Research ArticleMultiple Sclerosis

Teriflunomide treatment for multiple sclerosis modulates T cell mitochondrial respiration with affinity-dependent effects

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Science Translational Medicine  01 May 2019:
Vol. 11, Issue 490, eaao5563
DOI: 10.1126/scitranslmed.aao5563
  • Fig. 1 CD4+and CD8+T cell changes in teriflunomide-treated patients with RRMS from the TERIDYNAMIC study.

    (A and B) CD4+ T cell subpopulations in patients with RRMS after 3 months (3M) and 6 months (6M) of teriflunomide (TF) treatment from the TERIDYNAMIC clinical study (table S1). Box plots represent the interquartile range (IQR) with the horizontal line indicating the median and error bars showing maximum and minimum values. P values were calculated from linear mixed model on change from baseline. (A) Absolute cell numbers of TH1 (n = 32), TH2 (n = 32), TH17 (n = 32), and total Tregs (n = 37). (B) Frequencies of iTregs (n = 37). (C and D) Global TCR repertoire analysis of CD4+ and CD8+ T cells from HCs (n = 10) and treatment-naïve patients (n = 14) (table S3). Graphs display numbers of unique clones and sample overlap of CD4+ (C) and CD8+ (D) T cells. For changes at baseline between HCs and patients, P values were calculated using a Wilcoxon rank sum test. (E and F) Global TCR repertoire analysis of CD4+ and CD8+ T cells from patients at baseline and after 3 and 6 months of teriflunomide treatment (n = 15) from the TERIDYNAMIC clinical study. Graphs display numbers of unique clones and sample overlap of CD4+ (E) and CD8+ (F) T cells. P values were calculated from linear mixed model on change from baseline. (G and H) Global TCR repertoire properties of CD4+ T cells from patients at baseline and upon treatment with DMF for 6 months (n = 14), IFN-β for 12 months (12M) (n = 10), and GLAT for 12 months (n = 10) (table S10). Graphs display numbers of unique clones (G) and sample overlap (H). For changes from baseline and (C), P values were calculated using a paired Student’s t test. Horizontal lines indicate mean, and error bars show SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 2 Antigen affinity–dependent effect of teriflunomide on CD4+and CD8+T cells.

    (A) Proliferation of CD4+ T cells from 2D2 mice upon stimulation with NFM15–35 (high-affinity) or MOG35–55 (low-affinity) peptide-loaded dendritic cells in the presence (+TF) or absence (w/o) of teriflunomide at day 3. Data are representative of four independent experiments. (B) Percentages of proliferated CD4+ T cells from 2D2 mice upon stimulation with NFM15–35 or MOG35–55 peptide-loaded dendritic cells in the presence or absence of teriflunomide at day 3 (n = 4). ns, not significant. (C) Absolute cell numbers of CD4+ T cells from 2D2 mice upon stimulation with NFM15–35 or MOG35–55 peptide-loaded dendritic cells in the presence or absence of teriflunomide at day 3 (n = 5). Data were normalized to cells without treatment. (D) Proliferation of OT-I CD8+ T cells upon stimulation with altered peptide ligands of OVA257–264 with different affinities: SIINFEKL (N4) > SIIQFEKL (Q4) > SIITFEKL (T4) loaded on splenocytes in the presence or absence of teriflunomide at day 3. Generation analysis was performed with the FlowJo proliferation tool. Data are representative of three independent experiments. (E) Percentages of proliferated OT-I CD8+ T cells upon stimulation with N4, Q4, or T4 loaded on splenocytes in the presence or absence of teriflunomide at day 3 (n = 3). (F) Absolute cell numbers of OT-I CD8+ T cells upon stimulation with N4, Q4, or T4 loaded on splenocytes in the presence or absence of teriflunomide at day 3 (n = 3). Data were normalized to cells without treatment. (G) Proliferation of OT-I (high-affinity TCR) and OT-III (low-affinity TCR) CD8+ T cells upon stimulation with OVA257–264 peptide-loaded splenocytes in the presence or absence of teriflunomide at day 3. Generation analysis was performed with the FlowJo proliferation tool. Data are representative of three independent experiments. (H) Percentages of proliferated OT-I and OT-III CD8+ T cells upon stimulation with OVA257–264 peptide-loaded splenocytes in the presence or absence of teriflunomide at day 3 (n = 3). (I) Absolute numbers of OT-I and OT-III CD8+ upon stimulation with OVA257–264 peptide-loaded splenocytes in the presence or absence of teriflunomide at day 3 (n = 2). Data were normalized to cells without treatment. All data are displayed as means ± SEM. Statistical analysis was conducted by Student’s t test and was defined as *P < 0.05 and **P < 0.01.

