Research ArticleTUMOR IMMUNOLOGY

Impaired enolase 1 glycolytic activity restrains effector functions of tumor-infiltrating CD8+ T cells

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Science Immunology  25 Jan 2019:
Vol. 4, Issue 31, eaap9520
DOI: 10.1126/sciimmunol.aap9520
  • Fig. 1 CD8+ TILs are metabolically less active than acute Teff, but more active than quiescent CD8+ T cells.

    (A to F) Naive and antigen-specific Teff (day 5 and day 12) were FACS-sorted from the spleens of C57Bl/6 mice immunized with OVA protein, poly I:C, and anti-CD40, whereas antigen-experienced CD8+ TILs were FACS-sorted from B16cOVA melanoma tumors that developed subcutaneously in C57Bl/6 mice for 12 to 15 days. (A to C) Ex vivo T cell glycolytic metabolism as measured by the ECAR (A and C) or the amount of tritiated water ([3H]H2O) released from [3-3H]glucose during glycolysis (B). The basal ECAR was measured in unmanipulated cells, whereas the maximum ECAR was quantified after exposing cells to a mitochondrial ATP synthase inhibitor (1 μM oligomycin) during the extracellular flux assay. (D and E) Oxidative metabolism (OXPHOS) of the cells in (A) and (C) as quantified by OCR. Basal OCR was from sorted cells in the steady state, whereas maximum OCR was from cells in response to exposure to an uncoupler of mitochondrial OXPHOS (1 μM FCCP). (F) Intracellular ATP levels in sorted ex vivo naive CD8+ T cells, day 5 (d5) Teff, and d14 CD8+ TILs as measured by a luciferase-based ATP determination kit. Data in (A), (C), (D), and (E) are representative of at least three independent experiments where samples were sorted from n = 3 to 5 mice per group and then pooled in each experiment. Data used to generate the graph in (B) were compiled from two independent experiments where the indicated sorted T cell samples were pooled from n = 5 to 9 mice per group. Data in (F) are representative of three independent experiments with samples pooled from n = 3 to 5 mice per group in each experiment. Data in (A) and (D) show means ± SEM and are analyzed by unpaired Student’s t test. Data in (B), (C), (E), and (F) show means ± SEM and analyzed by one-way ANOVA, followed by Tukey’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 2 Glucose uptake is not limited in CD8+ TILs.

    (A) Flow cytometric analysis of GLUT1 protein expression by ex vivo naive CD8+ T cell, d5 Teff, and d14 CD8+ TILs generated as in Fig. 1. The numbers within representative dot plots describe the frequency of cells expressing GLUT1 for individual samples. Bar charts show the overall frequency (middle chart) and intensity of GLUT1 expression (GMFI, right chart) of GLUT1+ cells in CD8+CD44lo naive CD8+ T cells and OVA-specific CD8+CD44hi for d5 Teff and CD8+ TILs. (B) Glucose uptake potential of ex vivo d5 and d14 Teff and d14 CD8+ TILs as measured by flow cytometry after pulsing cells with the fluorescent glucose analog 2-NBDG. The frequency (middle chart) of 2-NBDG+ cells and amount of uptake (right chart) within the OVA-specific CD8+CD44hi cell population are shown. (C) Survival of B16cOVA tumor-bearing mice was assessed after adoptive transfer of in vitro–activated nontransgenic or GLUT1 transgenic OT-1 T cells 5 days after subcutaneous tumor injection. Data in (A) and (B) are representative of three to five independent experiments, with at least two mice per group in each experiment. Data in (C) are from n = 7 mice per group, and the experiment was repeated twice. Data show means ± SEM: one-way ANOVA, followed by Tukey’s multiple comparison test. *P < 0.05, ***P < 0.001.

  • Fig. 3 Glycolysis-regulating signaling molecules are expressed and active in CD8+ TILs.

