapamycin was added at different time points (left panel). Cells were subjected to BrdU staining at 24 h after addition of rapamycin. Data are representative of 3 (A,B,H), 1 (C; n=4 mice per group in the microarrays) or 2 (D–G) independent experiments. See also Figure S3 and Tables S1 and S2.
Figure 4
Figure 4. Raptor-mTORC1 signaling coordinates glycolysis, lipid genesis and oxidative phosphorylation of activated T cells
(A,B) Phosphorylation of S6K1, S6 and 4E-BP1 (A) or AKT, 4E-BP1, ERK and IκBα (B) in CD4+ T cells stimulated with α-CD3-CD28. (C) Glycolytic activity of CD4+ T cells stimulated with or without α-CD3-CD28 for 24 h. (D) Real-time PCR analysis of glycolytic genes in CD4+ T cells stimulated with α-CD3-CD28. (E,F) Immunoblot (E) and real-time PCR analysis (F) of c-Myc expression in CD4+ T cells stimulated with α-CD3-CD28. (G) De novo lipid biosynthesis of CD4+ T cells stimulated with or without α-CD3-CD28 for 24 h. (H) Expression of SREBP1 and SREBP2 full-length (Fl) and mature (M) forms in CD4+ T cells stimulated with α-CD3-CD28. Right, densitometric analysis of abundance of SREBP isoforms. (I) Real-time PCR analysis of Srebf1 and Srebf2 mRNA in CD4+ T cells stimulated with α-CD3-CD28. (J) Oxygen consumption rate (OCR) of CD4+ T cells stimulated with α-CD3-CD28 for 24 h. Data are representative of 2 independent experiments, and error bars represent the SEM. See also Figure S4.
Figure 5
Figure 5. Raptor deficiency impairs Th2 cell differentiation in vitro and suppresses allergic airway inflammation in vivo
(A,B) T cells were cultured under Th2 conditions for 5–6 d, followed by intracellular staining of IL-4 and IFN-γ upon PMA and ionomycin restimulation (A, left), bioplex measurement of IL-4 secretion upon restimulation with α-CD3 for 24 h (A, right), or real-time PCR analysis of Il4 and Il13 mRNA expression upon restimulation with α-CD3 for 5 h (B). (C) Intracellular staining of IL-4 in CFSE-labeled T cells cultured under Th2 conditions. (D) Histopathology of lung in OVA-challenged mice. Left, images of hematoxylin and eosin staining with 2× (upper) or 10× (bottom) of original magnification. Right, histological scores of the lung. (E) Quantification of eosinophils (Eos), neutrophils (Neu), dendritic cells (DC), and T cells (T) in lung and BAL fluid from OVA-challenged mice (n=4–7 mice per group). NS, not significant. (F) Intracellular staining of IL-4 in CFSE-labeled WT CD4+ cells cultured under Th2 conditions in the presence of mock or rapamycin (50 nM) added at different time points. (G) Intracellular staining of IL-4 in CD4+ T cells cultured under Th2 conditions in the presence of mock or rapamycin (50 nM). (H) Intracellular staining of IL-4 in CD4+ T cells cultured under Th2 conditions. Data are representative of 3 (A–C) or 2 (D–H) independent experiments, and error bars represent the SEM. See also Figure S5.
Figure 6
Figure 6. Raptor links glucose metabolism to cytokine receptor expression and Stat activation for the initiation of Th2 cell differentiation
(A) Intracellular staining of IL-4 in CFSE-labeled WT CD4+ cells cultured under Th2 conditions in the presence of mock or 2-DG added at the time of activation. (B) Intracellular staining of IL-4 and IFN-γ in WT CD4+ cells cultured under Th2 conditions in the presence of mock or 2-DG (0.3 mM) added at various times. (C) Expression of IL-4Rα on CD4+ cells cultured under α-CD3-CD28 alone or Th2 conditions for 24 h. (D–F) Flow cytometry of Stat6 phosphorylation (D), IL-2Rα (E), and Stat5 phosphorylation (F) in CD4+ cells cultured under Th2 conditions for 24 h. (G) Expression of IL-4Rα on CD4+ cells cultured under Th2 conditions in the presence of mock, 2-DG or rapamycin for 24 h. (H) Expression of IL-4Rα on CD4+ cells cultured under Th2 conditions in the presence of various doses of glucose. Mean fluorescent intensity (MFI) for the relevant staining is presented above the plots (C–F). Data are representative of 2 (A,B,G,H) or 3 (C–F) independent experiments, and error bars represent the SEM. See also Figure S6.
Figure 7
Figure 7. Raptor but not Rheb is essential for TCR and CD28-induced sustained mTORC1 activation and physiological effects
(A) Glycolytic activity of WT CD4+ T cells stimulated with α-CD3 or α-CD3-CD28 in the presence of IL-2, IL-4 or both for 24 h. (B) Phosphorylation of S6K1, S6 and 4E–BP1 in WT CD4+ T cells stimulated with α-CD3 or α-CD3-CD28. (C) BrdU staining of CD4+ T cells stimulated with α-CD3-CD28 for 22 h, followed by pulsing with BrdU for 2 h. (D) Intracellular staining of IL-4 and IFN-γ in CD4+ T cells cultured under Th2 conditions. (E) Phosphorylation of 4E-BP1, S6K1 and S6 in CD4+ T cells stimulated with α-CD3 or α-CD3-CD28. (F) Phosphorylation of S6K1, 4E-BP1 and AKT Ser473 in CD4+ T cells stimulated with α-CD3 or α-CD3-CD28. Data are representative of 1 (A) or 2 (B–F) independent experiments, and error bars represent the SEM. See also Figure S7.
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References

    1. Araki K, Turner AP, Shaffer VO, Gangappa S, Keller SA, Bachmann MF, Larsen CP, Ahmed R. mTOR regulates memory CD8 T-cell differentiation. Nature. 2009;460:108–112. - PMC - PubMed
    1. Benjamin D, Colombi M, Moroni C, Hall MN. Rapamycin passes the torch: a new generation of mTOR inhibitors. Nat Rev Drug Discov. 2011;10:868–880. - PubMed
    1. Bird JJ, Brown DR, Mullen AC, Moskowitz NH, Mahowald MA, Sider JR, Gajewski TF, Wang CR, Reiner SL. Helper T cell differentiation is controlled by the cell cycle. Immunity. 1998;9:229–237. - PubMed
    1. Chi H. Regulation and function of mTOR signalling in T cell fate decisions. Nat Rev Immunol. 2012;12:325–338. - PMC - PubMed
    1. Choo AY, Yoon SO, Kim SG, Roux PP, Blenis J. Rapamycin differentially inhibits S6Ks and 4E–BP1 to mediate cell-type-specific repression of mRNA translation. Proc Natl Acad Sci U S A. 2008;105:17414–17419. - PMC - PubMed

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