  • Fig. 3 Influence of DHODH interference on T cell metabolism.

    (A) Oxygen consumption rate (OCR) of α-CD3/CD28–stimulated 2D2 CD4+ T cells in the presence or absence of teriflunomide at day 3. (B) Bar graph of basal and maximal (Max.) respiration from unstimulated naïve (Naive) and α-CD3/CD28–stimulated (Stim) 2D2 CD4+ T cells in the presence or absence of teriflunomide at day 3. (C) OCR of α-CD3/CD28–stimulated OT-I CD8+ T cells in the presence or absence of teriflunomide at day 2. (D) Bar graph of basal and maximal respiration from unstimulated naïve and α-CD3/CD28–stimulated OT-I CD8+ T cells in the presence or absence of teriflunomide at day 2. (E) Extracellular acidification rate (ECAR) of α-CD3/CD28–stimulated 2D2 CD4+ T cells in the presence or absence of teriflunomide at day 3. (F) Bar graph of glycolysis and glycolytic (Glyc.) capacity from unstimulated naïve and α-CD3/CD28–stimulated 2D2 CD4+ T cells in the presence or absence of teriflunomide at day 3. (G) ECAR of α-CD3/CD28–stimulated OT-I CD8+ T cells in the presence or absence of teriflunomide at day 2. (H) Bar graph of glycolysis and glycolytic capacity from unstimulated naïve and α-CD3/CD28–stimulated OT-I CD8+ T cells in the presence or absence of teriflunomide at day 2. (I) Proliferation of OT-I CD8+ T cells upon stimulation with OVA257–264 peptide-loaded splenocytes in the presence of teriflunomide added at indicated time points and measured at day 3. FACS, fluorescence-activated cell sorting; h, hours. (J) Mitochondrial respiration and glycolytic capacity of OT-I CD8+ T cells upon stimulation with OVA257–264 peptide-loaded splenocytes in the presence of teriflunomide added at indicated time points and measured at day 2. (K) ECAR of α-CD3/CD28 stimulated OT-I CD8+ T cells at day 3. Rotenone (Rot) and antimycin A (AA) were added as indicated. Oligo, oligomycin; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; Gluc, glucose; 2-DG, 2-deoxyglucose; Ctrl, control. All data are representative of three independent experiments or at least n = 3 per group (I and J) and displayed as means ± SEM. Statistical analysis was conducted by Student’s t test and was defined as **P < 0.01 and ***P < 0.001.

  • Fig. 4 Impact of teriflunomide on high-affinity versus low-affinity T cell metabolism.