    (A) The signaling pathway downstream of TCR and cytokine signals that are known to promote the glucose uptake and glycolysis. (B to D) Flow cytometric analysis of p-PDK1 (Ser244), p-mTORC1 (Ser2448), and HIF1α expression in naive CD8+ T cells and OVA-specific d5 Teff, d14 Teff, and d14 CD8+ TILs that were prepared as in Fig. 1. (C) GMFI of pPDK1, p-mTORC1, and HIF1α in the CD8+CD44lo naive or OVA-specific CD8+CD44hi Teff and TILs expressing the protein of interest. (D) The frequency of p-PDK1+, p-mTORC1+, or HIF1α + cells within naive CD8+CD44lo and OVA-specific CD8+CD44hi Teff and TILs. (E) Phosphoflow cytometric analysis of mTORC1 activity via determination of the phosphorylated downstream targets (S6 and 4E-BP1) after 1-hour in vitro stimulation of acute Teff and CD8+ TILs with anti-CD3 (5 μg/ml). The CD8+ TIL in vitro stimulation was also conducted in the presence of 250 μM rapamycin to inhibit pS6 or 250 μM Torin-1 to inhibit p4E-BP1 for staining negative control. Data in (B) to (D) are representative of three to five independent experiments, with at least two mice per group in each experiment. Data in (E) are from samples pooled from n = 5 to 6 mice per group, and the experiment was repeated twice. Data show means ± SEM: one-way ANOVA, followed by Tukey’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 4 Weak enolase activity restrains glucose metabolism in glycolysis in CD8+ TILs.

    (A) Metabolomic analysis of the relative metabolite composition of d5 Teff and d14 CD8+ TILs normalized to that of naive CD8+ T cells. Metabolites were extracted from similar samples to that described in Fig. 1 and subjected to mass spectrometric analysis. 3-PG/2-PG, 3-phosphoglycerate and 2-phosphoglycerate. (B) The expression of enolase 1 transcripts in d5 Teff and d14 CD8+ TILs was measured by qPCR and ENOLASE1 protein was measured by Western blot (numbers indicate molecular weight). (C and D) Flow cytometric analysis of ENOLASE 1 protein expression in naive CD8 T cells, d5 Teff, and d14 CD8+ TILs. The frequencies of cells that express ENOLASE 1 within naive CD8+CD44lo and OVA-specific CD8+CD44hi Teff or CD8+ TILs are indicated. GMFI is derived from ENOLASE 1–expressing cells. (E) Fluorescence-based spectrophotometric analysis of enolase activity (PEP formation) measured in lysates prepared from FACS-sorted naive CD8+ T cells, acute Teff, and CD8+ TILs either directly ex vivo (left) or after in vitro stimulation with anti-CD3 and anti-CD28 for 4 days (right). Data are on samples pooled from n = 5 to 9 mice per group (A), representative from two independent experiments with samples pooled from at least six mice per group (B), and are representative of three independent experiments with at least three mice per group (C and D). Data in (E) are combined from multiple independent experiments with T cells pooled from 2 to 3 mice per group. Data show means ± SEM and are analyzed by unpaired Student’s t test (A, B, and E, right) or one-way ANOVA, followed by Tukey’s multiple comparison test (D and E, left). *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 5 Bypassing enolase restores metabolic function and effector activity in CD8+ TILs.

    (A and B) ECAR and OCR by FACS-sorted d5 Teff and d12 CD8+ TILs as measured by Seahorse in pyruvate-free or 2 mM pyruvate-containing XF minimal media. Basal ECAR and basal OCR were measured without any manipulation, whereas maximum ECAR and maximum OCR were determined after oligomycin or FCCP exposure, respectively. (C and D) Intracellular flow cytometric analysis of IFN-γ and TNFα production from Teff and CD8+ TILs treated for 4 hours with vehicle, 5 mM pyruvate, or 2 μM NaF during in vitro culture and stimulation with OVA257-pulsed antigen-presenting cells. (D) Fold increase in the proportion of CD8+ T cells expressing cytokines after culturing with 5 mM pyruvate or PEP (1 μg/ml; after partial permeabilization) for 4 hours. Data are representative of two to three independent experiments and show means ± SEM. Statistical analyses were done by unpaired Student’s t test (A, B, and D) and two-way ANOVA, followed by Tukey’s multiple comparison test (C). *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 6 Checkpoint molecule blockade supports CD8+ TIL enolase function.

