Research ArticleImmunometabolism

Immunometabolism regulates TCR recycling and iNKT cell functions

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Science Signaling  26 Feb 2019:
Vol. 12, Issue 570, eaau1788
DOI: 10.1126/scisignal.aau1788
  • Fig. 1 iNKT cells activate glycolysis after TCR stimulation.

    (A to D) Seahorse extracellular flux analysis of ECAR (A) and OCR (C) on iNKT cells activated by antibodies against CD3 and CD28, as indicated. Basal glycolysis, glycolytic capacity, basal respiration, and maximal respiration were calculated, as indicated in (A) and (C). Normalized values (B and D) are means ± SEM of seven to nine biological replicates pooled from two independent experiments. (E to G) Seahorse extracellular flux analysis of OCR and ECAR on iNKT cells activated as indicated by antibodies against CD3 and CD28 in normal medium (E) or in glucose-free medium (F) or in the presence of 2-DG (G). Data are means ± SEM of nine biological replicates pooled from three independent experiments. **P < 0.01 and ***P < 0.001 by Student’s t test.

  • Fig. 2 2-DG inhibits proliferation of iNKT cells and polarizes iNKT cells toward TH2 response.

    (A and B) Cytometric bead array (CBA) analysis of IFN-γ and IL-4 production by iNKT cells activated by immobilized mCD1d-PBS57 tetramer overnight with 2-DG, as indicated. Data (A) and relative IL-4/IFN-γ ratio (B) are means ± SEM of nine independent biological replicates pooled from three independent experiments. (C to E) Flow cytometry analysis of intracellular IFN-γ and IL-4 in hepatic iNKT cells activated by αGC in vivo with 2-DG, as indicated. Dot plots (C) are representative of three independent experiments. Quantified percent cytokine+ iNKT cells (D) and cytokine mean fluorescence intensity (E) are means ± SEM of seven mice per group. (F and G) CBA analysis of IFN-γ and IL-4 production by iNKT cells activated with antibodies against CD3 and CD28 in medium with or without glucose, as indicated. Data (F) and IL-4/IFN-γ ratio (G) are means ± SEM of 9 biological replicates pooled from three independent experiments. (H and I) CBA analysis of IFN-γ and IL-4 production by iNKT cells activated with antibodies against CD3 and CD28 with or without G6P. Data (H) and IL-4/IFN-γ ratio (I) are means ± SEM of nine biological replicates pooled from three independent experiments. (J and K) CBA analysis of IFN-γ and IL-4 production by iNKT cells activated with immobilized CD1d-PBS57 tetramer overnight in the presence of 2-DG and pyruvate replenishment, as indicated. Data (J) and IL-4/IFN-γ ratio (K) are means ± SEM of nine biological replicates pooled from three independent experiments. (L) Flow cytometry analysis of surface CD69, CD25, and CD40L abundance on iNKT cells activated with immobilized CD1d-PBS57 tetramer overnight and 2-DG, as indicated. Data are means ± SEM of nine independent replicates pooled from three independent experiments. (M) Flow cytometry analysis of Ki67 expression in iNKT cells activated by immobilized mCD1d-PBS57 tetramer for 2 days with or without 2-DG. Data are means ± SEM of nine biological replicates pooled from three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test.

  • Fig. 3 2-DG inhibits iNKT cell TCR signaling.

    (A and B) Flow cytometry analysis of intracellular IFN-γ and IL-4 in iNKT cells activated with PMA + ionomycin (P + I) and 2-DG for 4 hours, as indicated. Frequency data (A) and cytokine mean fluorescence intensity (MFI) (B) are means ± SEM of nine biological replicates pooled from three independent experiments. (C and D) TIRFM analysis of the distribution (C) and amount (D) of surface TCR at the synapses of iNKT cells activated with CD1d-PBS57 tetramer in the presence of phosphate-buffered saline (PBS) control or 2-DG at indicated time points. Images (C) are representative of six independent experiments (C). Quantified values are means ± SEM of 60 cells per group pooled from six independent experiments (D). Scale bars, 5 μm. A.U., arbitrary units. (E and F) Western blot analysis of phosphorylation of pLCK-Tyr394, pLAT-Tyr191, pPKCθ-Ser676, pP38 MAPK-Thr180/Tyr182, and pErk1/2-Thr202/Tyr204 in lysates of iNKT cells activated with antibodies against CD3 and CD28 for 45 min with or without 2-DG. Blots (E) are representative of three independent experiments. Quantified band intensity values (F) are means ± SEM pooled from three independent experiments. (G) CBA analysis of inhibition of IFN-γ and IL-4 production by 2-DG compared to PBS control added to iNKT cells after activating for 2 and 8 hours. Data are means ± SEM of nine biological replicates pooled from three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test or by Wilcoxon test.

