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Metabolic maintenance of cell asymmetry following division in activated T lymphocytes

Nature volume 532, pages 389–393 (2016)Cite this article

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Abstract

Asymmetric cell division, the partitioning of cellular components in response to polarizing cues during mitosis, has roles in differentiation and development1. It is important for the self-renewal of fertilized zygotes in Caenorhabditis elegans and neuroblasts in Drosophila, and in the development of mammalian nervous and digestive systems1. T lymphocytes, upon activation by antigen-presenting cells (APCs), can undergo asymmetric cell division, wherein the daughter cell proximal to the APC is more likely to differentiate into an effector-like T cell and the distal daughter is more likely to differentiate into a memory-like T cell2. Upon activation and before cell division, expression of the transcription factor c-Myc drives metabolic reprogramming, necessary for the subsequent proliferative burst3. Here we find that during the first division of an activated T cell in mice, c-Myc can sort asymmetrically. Asymmetric distribution of amino acid transporters, amino acid content, and activity of mammalian target of rapamycin complex 1 (mTORC1) is correlated with c-Myc expression, and both amino acids and mTORC1 activity sustain the differences in c-Myc expression in one daughter cell compared to the other. Asymmetric c-Myc levels in daughter T cells affect proliferation, metabolism, and differentiation, and these effects are altered by experimental manipulation of mTORC1 activity or c-Myc expression. Therefore, metabolic signalling pathways cooperate with transcription programs to maintain differential cell fates following asymmetric T-cell division.

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Figure 1: c-Myc asymmetrically segregates to the proximal daughter cell in activated CD8 T lymphocytes.
Figure 2: Amino acid metabolism is necessary for the maintenance of c-Myc asymmetry in activated CD8 T cells.
Figure 3: mTORC1 activity is required for the maintenance of c-Myc asymmetry in activated CD8 T cells.
Figure 4: T cell proliferation, metabolism, and fate are directed by differential expression of c-Myc protein.

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  • 20 April 2016

    The text of the first paragraph has been corrected.

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Acknowledgements

We thank R. Cross, G. Lennon, and P. Ingle for cell sorting, P. Thomas for assistance with influenza infections, H. Chi for help with Listeria infections, and I. Pawlikowska for help with statistical analyses. This work was supported by ALSAC and grants from the US National Institutes of Health.

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Authors and Affiliations

  1. Department of Immunology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, 38105, Tennessee, USA

    Katherine C. Verbist, Cliff S. Guy, Sandra Milasta, Swantje Liedmann, Marcin M. Kamiński & Douglas R. Green

  2. Center for Childhood Cancer and Blood Disease, The Research Institute at Nationwide Children’s Hospital, Columbus, 43205, Ohio, USA

    Ruoning Wang

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  1. Katherine C. Verbist
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  2. Cliff S. Guy
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  3. Sandra Milasta
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  5. Marcin M. Kamiński
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  6. Ruoning Wang
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  7. Douglas R. Green
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Contributions

K.C.V. conceived the project, designed and performed most experiments, interpreted results, and co-wrote the manuscript. C.S.G. guided imaging, performed live imaging experiments, and performed experiments with TSC1 flox/flox animals. S.M. provided technical assistance with Seahorse XF24 assays and helped with several in vivo studies. S.L. performed some imaging experiments with amino acid transports, performed experiments with high IL-2, and helped with several in vivo studies. M.M.K. performed and analysed all gene expression analyses. R.W. provided intellectual contributions. D.R.G. conceived the project, supervised experimental designs, interpreted results, and co-wrote the manuscript.

Corresponding author

Correspondence to Douglas R. Green.