    (A and B) OCR (A) and ECAR (B) of OT-I (high-affinity TCR) and OT-III (low-affinity TCR) CD8+ T cells upon stimulation with OVA257–264 peptide-loaded splenocytes at day 2. Bar graphs display OCR of basal respiration and maximal respiration (A) or ECAR of glycolysis and glycolytic capacity (B). (C) Kinetic analysis of maximal respiration and glycolytic capacity of OT-I and OT-III CD8+ T cells upon stimulation with OVA257–264 peptide-loaded splenocytes at indicated time points. (D) XF phenogram of OT-I and OT-III CD8+ T cells upon stimulation with OVA257–264 peptide-loaded splenocytes at day 2. (E and F) OCR (E) and ECAR (F) of 2D2 CD4+ T cells upon stimulation with NFM15–35 (high-affinity) or MOG35–55 (low-affinity) peptide-loaded dendritic cells at day 3. Bar graphs display OCR of basal respiration and maximal respiration (E) or ECAR of glycolysis and glycolytic capacity (F). (G) XF PhenoGram of 2D2 CD4+ T cells upon stimulation with NFM15–35 or MOG35–55 peptide-loaded dendritic cells at day 3. All data are representative of three independent experiments and displayed as means ± SEM. Statistical analysis was conducted by Student’s t test and was defined as *P < 0.05 and ***P < 0.001.

  • Fig. 5 Affinity-dependent gene expression.

    (A) Proliferation of OT-I (high-affinity TCR) and OT-III (low-affinity TCR) CD8+ T cells upon stimulation with OVA257–264 peptide-loaded splenocytes at indicated time points. Data are representative of at least two independent experiments. (B) Statistical analysis of data depicted in (A). (C) Expression of transcription factor IRF4 in the nucleus of OT-I and OT-III CD8+ T cells after antigen-specific activation in the presence or absence of teriflunomide for 3 hours (at least n = 3). Graph displays mean fluorescence intensity (MFI) analyzed by flow cytometry. (D) MFI of c-Myc in the nucleus of OT-I and OT-III CD8+ T cells after antigen-specific activation in the presence or absence of teriflunomide for 1 hour (n = 4). (E) Expression of phosphorylated S6 (phospho-S6) protein, a downstream target of mTOR, in OT-I and OT-III CD8+ T cells after antigen-specific activation in the presence or absence of teriflunomide for 3 hours (at least n = 3). Graph displays MFI analyzed by flow cytometry. (F) Heat map of the expression of 84 glycolytic and 84 mitochondrial respiration genes assessed in OT-I and OT-III CD8+ T cells after antigen-specific activation for 12 hours in the presence or absence of teriflunomide (at least n = 3). (G) Principal components analysis (PCA) of data depicted in (F). (H) Venn diagram of all differentially expressed genes (OT-I versus OT-III). The orange area indicates the overlap between TCR affinity–regulated genes and stimulation dependent–regulated genes. All data are displayed as means ± SEM. Statistical analysis was conducted by Student’s t test or one-way analysis of variance (ANOVA) (C to E) and was defined as *P < 0.05, **P < 0.01, and ***P < 0.001. Unstim, unstimulated.

  • Fig. 6 Effect of DHODH inhibition on mitochondrial content and function.

    (A) Relative mitochondrial DNA (mtDNA)/nuclear DNA (nDNA) ratio of OT-I (high-affinity TCR) and OT-III (low-affinity TCR) CD8+ T cells upon stimulation with OVA257–264 peptide-loaded splenocytes in the presence or absence of teriflunomide at day 2 (n = 3). Data were normalized to OT-I without. (B) OT-I and OT-III CD8+ T cells were activated with α-CD3/2CD8 in the presence or absence of teriflunomide for 2 days. Cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue) and MitoTracker green (MT green, green). Scale bars, 20 μM. (C) Quantification of MT green intensity with ImageJ software. Dots represent the intensity of single images with the same size and exposure time. (D) Histochemistry of complex IV activity of OT-I CD8+ T cells unstimulated or activated with α-CD3/CD28 in presence or absence of teriflunomide at day 2. Scale bars, 10 μM. Graph data display quantification of densitometric mean of individual complex IV puncta. (E) Activity of complexes I, II/III, and IV of the mitochondrial respiration chain in OT-I CD8+ T cells activated with α-CD3/CD28 in the presence or absence of teriflunomide for 2 days (n = 5). Cells were permeabilized before measurement, and substrates and inhibitors of mitochondrial respiration chain complexes were successively added. Scheme shows simplified illustration of the mitochondrial respiration chain (complexes I to IV) with all substrates (green) and inhibitors (red). Succ, succinate; TMPD, tetramethylphenylendiamin; Asc, ascorbate; Cyt c, cytochrome c. All data are displayed as means ± SEM. Statistical analysis was conducted by Student’s t test (A and C), one-way ANOVA (D), or two-way ANOVA (E) and was defined as *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 7 Relevance of DHODH inhibition in vivo.