  • Fig. 4 Glycolysis promotes TCR recycling.

    (A) Flow cytometry analysis of total surface and intracellular TCR in iNKT cells activated with CD1d-PBS57 tetramer and PBS or 2-DG for the indicated times. Data are means ± SEM of nine biological replicates pooled from three independent experiments. (B and C) Flow cytometry analysis of TCR internalization (B) and recycling (C) in iNKT cells activated with CD1d-PBS57 tetramer and PBS or 2-DG for the indicated times. Data are means ± SEM of 12 biological replicates pooled from four independent experiments. (D) Flow cytometry analysis of TCR recycling in iNKT cells activated with CD1d-PBS57 tetramer and PBS, 2-DG, or 2-DG plus pyruvate for the indicated times. Data are means ± SEM of nine biological replicates pooled from three independent experiments. (E to G) TIRFM analysis of the distribution (E), density (F), and mean fluorescence intensity (G) of intracellular TCR in iNKT cells activated with CD1d-PBS57 tetramer and 2-DG as indicated for 45 min. Images (E) are representative of three independent experiments with threshold images are inserted. Quantified values (F and G) are means ± SEM of 100 cells per group pooled from three independent experiments. (H and I) TIRFM analysis of the distribution (H) and amount (I) of surface TCR at the synapses of iNKT cells activated with CD1d-PBS57 tetramer and G6P as indicated for 45 min. Images (H) are representative of three independent experiments. Quantified values (I) are means ± SEM of 60 cells per group pooled from three independent experiments. (J to L) TIRFM analysis of the distribution (J), density (K), and mean fluorescence intensity (L) of intracellular TCR in iNKT cells activated with CD1d-PBS57 tetramer and G6P as indicated for 45 min. Images (J) are representative of three independent experiments. Quantified values (K and L) are means ± SEM of 80 cells per group pooled from three independent experiments. Scale bars, 5 μm. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test.

  • Fig. 5 Dissociation of HK-II from mitochondria inhibits IFN-γ production and TCR recycling in iNKT cells.

    (A and B) TIRFM analysis of the distribution (A) and colocalization (B) of mitochondrial marker ATPB and HK-II in iNKT cells activated with CD1d-PBS57 tetramer and HKVBD, 2-DG, or G6P as indicated for 45 min. Images (A) are representative of three independent experiments. Quantified values (B) are means ± SEM of 60 cells per group pooled from three independent experiments. (C and D) CBA analysis of IFN-γ and IL-4 production by iNKT cells activated with CD1d-PBS57 tetramer and HKVBD peptide, as indicated. Data (C) and IL-4/IFN-γ ratio (D) are means ± SEM of nine biological replicates pooled from three independent experiments. (E and F) TIRFM analysis of the distribution (E) and amount (F) of surface TCR at the synapses of iNKT cells activated with CD1d-PBS57 tetramer and HKVBD peptide as indicated for 45 min. Images (E) are representative of three independent experiments. Quantified data (F) are means ± SEM of 60 cells per group pooled from three independent experiments. (G to I) TIRFM analysis of the distribution (G), density (H), and mean fluorescence intensity (I) of intracellular TCR in iNKT cells after activation with CD1d-PBS57 tetramer and HKVBD peptide for 45 min. Images (G) are representative of three independent experiments. Quantified data (H and I) are means ± SEM of 80 cells per group pooled from three independent experiments. Scale bars, 5 μm. *P < 0.05 and ***P < 0.001 by Student’s t test.

  • Fig. 6 Dissociation of HK-II from mitochondria inhibits mTORC2 activation.

    (A and B) TIRFM analysis of the distribution (A) and colocalization (B) of the mitochondrial marker ATPB and mTOR in iNKT cells activated with CD1d-PBS57 tetramer and HKVBD peptide, 2-DG, or G6P as indicated for 45 min. Images (A) are representative of three independent experiments. Quantified values are means ± SEM of 90 cells per group pooled from three independent experiments. Scale bars, 5 μm. (C and D) Western blot analysis of mTOR and rictor in crude mitochondrial fractions from expanded iNKT cells activated with antibodies against CD3 and CD28, and HKVBD, 2-DG, or G6P as indicated for 45 min. Blots (C) are representative of three independent experiments. Quantified band intensity values (D) are means ± SEM pooled from three independent experiments. (E and F) Western blot analysis of pAKT-Ser473 and pPKCθ-Ser676 in lysates from iNKT cells activated with antibodies against CD3 and CD28, and HKVBD, 2-DG, or G6P as indicated for 45 min. Blots (E) are representative of three independent experiments. Quantified band intensity values (F) are means ± SEM polled from three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test or by Wilcoxon test.

