Abstract
Sustained glucose and glutamine transport are essential for activated T lymphocytes to support ATP and macromolecule biosynthesis. We found that glutamine and glucose also fuel an indispensable dynamic regulation of intracellular protein O-GlcNAcylation at key stages of T cell development, transformation and differentiation. Glucose and glutamine are precursors of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a substrate for cellular glycosyltransferases. Immune-activated T cells contained higher concentrations of UDP-GlcNAc and increased intracellular protein O-GlcNAcylation controlled by the enzyme O-linked-β-N-acetylglucosamine (O-GlcNAc) glycosyltransferase as compared with naive cells. We identified Notch, the T cell antigen receptor and c-Myc as key controllers of T cell protein O-GlcNAcylation via regulation of glucose and glutamine transport. Loss of O-GlcNAc transferase blocked T cell progenitor renewal, malignant transformation and peripheral T cell clonal expansion. Nutrient-dependent signaling pathways regulated by O-GlcNAc glycosyltransferase are thus fundamental for T cell biology.
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Acknowledgements
We acknowledge A. Whigham and R. Clarke for cell sorting facilities, V. Borodkin for generating inhibitors, S. Thompson for assistance with the in vivo Listeria infection, E. Emslie for genotyping and managing mouse colonies, and members of the D.A.C. laboratory for critical reading of the manuscript. We thank M.A. Ferguson for help with measurement of sugar nucleotides, H. Shen (University of Pennsylvania) for the attenuated Listeria strain and J. Zúñiga-Pflücker (University of Toronto) for OP9-DL1 cells. We are grateful for facilities provided by the Biological Resources unit, University of Dundee. This work was supported by the Wellcome Trust (Principal Research Fellowship 097418/Z/11/Z to D.A.C.), and by Tenovus Scotland (M.S.).
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M.S. designed and performed most of the experiments. S.P., K.M.G. and L.V.S. performed the experiments and provided intellectual input. S.D. performed key UDP-GlcNAc measurements. D.M.F.v.A. contributed advice and reagents. D.A.C. and M.S. designed the project and wrote the manuscript.
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Supplementary Figure 1 Ogtfl/flLck-Cre+ mice have failed thymocyte development.
(a) Counts of TCRβ-expressing T cells in spleens (left) and inguinal LNs (right) from Ogtwt/wtLck-Cre+ (wt) and Ogtfl/Y or fl/flLck-Cre+ (KO) mice (n=8 each). ** P< 0.01 (Student's t-tests).
(b) MFI of Notch1, Notch2 and IL-7R analysed by flow cytometry is plotted for DN3, DN4 and DP subsets from OgtwtLck-Cre+ (wt) and Ogtfl/YLck-Cre+ (KO) mice. Mean and SD from staining of 3 mice is shown.
(c) CD25 and CD44 profiling of Thy1+ CD4-CD8- (DN) thymocytes from OgtwtLck-Cre+ (WT) and Ogtfl/YLck-Cre+ (KO) mice. Total cell numbers in each thymic DN subset are shown to the right (n=8 for WT and KO mice).
Supplementary Figure 2 Impaired survival of DP thymocytes in Ogtfl/flCD4-Cre+ mice.
(a) Gating strategy for populations shown in Fig. 6a.
(b) Total thymocytes numbers from Ogtwt/wtCd4-Cre+ (WT) and Ogtfl/flCd4-Cre+ (OGT-ko) mice (n=7 for each).
(c) Representative flow cytometric plots of TCRβ+ cells (T cells) vs B220+ cells (B cells) in axillary LNs and counts of abT cells in Ogtwt/wtCd4-Cre+ (WT) and Ogtfl/flCd4-Cre+ (KO) mice (n=9 each).
Supplementary Figure 3 Analyses of OGT deletion in Ogtfl/flTamox-Cre mice.
CTL generated from Ogtfl/flTamox-Cre+ mice were treated with 4-OHT at indicated concentrations on day 5 of culture to delete the floxed OGT alleles. Immunoblot analyses on days 6 and 7 of culture showed loss of OGT (long and short exposure times) and global O-GlcNAcylation of the proteins. SMC1 was used as the loading control.
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Swamy, M., Pathak, S., Grzes, K. et al. Glucose and glutamine fuel protein O-GlcNAcylation to control T cell self-renewal and malignancy. Nat Immunol 17, 712–720 (2016). https://doi.org/10.1038/ni.3439
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DOI: https://doi.org/10.1038/ni.3439
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