    (A) Clinical EAE score of C57BL/6 mice treated orally with leflunomide (+LF, precursor of teriflunomide) or vehicle from 3 days before immunization throughout the disease course. Active MOG-EAE was induced in C57BL/6 mice (at least n = 13 per group), and the clinical disease score was assessed daily. (B and C) Absolute numbers of CD4+ T cells (B), CD4+ IFN-γ+, and CD4+ IL-17A+ T cells (C) from the CNS of immunized mice from (A) at day 14 analyzed by flow cytometry. (D) Absolute numbers of CD4+ IFN-γ+ and CD4+ IL-17A+ T cells from the spleen of immunized mice from (A) at day 14 analyzed by flow cytometry. (E) Tetramer staining and summary graph of MOG35–55-IAb+ CD4+ T cells isolated from the CNS of immunized mice treated with leflunomide or vehicle at day 14 (n = 7 to 8 per group). (F and G) Adhesion frequency of MOG35–55-specific CD4+ T cells isolated from the CNS of mice treated with leflunomide or vehicle at day 10 (n = 12 per group). T cells were tested for adhesion to MOG38–49-IAb or negative control hCLIP103–117-IAb. Receptor (R) density was assessed by flow cytometry (CD4+ T cells, 18 R/μm2; pMHC MOG38–49 -IAb, 770 R/μm2; and pMHC hCLIP103–117 -IAb, 393 R/μm2). (H and I) Treatment-naïve HLA-DR4+ patients with RRMS at baseline and during at least 6 months of teriflunomide treatment (n = 3) were analyzed for frequencies of myelin-specific T cells by using DRB1*0401/MOG97–109 and DRB1*0401/PLP180–199 tetramers and the DRB1*0401/CLIP87–101 control tetramer (table S8). FACS plots show one representative example. Bar graphs display the relative ratio of MOG97–109+ (H) or PLP180–199+ (I) CD4+ T cells to CLIP87–101+ CD4+ T cells. Data are displayed as means ± SEM. Statistical analysis was conducted by two-way ANOVA (A) or Student’s t test and was defined as *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 8 Metabolic profiles of T cells from patients with RRMS and HCs.

    (A and B) Basal and maximal respiration (A) or glycolysis and glycolytic capacity (B) of human CD4+ T cells from HCs (n = 24), treatment-naïve patients without (n = 25) and with relapse (n = 24) upon short-term stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (Iono) for 2.5 hours (table S11). (C and D) Activation-induced increase in mitochondrial respiration (C) and glycolysis (D) was calculated from human CD4+ T cells from the cohort depicted in (A) and (B) either left unstimulated or upon short-term stimulation for 2.5 hours with PMA/ionomycin before measurement of OCR or ECAR (stimulated OCR or ECAR values/unstimulated OCR or ECAR values). (E) CD4+ T cells from treatment-naïve patients at baseline and after at least 6 months of teriflunomide treatment (n = 14) were analyzed for maximal respiration and glycolytic capacity upon short-term stimulation with PMA/ionomycin for 2.5 hours (table S9). (F) OCR and ECAR values of CD4+ T cells from one representative treatment-naïve patients with RRMS at baseline and during teriflunomide treatment from (E). (G) Maximal respiration and glycolytic capacity of human CD4+ T cells from HCs (n = 12) and patients (n = 11) activated with α-CD3/CD28 in the presence or absence of teriflunomide for 3 days (table S12). (H) OCR and ECAR values of CD4+ T cells from one representative patient with RRMS from (G). All data are displayed as means ± SEM. Statistical analysis was conducted by one-way ANOVA (A to D) or Student’s t test (E to G) and was defined as *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Table 1 Evolution and change from baseline in the percentages and absolute counts of overall lymphocyte populations and CD4+T cell subsets in PBMCs.