  • Fig. 7 AKT and PKCθ promote TCR recycling and IFN-γ production in iNKT cells.

    (A and B) CBA analysis of IFN-γ and IL-4 production by iNKT cells activated with CD1d-PBS57 tetramer and PKCθ inhibitor sotrastaurin (Sotra), as indicated. Data (A) and IL-4/IFN-γ ratio (B) are means ± SEM of nine biological replicates pooled from three independent experiments. (C and D) TIRFM analysis of the distribution (C) and amount (D) of surface TCR at the synapses of iNKT cells activated with CD1d-PBS57 tetramer and Sotra as indicated for 45 min. Images (C) are representative of three independent experiments. Quantified values (D) are means ± SEM of 120 cells per group pooled from three independent experiments. (E to G) TIRFM analysis of the distribution (E), density (F), and mean fluorescence intensity (G) of intracellular TCR in iNKT cells activated with CD1d-PBS57 tetramer and Sotra as indicated for 45 min. Images (E) are representative of three independent experiments. Quantified values (F and G) are means ± SEM of 60 cells per group pooled from three independent experiments. (H and I) CBA analysis of IFN-γ and IL-4 by iNKT cells activated with CD1d-PBS57 tetramer and Akt inhibitor MK2206 as indicated. Data production (H) and IL-4/IFN-γ ratio (I) are means ± SEM of nine biological replicates pooled from three independent experiments. (J to N) TIRFM analysis of distribution (J) and amount (K) of surface TCR, or the distribution (L), density (M), and mean fluorescence intensity (N) of intracellular TCR at the synapses of iNKT cells activated with CD1d-PBS57 tetramer and MK2206 as indicated for 45 min. Images (J and L) are representative of three independent experiments. Quantified values (K, M, and N) are means ± SEM of 60 cells per group pooled from three independent experiments. Scale bars, 5 μm. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/12/570/eaau1788/DC1

    Fig. S1. Reduced respiratory capacity but increased glycolytic activity in iNKT cells than in CD4 T cells.

    Fig. S2. Metabolic reprogramming after long-term activation.

    Fig. S3. 2-DG inhibits IFN-γ production and TCR recycling in iNKT cells activated by anti-CD3 plus anti-CD28.

    Fig. S4. 2-DG does not reduce cell numbers of iNKT cells.

    Fig. S5. Influences of 2-DG on proliferation and activation markers of iNKT cells activated by PMA plus ionomycin.

    Fig. S6. Shortening TCR signaling inhibits IFN-γ production.

    Fig. S7. 2-DG inhibits TCR signaling proteins even after TCR engagement.

    Fig. S8. 2-DG inhibits gene transcription.

    Fig. S9. 2-DG does not influence accumulation of TCR at synapses of CD4 T cells.

    Fig. S10. Inhibitors do not influence the expression of HK-II.

    Fig. S11. Inhibition of glycolysis reduces translocation of mTOR to lysosomes.

    Fig. S12. PEP and d-mannose do not restore IFN-γ production in 2-DG–treated iNKT cells.

    Table S1. List of primer sequences.

  • This PDF file includes:

    • Fig. S1. Reduced respiratory capacity but increased glycolytic activity in iNKT cells than in CD4 T cells.
    • Fig. S2. Metabolic reprogramming after long-term activation.
    • Fig. S3. 2-DG inhibits IFN-γ production and TCR recycling in iNKT cells activated by anti-CD3 plus anti-CD28.
    • Fig. S4. 2-DG does not reduce cell numbers of iNKT cells.
    • Fig. S5. Influences of 2-DG on proliferation and activation markers of iNKT cells activated by PMA plus ionomycin.
    • Fig. S6. Shortening TCR signaling inhibits IFN-γ production.
    • Fig. S7. 2-DG inhibits TCR signaling proteins even after TCR engagement.
    • Fig. S8. 2-DG inhibits gene transcription.
    • Fig. S9. 2-DG does not influence accumulation of TCR at synapses of CD4 T cells.
    • Fig. S10. Inhibitors do not influence the expression of HK-II.
    • Fig. S11. Inhibition of glycolysis reduces translocation of mTOR to lysosomes.
    • Fig. S12. PEP and d-mannose do not restore IFN-γ production in 2-DG–treated iNKT cells.
    • Table S1. List of primer sequences.

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