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Asymmetric inheritance of mTORC1 kinase activity during division dictates CD8+ T cell differentiation

Nature Immunology volume 17, pages 704–711 (2016)Cite this article

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Abstract

The asymmetric partitioning of fate-determining proteins has been shown to contribute to the generation of CD8+ effector and memory T cell precursors. Here we demonstrate the asymmetric partitioning of mTORC1 activity after the activation of naive CD8+ T cells. This results in the generation of two daughter T cells, one of which shows increased mTORC1 activity, increased glycolytic activity and increased expression of effector molecules. The other daughter T cell has relatively low mTORC1 activity and increased lipid metabolism, expresses increased amounts of anti-apoptotic molecules and subsequently displays enhanced long-term survival. Mechanistically, we demonstrate a link between T cell antigen receptor (TCR)-induced asymmetric expression of amino acid transporters and RagC-mediated translocation of mTOR to the lysosomes. Overall, our data provide important insight into how mTORC1-mediated metabolic reprogramming affects the fate decisions of T cells.

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Figure 1: mTORC1 activity is asymmetrically inherited in dividing CD8+ T cells after TCR stimulation.
Figure 2: Asymmetric inheritance of mTORC1 results in CD8+ daughter T cells with distinct metabolic capabilities.
Figure 3: Asymmetric inheritance of mTORC1 results in CD8+ daughter T cells with different in vivo survival.
Figure 4: The differential RagC-mediated translocation of mTOR to the lysosome contributes to the asymmetric division of CD8+ T cells.
Figure 5: TCR-induced mTOR localization to the lysosome is dependent on differential amino-acid-transporter expression.

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Acknowledgements

We thank members of the Powell lab and C. Gamper for critical discussion of the manuscript; B. Smith and T. Stephens for their technical support in microscopy studies; M. Gambello (Emory University, Atlanta, Georgia, USA) for Tsc2loxP mice; and P. Worley (Johns Hopkins University, Baltimore, Maryland, USA) for RhebloxP mice. This work was supported by the NIH (grants AI072677, AI77610 and AI091481 to J.D.P., and grants S10 OD016374 and S10 RR024550 to the JHUSOM Microscope Facility).

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Author notes

  1. Kristen N Pollizzi, Ying-Chun Lo & Adam T Waickman

    Present address: Present addresses: Department of Research Oncology, MedImmune LLC, Gaithersburg, Maryland, USA (K.N.P.); Department of Pathology, Yale School of Medicine, New Haven, Connecticut, USA (Y.-C.L.); Experimental Immunology Branch, National Cancer Institute, NIH, Bethesda, Maryland, USA (A.T.W.).,

  2. Kristen N Pollizzi, Im-Hong Sun and Chirag H Patel: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Oncology, Sidney-Kimmel Comprehensive Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Kristen N Pollizzi, Im-Hong Sun, Chirag H Patel, Ying-Chun Lo, Min-Hee Oh, Adam T Waickman, Ada J Tam, Richard L Blosser, Jiayu Wen, Greg M Delgoffe & Jonathan D Powell

  2. Department of Immunology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Greg M Delgoffe

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Contributions

K.N.P., I.-H.S., C.H.P. and J.D.P. planned the experiments, analyzed the data and wrote the manuscript; K.N.P., I.-H.S., C.H.P., Y.-C.L., M.-H.O., A.T.W. and J.W. performed the experiments; G.M.D. and A.T.W. contributed to the experimental design, execution and data interpretation; A.J.T. and R.L.B. provided support for flow cytometry experiments; G.M.D. and J.D.P. conceived the idea for the study; K.N.P., I.-H.S., C.H.P., Y.-C.L., M.-H.O., G.M.D. and J.D.P. provided critical review of the manuscript.

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Correspondence to Jonathan D Powell.

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Integrated supplementary information

Supplementary Figure 1 Polarized TCR activation induces asymmetric division of CD8+ T cells with phenotypical and functional differences.

(a-c) As in Fig. 1A, eFluor 450-labeled OT-I cells were adoptively transferred (i.v.) into WT mice and infected with LM-OVA (i.v.). Flow cytometry plots showing CD8 expression (a) and p-S6, CD98, T-bet, and Myc (b) between CD8hi and CD8lo T cells in 1st division and undivided cells. (c) Flow cytometry analysis of Eomes and CD44 expression shown from first division CD8hi (grey) and CD8lo (red) T cells. Naïve cells are shown in black dotted line. (d) Immunoblot analysis of p-4E-BP1 and p-ERK between sorted CD8hi and CD8lo T cells. (e) Flow cytometry of activation markers in CD8hi (grey) and CD8lo (red) T cells from first division after in vitro activation. (f) Confocal images of dividing CD8+ T cells in contact with bone marrow derived APC. Proximal cell in contact is shown on top of the APC. (g) qRT-PCR analysis of mRNA relative expression of Ifng and Prf1 in sorted CD8hi and CD8lo obtained from in vitro activation, n=5 (Ifng), 6 (Prf1). (h) Supernatant was collected from CD8hi and CD8lo T cells 5 h post-sorting. Amount of IFN-γ was measured from the supernatant by ELISA, n=6 (IFN-γ). *P < 0.05; ***P < 0.0005; ****P < 0.0001 (Mann-Whitney t test (g,h). Data are summary plots from at least 2 independent experiments (a-f) or compilation of 2 independent experiments (g,h). Scale bars, 10µm.

Supplementary Figure 2 Polarized T cell activation initiates asymmetric division of CD8hi and CD8lo daughter T cells.

Confocal images of CD8+ T cells activated for 36 h with polarized plate-bound stimuli (top) or activated for 5 h by PMA + ionomycin and rested for additional 31 h (bottom). T Cells were stained with anti-CD8 and anti-p-S6. Statistical analysis was performed comparing p-S6 MFI between CD8hi and CD8lo T cells, n=18 (Platebound) and 19 (PMA + Ionomycin). *P < 0.05; NS, not significant (Wilcoxon rank test). Data are representative of at least 2 independent experiments. Scale bars, 10µm.

Supplementary Figure 3 CD98 accumulates at the immunological synapse.

OT-I CD8+ T cells were activated in the presence of OVA-I peptide-pulsed bone marrow derived APCs for 19 h. Confocal image of APCs and CD8+ T cells stained with anti-CD98 and anti-talin. Results are representative of 2 independent experiments. Scale bar, 10µm.

Supplementary Figure 4 mTORC1 activity does not control differential expression of amino acid transporters in an in vivo infection model.

Flow cytometry of LFA-1, CD98, and p-S6 expression gated on adoptive transferred (i.v.) WT or T-Rheb–/– OT-I CD90.1+ CD8+ T cells from LM-OVA-infected WT hosts from first division indicated by proliferation dye. Data are representative of 2 independent experiments.

Supplementary Figure 5 Model of asymmetric division.

CD8hi proximal T cells have increased levels of amino acid transporter SLC7A5, resulting in an increased influx of amino acids that correlates with increased mTOR activity. This supports robust effector function in CD8hi daughter cells. In contrast, CD8lo distal cells have lower levels of mTOR activity and are destined to become memory cells.

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Pollizzi, K., Sun, IH., Patel, C. et al. Asymmetric inheritance of mTORC1 kinase activity during division dictates CD8+ T cell differentiation. Nat Immunol 17, 704–711 (2016). https://doi.org/10.1038/ni.3438

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