    Per-protocol predefined population, people with RRMS treated with 14 mg of teriflunomide. Peripheral blood mononuclear cells (PBMCs) isolated and analyzed by flow cytometry at baseline and 6 months. Data are represented as mean (SD) or median (IQR). Change from baseline represented as least squares means (LSM) change (SEM) or median (IQR). P values from linear mixed model analysis of month 6 with baseline. N/A, not applicable

    LymphocytesCD4+ T cellsCD8+ T cellsCD19+ B cellsRatio CD4/CD8
    Absolute counts
    (cells/μl)
    Baseline
    Month 6
    Δ from baseline
    P value
    1882.8 (651)
    1587.9 (451)
    −284.94 (47.3)
    <0.001
    1036.1 (449.7)
    913.9 (383.6)
    −101.14 (30.91)
    0.002
    154.3 (97.7; 203.9)
    118.4 (67.5; 233.9)
    −34.7 (−55.5;−5.3)
    0.005
    N/AN/A
    Lymphocytes
    (% PBMC)
    CD4+ T cells
    (% CD3+ cells)
    CD8+ T cells
    (% CD3+ cells)
    CD19+ B cells
    (% lymphocytes)
    Ratio CD4/CD8
    Frequencies
    Baseline
    Month 6
    Δ from baseline
    P value
    79.5 (9.1)
    76.7 (11.3)
    −3.05 (1.43)
    0.040
    82.9 (74.9; 88.2)
    85.3 (79.1; 91.2)
    1.6 (−0.5; 3.7)
    0.006
    12.5 (8.9; 18.6)
    10.9 (6.7; 16.9)
    −1.0 (−3.5; 0.4)
    0.009
    10.6 (7.7; 14.9)
    7.7 (6.4; 11.8)
    −2.0 (−4.1; 0)
    0.001
    6.8 (4.2; 9.9)
    7.8 (4.8; 12.8)
    0.6 (−0.2; 3.9)
    0.008

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/490/eaao5563/DC1

    Materials and Methods

    Fig. S1. Changes in T cell subsets in patients with RRMS on teriflunomide treatment.

    Fig. S2. Influence of teriflunomide on proliferation and cytokine production.

    Fig. S3. Impact of antigen affinities and DHODH interference on proliferation.

    Fig. S4. Metabolic assessment of T cells under teriflunomide treatment.

    Fig. S5. Influence of DHODH inhibition on T cell metabolism, on gene expression of OT-I and OT-III T cells, on TH1 differentiation and proliferation, and on Treg subpopulations in EAE.

    Fig. S6. Metabolism of T cells from patients with RRMS and HCs.

    Fig. S7. Comparison of freshly isolated versus frozen PBMCs from HCs regarding immune cell subset composition and metabolism.

    Fig. S8. Additional information supporting methodology.

    Table S1. Demographics and baseline disease characteristics TERIDYNAMIC trial.

    Table S2. Differentially regulated genes depicted in Fig. 5I.

    Table S3. Demographics and baseline disease characteristics of HCs and treatment-naïve patients analyzed for TCR repertoire changes.

    Table S4. Demographics and baseline disease characteristics of treatment-naïve patients before and during teriflunomide treatment analyzed for TCR repertoire changes.

    Table S5. Demographics and baseline disease characteristics of treatment-naïve patients before and during teriflunomide treatment analyzed for immune cell phenotyping.

    Table S6. Demographics and baseline disease characteristics of patients before and during teriflunomide treatment for at least 6 months analyzed for suppressive capacity of Tregs.

    Table S7. Demographics and baseline disease characteristics of patients before and during teriflunomide treatment for at least 6 months analyzed for cytokine expression of Tregs.

