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Metabolic control of the scaffold protein TKS5 in tissue-invasive, proinflammatory T cells

Nature Immunology volume 18, pages 1025–1034 (2017)Cite this article

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Abstract

Pathogenic T cells in individuals with rheumatoid arthritis (RA) infiltrate non-lymphoid tissue sites, maneuver through extracellular matrix and form lasting inflammatory microstructures. Here we found that RA T cells abundantly express the podosome scaffolding protein TKS5, which enables them to form tissue-invasive membrane structures. TKS5 overexpression was regulated by the intracellular metabolic environment of RA T cells—specifically, by reduced glycolytic flux that led to deficiencies in ATP and pyruvate. ATPlopyruvatelo conditions triggered fatty acid biosynthesis and the formation of cytoplasmic lipid droplets. Restoration of pyruvate production or inhibition of fatty acid synthesis corrected the tissue-invasiveness of RA T cells in vivo and reversed their proarthritogenic behavior. Thus, metabolic control of T cell locomotion provides new opportunities to interfere with T cell invasion into specific tissue sites.

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Figure 1: Hypermotility and tissue-invasiveness of RA T cells.
Figure 2: TKS5 expression is metabolically regulated.
Figure 3: CD4+ T cells from individuals with RA accumulate cytoplasmic LDs.
Figure 4: RA CD4+ T cells favor the anabolic lipogenesis program.
Figure 5: LD accumulation is regulated by glycolytic flux and pyruvate availability.
Figure 6: Reduced glycolytic flux renders CD4+ T cells tissue-invasive and proinflammatory.
Figure 7: Inhibition of FA synthesis or acceleration of pyruvate generation corrects the tissue-invasive and arthritogenic behavior of RA T cells.

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Acknowledgements

This work was supported by the US National Institutes of Health (grants R01 AR042527, R01 HL 117913, R01 AI108906 and P01 HL129941 to C.M.W.; grants R01 AI108891, R01 AG045779, U19 AI057266 and I01 BX001669 to J.J.G.).

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Authors and Affiliations

  1. Division of Immunology and Rheumatology, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA

    Yi Shen, Zhenke Wen, Yinyin Li, Jison Hong, Jörg J Goronzy & Cornelia M Weyand

  2. Division of Rheumatology, Mayo Clinic College of Medicine, Rochester, Minnesota, USA

    Eric L Matteson

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Contributions

C.M.W., Y.S. and J.J.G. designed the study and analyzed the data. Y.S., Z.W. and Y.L. performed experiments. Y.S. created all of the figures. E.L.M. and J.H. were responsible for subject selection, evaluation and recruitment. C.M.W., Y.S. and J.J.G. wrote the manuscript.

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Correspondence to Cornelia M Weyand.

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Integrated supplementary information

Supplementary Figure 1 Genetic manipulation of TKS5 expression in human T cells.

(a, b) CD4+CD45RA+ T cells from RA patients were transfected with SH3PXD2A siRNA or control siRNA on day 2 after stimulation. SH3PXD2A transcripts were quantified 24 h later in control and SH3PXD2A siRNA-transfected cells by RT-PCR. Data are from ten independent experiments. TKS5 protein expression was measured in T cells transfected with control or SH3PXD2A siRNA by flow cytometry. (c, d) CD4+CD45RA+ T cells from healthy donors were transfected with control plasmids or a flag-SH3PXD2A construct. After 24 h, transfection efficiency was monitored by qPCR (n = 14) (c) and western blotting (d). Results are given as mean ± s.e.m. **P < 0.01 (Mann-Whitney U-test).

Supplementary Figure 2 Dose–response analysis for pharmacological inhibitors.

CD4+CD45RA+ T cells were isolated from healthy individuals and stimulated in the absence or presence of the PFKFB3 inhibitor 3PO (a) or the PKM2 inhibitor Shikonin (b). CD4+CD45RA+ T cells from RA patients were incubated with C75 (c), an inhibitor of fatty acid synthase, at concentrations indicated. Expression of the SH3PXD2A gene was assessed by RT-PCR (n = 6 samples). Results are mean ± s.e.m. *P < 0.05. **P < 0.01 (Mann-Whitney U-test).