Extended data figures and tables

Extended Data Figure 1 Characterization of c-Myc asymmetry.

a, Gating strategy for CD8high and CD8low cells. CellTrace-Violet-labelled naive T cells were activated with anti-CD3, anti-CD28, and ICAM for 36 h. CD8 high or low cells were identified as activated, undivided (U), first division (1), or second division (2) on the basis of dilution of CellTrace Violet. Frequencies of cells in each population are presented in table below. b, Time course of CellTrace Violet dilution in naive, T cells activated with anti-CD3, anti-CD28, and ICAM for the indicated time points. c, Schematic representation of staining patterns for interpretation of data represented in Fig. 1c–h. Black lines indicate axes on graphs. d, Frequencies of asymmetric cell division were determined by analysing 92 conjoined daughter cells from cultures activated with anti-CD3, anti-CD28, and ICAM and 123 conjoined daughter cells from cultures activated with SIINFEKL-pulsed BMDCs. T cells were analysed for c-Myc–GFP intensity in both daughter cells. Using 1.5-times brightness in one daughter versus the other as a cut off, each pair was assigned either asymmetric or not, and the frequency of asymmetric pairs is plotted.

Extended Data Figure 2 Representative confocal images and single stains.

For data in Fig. 1. a, Fig. 1b, c; b, Fig. 1d, e; c, Fig. 1f; d, Fig. 1g; e, Fig. 1h.

Extended Data Figure 3 c-Myc expression becomes asymmetric in late telophase.

Overlay, β-tubulin (blue), Hoechst 33258 (grey), c-Myc–GFP (green), and phalloidin (red) for each mitotic phase indicated on the left as identified by chromatin and tubulin staining patterns.

Extended Data Figure 4 Asymmetric assortment of mRNA for c-Myc target genes in activated T cells.

CellTrace-Violet-labelled T cells were stimulated for 36 h with anti-CD3, anti-CD28, and ICAM. a–c, First division (a, c) or undivided (a–c) c-Mychigh (H) and c-Myclow (L) T cells were sorted on the basis of c-Myc–GFP expression (b is undivided cells only), and RNA was extracted from each population. Fold change in gene expression for the indicated primers is quantified using the 2−ΔΔCt method relative to β-2-microglobulin across three (a, c) or four (b) independent experiments (mean ± s.d.).

Extended Data Figure 5 Cytokine signalling does not influence c-Myc asymmetry and asymmetric assortment of CD98.

a, Mean fluorescent intensity (MFI) of c-Myc–GFP (left panel) or CD98 (right panel) in CD8high (shaded bars) and CD8low (open bars) in the first division after 35 h of activation by anti-CD3, anti-CD8, and ICAM and the indicated treatment for 1 h. All differences were determined significant (P < 0.05) by unpaired Student’s t-test. b, Flow cytometric analysis of c-Myc GFP and CD98 in CD8high (green histograms), CD8low (grey histograms), or IL-7 (5 ng ml−1) rested unactivated (gold histograms) T cells in control conditions or activated in the presence of 10 ng ml−1 IL-2 (0–36 h after activation). Representative flow plots are on the left, and quantification of the mean fluorescence intensities of c-Myc and CD98-APC are on the right. Experiment representative of three independent experiments. c, Representative confocal image and single stain images for Fig. 2d, e. OT-I T cells stimulated on SIINFEKL-pulsed BMDCs d, Quantification of CD98 and c-Myc asymmetry in T cells stimulated with anti-CD3, anti-CD28, and ICAM for 36 h. 100% concordance of markers (P = 0.0039 Two-Tailed Binomial Test); R2 = 0.2304, P = 0.2304 linear regression. e, Polarization of SLC3A2 in two representative confocal images in antibody-stimulated OT-I Tg T cells (see methods). f, Asymmetric index (difference in RFP Intensity in proximal (P) and distal (D) sides of cell/sum of RFP intensities in proximal and distal sides of cell) for SLC3A2 staining in activated, undivided CD8 T cells (each point represents an activated T cell). Error bars, mean ± s.d.

Extended Data Figure 6 Amino acid transporter SLC1A5, but not SLC1A3, asymmetrically assorts in activated T cells.

a, b, Representative confocal image, quantification, and single-stain images for SLC1A5; 88.9% both bright in proximal daughter (x2 = 19.89, DF = 3, P = 0.0002, chi-square goodness of fit test); R2 = 0.2961, P = 0.1299, linear regression (a); and SLC1A3 100% c-Myc bright in proximal daughter, 62.5% SLC1A3 bright in distal daughter (x2 = 9, DF = 3, P = 0.0293 chi-square goodness of fit test); R2 = 0.07944, P = 0.4989 linear regression (b) for OT-I CD8 T cells co-cultured with SIINFEKL-pulsed BMDCs for 36 h.