    (A to G) B6 mice were subcutaneously injected with B16cOVA tumor cells and treated with either control, a combination of checkpoint inhibitor (CPi: anti–PD-1, anti–CTLA-4, and anti–TIM-3) antibodies, or individual antibodies on days 8, 11, and 14. (G) FTY-720 treatment was continuously provided through the drinking water starting on day 8 after tumor injection. All tumors were harvested on day 15 after injection. (A) The absolute numbers of antigen-experienced CD8+ TILs per square millimeter of tumor. (B) Tumor growth over time. (D) The frequency (left and middle) and intensity of expression (right) of enolase within OVA-specific CD8+CD44hi populations from control or combined CPi-treated mice. (E to G) Fluorescence-based spectrophotometric direct enolase activity (PEP formation) measurement in the lysates prepared from FACS-sorted antigen-experienced CD8+ TILs from control, combined-CPi (E and G), or individual-CPi–treated (F) mice in the absence (E and F) or presence of FTY-720 (G). The enolase activity in (E) and (F) was normalized to the average of the control replicates for CD8+ TILs to allow cross-experiment comparison. (H and I) Analysis of ENOLASE 1 expression and activity in ex vivo human CD8+ TILs using human healthy donor PBMCs that were in vitro stimulated with anti-CD3 and anti-CD28 for 3 days as a positive control. (H) Frequency of ENOLASE 1–positive cells within CD8+CD45RO+ human Teff and TILs. (I) Fluorescence-based spectrophotometric analysis of the activity of enolase in lysates prepared from FACS-sorted CD8+CD45RO+ human Teff and TILs. Data are representative of three independent experiments (A to D), combined from three to five independent experiments with samples pooled from at least three mice per group (E and F), combined from two independent experiments with at least three mice per group (G), or from two healthy donor PBMCs and three melanoma patient TILs (H and I). Data show means ± SEM and are analyzed by unpaired Student’s t test. **P < 0.01, ***P < 0.001.

  • GeneForward primersReverse primers
    RPS18S5′-ATGCGGC GGCGTTATTCC-3′5′-GCTATCAATCGTT-CAATCCTGTCC-3′
    GLUT15′-CAGTTCGGCTAT-AACACTGGTG-3′5′-GCCCCCGAC-AGAGAAGATG-3′
    mGlut35′-TAAACCAGCTGG-GCATCGTTGTTG-3′5′-AATGATGGTTAAG-CCAAGGAGCCC-3′
    Hk25′-TGATCGCCTGCTT-ATTCACGG-3′5′-AACCGCCTAGA-AATCTCCAGA-3′
    Pfkm5′-GGAAAGGAAGACA-GAGTGGGAGGC-3′5′-CAGATCGACCTCAA-CAGTGGGATTC-3′
    Pfkp5′-GGTACAGATTC-AGCCCTGCACC-3′5′-GTCGGCACCG-CAAGTCAAGG-3′
    GAPDH5′-GTCGGTGTGA-ACGGATTTG-3′5′-TAGACTCCACGA-CATACTCAGCA-3′
    Pgm35′-CCCAGCATCTCGAT-CATATCATGTTTCG-3′5′-GTCCTGCTCC-TCCGCACTGG-3′
    Eno15′-GGAAAGGAAGAC-AGAGTGGGAGGC-3′5′-CAGATCGACCTCA-ACAGTGGGATTC-3′
    Ldha5′-CATTGTCAAGTAC-AGTCCACACT-3′5′-TTCCAATTACTC-GGTTTTTGGGA-3′
    Ldhb5′-GGACAAGTGGG-TATGGCATGTG-3′5′-CCGTCACCACC-ACAATCTTAGA-3′
    MCT45′-TCACGGGTTT-CTCCTACGC-3′5′-GCCAAAGCG-GTTCACACAC-3′

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/31/eaap9520/DC1

    Fig. S1. B16cOVA-infiltrating CD8+ TILs exhibit phenotypic and functional markers of exhaustion.

    Fig. S2. CD8+ TILs express high levels of glucose transporters.

    Fig. S3. The abundance of glycolytic enzymes and metabolites suggests that PEP deficiency of CD8+ TILs is driven by lack of strong enolase activity.

    Fig. S4. Inhibition of the enolase activity in T cells limits cytokine production.

    Fig. S5. Enolase activity contributes to the effector function of CPi-treated CD8+ TILs.

    Fig. S6. Human melanoma TILs have low function and enolase activity.

    Table S1. Raw data file.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. B16cOVA-infiltrating CD8+ TILs exhibit phenotypic and functional markers of exhaustion.
    • Fig. S2. CD8+ TILs express high levels of glucose transporters.
    • Fig. S3. The abundance of glycolytic enzymes and metabolites suggests that PEP deficiency of CD8+ TILs is driven by lack of strong enolase activity.
    • Fig. S4. Inhibition of the enolase activity in T cells limits cytokine production.
    • Fig. S5. Enolase activity contributes to the effector function of CPi-treated CD8+ TILs.
    • Fig. S6. Human melanoma TILs have low function and enolase activity.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Raw data file.

    Files in this Data Supplement:

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