Supplementary Figure 3 Effect of metabolic interference on intracellular pyruvate, ATP and SH3PXD2A expression.

CD4+CD45RA+ T cells were stimulated and treated with pharmacologic inhibitors as in Fig. 5. ML265 or pyruvate was added to the cultures at the indicated concentrations. (a, b) Pyruvate and intracellular ATP were measured in T cells from 8 donors. Vehicle (Veh), shikonin (SK). All data are mean ± s.e.m. *P < 0.05. **P < 0.01 (paired t-test). (c, d) Dose response analysis for RA T cells (n = 5) treated with ML265 or exogenous pyruvate. SH3PXD2A transcripts were measured by RT-PCR. Data are mean ± s.e.m. (paired t-test). Non-significant (ns).

Supplementary Figure 4 Metabolic interference and induction of lipid droplets does not affect PPAR-γ expression.

CD4+CD45RA+ T cells from 3 healthy controls were stimulated and cultured with (a, b) the glycolytic flux inhibitors (3PO, 200 nM or Shikonin, SK, 250 nM). (c, d) Alternatively, glycolytic flux was enhanced in RA T-cells by treating with ML265 or adding exogenous pyruvate (ML265, 10 μM or pyruvate, PY, 1 μM) as described in Fig.5. After 3 days, PPAR-γ expression levels were assessed by flow cytometry. Data are mean ± s.e.m. from three experiments (Mann-Whitney U-test). Non-significant (ns).

Supplementary Figure 5 Knockdown of PFKFB3 expression in human T cells.

(a) CD4+CD45RA+ T cells were transfected with PFKFB3 siRNA or control siRNA on day 2 after stimulation. PFKFB3 transcripts were quantified 24 h later by RT-PCR. Data are from twelve independent experiments. Results are given as mean ± s.e.m. *P < 0.05 (Mann-Whitney U-test). (b)To test for the durability of the PFKFB3 knockdown, transcripts were quantified at 6, 24, 48, 72 and 96 h after transfection (n = 6). Data are shown as fold change compared to control siRNA.

Supplementary Figure 6 Toxicity of the FAS inhibitor C75 in vivo.

CD4+CD45RA+ T cells were isolated from healthy controls and adoptively transferred into human synovium-NSG chimeric mice. Mice were randomly assigned to one of four treatment arms and received IP injections every other day: control arm (vehicle); C75 (1 mg/kg), C75 (5 mg/kg) or C75 (10 mg/kg). Human cells in the peripheral blood and the spleens of the mice were enumerated and stained with anti-CD3, anti-CD4 and anti-CD8 ab. Frequencies of the CD3+CD4+ population were determined by flow cytometry. Data are mean ± s.e.m. from three experiments (Mann-Whitney U-teat). Non-significant (ns).

Supplementary Figure 7 Purity of CD4+CD45RA+ T cell populations.

CD4+CD45RA+ T cells were purified as described in the Methods section from 10 healthy-RA pairs. To identify memory CD4+ T cells which have reverted to a naïve phenotype, CD4+CD45RA+ T cell populations from healthy individuals and RA patients were analyzed for: (a) CD4+CD45RA+CD28+CD95+ T cells. Representative histograms from a healthy control and a RA patient. (b) CD4+CD45RA+IFN-γ+ T cells. Intracellular IFN-γ was detected by flow cytometry 3 h after stimulation with PMA (50 ng/ml) plus ionomycin (1 μg/ml). Representative data from 10 experiments.

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Shen, Y., Wen, Z., Li, Y. et al. Metabolic control of the scaffold protein TKS5 in tissue-invasive, proinflammatory T cells. Nat Immunol 18, 1025–1034 (2017). https://doi.org/10.1038/ni.3808

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