    Table S8. Demographics and baseline disease characteristics of treatment-naïve patients before and during teriflunomide treatment for at least 6 months analyzed for myelin-specific T cell frequencies.

    Table S9. Demographics and baseline disease characteristics of treatment-naïve patients before and during teriflunomide treatment for at least 6 months analyzed for metabolic activity.

    Table S10. Demographics and baseline disease characteristics of treatment-naïve patients before and during treatment with DMF, IFN-β, or GLAT analyzed for TCR repertoire changes.

    Table S11. Demographics and baseline disease characteristics of HCs and treatment-naïve patients analyzed for metabolic activity.

    Table S12. Demographics and baseline disease characteristics of HCs and patients analyzed for metabolic activity after 72 hours of in vitro stimulation.

    Table S13. Demographics of HCs analyzed for immune cell phenotyping of freshly isolated versus frozen PBMCs.

    Table S14. Demographics of HCs analyzed for metabolic differences of T cells isolated from fresh blood or frozen PBMCs.

    Data file S1. Primary data.

    References (3638)

  • The PDF file includes:

    • Material and Methods
    • Fig. S1. Changes in T cell subsets in patients with RRMS on teriflunomide treatment.
    • Fig. S2. Influence of teriflunomide on proliferation and cytokine production.
    • Fig. S3. Impact of antigen affinities and DHODH interference on proliferation.
    • Fig. S4. Metabolic assessment of T cells under teriflunomide treatment.
    • Fig. S5. Influence of DHODH inhibition on T cell metabolism, on gene expression of OT-I and OT-III T cells, on TH1 differentiation and proliferation, and on Treg subpopulations in EAE.
    • Fig. S6. Metabolism of T cells from patients with RRMS and HCs.
    • Fig. S7. Comparison of freshly isolated versus frozen PBMCs from HCs regarding immune cell subset composition and metabolism.
    • Fig. S8. Additional information supporting methodology.
    • Table S1. Demographics and baseline disease characteristics TERIDYNAMIC trial.
    • Table S2. Differentially regulated genes depicted in Fig. 5I.
    • Table S3. Demographics and baseline disease characteristics of HCs and treatment-naïve patients analyzed for TCR repertoire changes.
    • Table S4. Demographics and baseline disease characteristics of treatment-naïve patients before and during teriflunomide treatment analyzed for TCR repertoire changes.
    • Table S5. Demographics and baseline disease characteristics of treatment-naïve patients before and during teriflunomide treatment analyzed for immune cell phenotyping.
    • Table S6. Demographics and baseline disease characteristics of patients before and during teriflunomide treatment for at least 6 months analyzed for suppressive capacity of Tregs.
    • Table S7. Demographics and baseline disease characteristics of patients before and during teriflunomide treatment for at least 6 months analyzed for cytokine expression of Tregs.
    • Table S8. Demographics and baseline disease characteristics of treatment-naïve patients before and during teriflunomide treatment for at least 6 months analyzed for myelin-specific T cell frequencies.
    • Table S9. Demographics and baseline disease characteristics of treatment-naïve patients before and during teriflunomide treatment for at least 6 months analyzed for metabolic activity.
    • Table S10. Demographics and baseline disease characteristics of treatment-naïve patients before and during treatment with DMF, IFN-β, or GLAT analyzed for TCR repertoire changes.
    • Table S11. Demographics and baseline disease characteristics of HCs and treatment-naïve patients analyzed for metabolic activity.
    • Table S12. Demographics and baseline disease characteristics of HCs and patients analyzed for metabolic activity after 72 hours of in vitro stimulation.
    • Table S13. Demographics of HCs analyzed for immune cell phenotyping of freshly isolated versus frozen PBMCs.
    • Table S14. Demographics of HCs analyzed for metabolic differences of T cells isolated from fresh blood or frozen PBMCs.
    • References (3638)

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    Other Supplementary Material for this manuscript includes the following:

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