Extended Data Figure 7 Regulation of p-mTOR and mTORC1 signalling by c-Myc.

a, b, Quantification of p-mTOR staining and c-Myc–GFP for OT-I CD8 T cells co-cultured with SIINFEKL-pulsed BMDCs 80.9% both bright in proximal daughter (x2 = 25.67, DF = 3, P < 0.0001 chi-square goodness of fit test); R2 = 0.2307, P = 0.0275, linear regression (a); or T cells stimulated for 36 h with anti-CD3, anti-CD28, and ICAM 82.4% concordance of markers (P = 0.0013, two-tailed binomial test); R2 = 0.1204, P = 0.1725, linear regression (b). Asymmetry as assessed by fluorescence intensity is expressed as (proximal − distal)/total (a), or values from (c-Mychigh – c-Myclow)/total (b). c, Western blot analysis of CD8 T cells activated with anti-CD3, anti-CD28, and ICAM for 6 h without treatment (ctrl) or with 1 μM or 5 μM of the bromodomain inhibitor JQ1. Data are representative of three independent experiments.

Extended Data Figure 8 Asymmetric assortment of mTORC1 activity with c-Myc–GFP.

a, b, Representative confocal images for quantifications of c-Myc–GFP and pS6 staining corresponding to Fig. 3a, b (a) or c-Myc–GFP and p70S6K corresponding to Fig. 3c (b) in OT-I T cells co-cultured with SIINFEKL-pulsed BMDCs. c, d, Quantifications for c-Myc–GFP and pS6 100% both bright in proximal daughter (x2 = 19.89, DF = 3, P = 0.0002, chi-square goodness of fit test); R2 = 0.9457, P = 0.0055, linear regression (c); or p70S6K 100% both bright in proximal daughter (x2 = 14.14, DF = 3, P = 0.0027, chi-square goodness of fit test); R2 = 0.4875, P = 0.0026, linear regression (d) staining in T cells stimulated on anti-CD3, anti-CD28, and ICAM for 36 h. e, Representative confocal images for quantifications of c-Myc–GFP and pFOXO1 staining corresponding to Fig. 3d, e. T cells stimulated with anti-CD3, anti-CD28, and ICAM for 36 h.

Extended Data Figure 9 First-division CD8high c-Myc–GFPhigh CD8 T cells are more glycolytic and exhibit more glutaminolysis and pentose phosphate pathway activity than first division CD8low c-Myc–GFPlow CD8 T cells but have decreased FAO.

Undivided or first-division antibody-activated T cells were sorted on c-Myc expression. 1.6 million T cells from each group were analysed via UHPLC/MS/MS for unbiased metabolomic profiling performed by Metabolon, Inc. a–d, Metabolic pathway schematic (a) was generated by Metabolon Inc., as were graphs (b) for select metabolites in pathway, representing data for DNA-normalized data. Metabolic pathway schematics and graphs were also generated by Metabolon Inc. for select metabolites in the pathway, representing data for DNA-normalized data for nucelotide biosynthesis (c) and fatty acid oxidation (d).

Extended Data Figure 10 c-Mychigh CD8 T cells are more glycolytic than CD8low CD8 T cells without asymmetric distribution of mitochondria.

a, The extracellular acidification rate (ECAR) for sorted c-Mychigh (circles) and c-Myclow (squares) T cells from the first division after 36 h of activation with anti-CD3, anti-CD28, and ICAM as measured by a Seahorse Bioflux analyser during exposure to the indicated compounds are represented. Values are paired across three independent experiments. b, The oxygen consumption rate (top panels) and ECAR (bottom panels) for basal respiration in complete RPMI with glucose of sorted c-Mychigh (shaded bars) and c-Myclow (open bars) CD8 T cells from the first-division after 36 h of activation by anti-CD3, anti-CD28, and ICAM, as measured by a Seahorse Bioflux analyser across three independent experiments. c, Quantification of mitochondrial DNA in sorted first-division c-Myc low and high OT-I CD8 T cells activated on anti-CD3, anti-CD28 and ICAM for 36 h. Mean + s.d. are plotted for three technical replicates of n = 2 mice per group. d, Overlay and individual channel images of β-tubulin (blue), DAPI (grey), c-Myc–GFP (green), and TOM20 (red) of an OT-I CD8 T cell activated with anti-CD3, anti-CD28 and ICAM for 36 h.

Supplementary information

Supplementary Information

This file contains additional Supplementary Methods and References. (PDF 213 kb)

Asymmetric assortment of c-Myc in a dividing CD8+ T cell.

T cells from c-Myc-GFP knockin mice were stimulated by culture on plate bound anti-CD3, anti-CD28, and ICAM (see methods), then fixed. Shown is a 3D reconstruction turned about the x-axis of volumetric representations of compiled images taken at 0.4 microns. Beta tubulin is pseudo-coloured blue, c-Myc-GFP green, and CD8 red. (MP4 119 kb)

Asymmetric assortment of c-Myc in a dividing CD8+ T cell.

T cells from c-Myc-GFP knockin mice were stimulated by culture on plate bound anti-CD3, anti-CD28, and ICAM (see methods), then fixed. Shown is a 3D reconstruction turned about the x-axis of volumetric representations of compiled images taken at 0.4 microns. Beta tubulin is pseudo-coloured blue, c-Myc-GFP green, and CD8 red. (MP4 90 kb)

Asymmetric assortment of c-Myc in a dividing CD8+ T cell.

T cells from c-Myc-GFP knockin mice were stimulated by culture on plate bound anti-CD3, anti-CD28, and ICAM (see methods), then fixed. Shown is a 3D reconstruction turned about the x-axis of volumetric representations of compiled images taken at 0.4 microns. Beta tubulin is pseudo-coloured blue, c-Myc-GFP green, and CD8 red. (MP4 124 kb)

Asymmetric assortment of c-Myc in a dividing CD8+ T cell.

OT-I, c-Myc-GFP knockin T cells were stimulated by culture with SIINFEKL-pulsed BMDC (see methods), then fixed. Shown is a 3D reconstruction turned about the x-axis of volumetric representations of compiled images taken at 0.4 microns. Beta tubulin is pseudo-coloured blue, c-Myc-GFP green, and CD8 red. (MP4 199 kb)

Asymmetric assortment of c-Myc in a dividing CD8+ T cell.

OT-I, c-Myc-GFP knockin T cells were stimulated by culture with SIINFEKL-pulsed BMDC (see methods), then fixed. Shown is a 3D reconstruction turned about the x-axis of volumetric representations of compiled images taken at 0.4 microns. Beta tubulin is pseudo-coloured blue, c-Myc-GFP green, and CD8 red. (MP4 163 kb)

Asymmetric assortment of c-Myc in a dividing CD8+ T cell

OT-I, c-Myc-GFP knockin T cells were stimulated by culture with SIINFEKL-pulsed BMDC (see methods), then fixed. Shown is a 3D reconstruction turned about the x-axis of volumetric representations of compiled images taken at 0.4 microns. Beta tubulin is pseudo-coloured blue, c-Myc-GFP green, and CD8 red. (MP4 229 kb)

Time-lapse of asymmetric assortment of c-Myc in a dividing CD8+ T cell.

Compilation of images taken every 2 minutes of a dividing OT-I, c-Myc-GFP knockin T cell after co-culture with SIINFEKL-pulsed BMDCs. Cell Trace Violet, blue; c-Myc-GFP, green. Tiles in Figure 2A were taken from every other still in this compilation. (MP4 402 kb)

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Verbist, K., Guy, C., Milasta, S. et al. Metabolic maintenance of cell asymmetry following division in activated T lymphocytes. Nature 532, 389–393 (2016). https://doi.org/10.1038/nature17442

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