Research ArticleSystemic Lupus Erythematosus

Normalization of CD4+ T cell metabolism reverses lupus

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Science Translational Medicine  11 Feb 2015:
Vol. 7, Issue 274, pp. 274ra18
DOI: 10.1126/scitranslmed.aaa0835
  • Fig. 1. CD4+ T cells from TC mice show an enhanced metabolism.

    (A to D) ECAR (A), OCR (B and C), and SRC (D) measured in total CD4+ T cells from 2- and 9-month-old B6 and TC mice. (B) Representative OCR in 9-month-old B6 and TC CD4+ T cells. FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. (E) Extracellular lactate production from 3-month-old B6 and TC CD4+ T cells. (F) ATP production by B6 and TC CD4+ T cells stimulated with PMA/ionomycin (IO) or anti-CD3/CD28. RQ, relative quantity. (G and H) ECAR (G) and OCR (H) in Tn and Tem from 9-month-old B6 and TC mice. (I and J) ECAR (I) and OCR (J) in B6 and TC Tn after 24-hour stimulation with anti-CD3/CD28. n = 3 to 6.

  • Fig. 2. CD4+ T cells from TC mice show an increased mTORC1 activity.

    (A) S6 and 4E-BP1 phosphorylation and expression of CD98 and CD71 in total CD4+ T cells as well as Tn, Tem, and Tcm subsets from 2-month-old mice. n = 3 to 4. (B and C) ECAR (B) and OCR (C) in B6 CD4+ T cells stimulated with anti-CD3/CD28 with or without rapamycin (Rapa) (100 nM) for 24 hours. Representative graphs of two independent assays each performed with n = 7 technical replicates.

  • Fig. 3. Metabolic modulators normalized TC CD4+ T cell effector functions in vitro.

    (A to D) ECAR (A and C) and OCR (B and D) in B6 CD4+ T cells stimulated with anti-CD3/CD8 for 24 hours in the presence of 2DG or Met. Representative graphs of three assays each performed with technical replicates. (E) IFN-γ production in CD4+ T cells stimulated with PMA/ionomycin/GolgiPlug for 6 hours (Ctrl), in the presence of Met (2 mM), 2DG (5 mM), antimycin A/rotenone (A&R) (both 0.5 μM), or oligomycin (Olig) (1 μM). (F to H) IFN-γ production in CD4+ T cells cultured under TH1 condition for 3 days with Met [0 to 5 mM in (F) or 1 mM in (G)] or 2DG (1 mM) added from day 0 (d0) (F and G) or day 2 (d2) (H). (I and J) IL-2 production in CD4+ T cells stimulated with anti-CD3/CD28 for 3 days with 0 to 1 mM (I) or 1 mM (J). (G) Statistical significance of a two-way analysis of variance (ANOVA) between B6 and TC. n = 3 to 8.

  • Fig. 4. Treatment with Met + 2DG for 3 months reversed disease in 7-month-old B6 and TC mice.

    (A and B) ECAR (A) and OCR (B) in CD4+ T cells. (C) Spleen weight (representative spleens on the right of 14 for TC and 5 for B6). (D) Serum anti-dsDNA IgG in TC mice (two-way ANOVA). (E) Initial (I) and terminal (T) serum ANA from TC mice. Representative images of 14 sera per group and ANA intensity quantification, with each linked symbol representing a mouse. Untreated B6 mice are shown as control (paired t tests). (F) Autoantibody microarray analysis of terminal sera (IgG). ssDNA, single-stranded DNA; H2A, histone 2A; H2B, histone 2B. (G) Immune complex deposition in TC glomeruli. Representative images of 14 kidneys per group with C3 and IgG2a deposits (left) and C3 intensity (three to six glomeruli per mouse). (H) Renal pathology assessed by severity rank (median and interquartile range, two-tailed Mann-Whitney test) and GN score distribution (χ2 test). (I) Representative glomeruli (periodic acid–Schiff stain) from untreated mice (left) showing large subendothelial deposits (arrows) and from treated mice (right) showing open capillaries and reduced hypercellularity (stars) (scale bars, 25 μm). n = 14 TC and 5 B6 mice per group.

  • Fig. 5. A 3-month Met + 2DG treatment normalized CD4+ T cell phenotypes in aged TC mice.

    (A to C) Percentage of total splenic CD4+ T cells (A), CD69+ (B), and Tem (C) CD4+ T cells in B6 and TC-treated and control (Ctrl) mice. (D) Representative CD4+-gated FACS (fluorescence-activated cell sorting) plots showing the CD62L+CD44 Tn and CD62CD44+ Tem subsets. (E to G) Frequency of Tfh (E) and Tfr (F) CD4+ T cells and representative CD4+-gated FACS plots showing the PD-1hiCXCR5hiBCL6+Foxp3Tfh and PD-1hiCXCR5hiBCL6+Foxp3+ Tfr subsets (G). (H) Frequency of GC CD19+ B cells. (I) IL-2 production in CD4+ T cells stimulated with anti-CD3/CD28 for 24 hours. (J to M) Effect of treatment on phosphorylation of S6 (pS6) (J) and 4E-BP1 (p4E-BP) (K), as well as expression of CD98 (L) and CD71 (M) in total, Tn, and Tem T cells. n = 4 to 14. MFI, mean fluorescence intensity.

  • Fig. 6. Met + 2DG treatment for 1 month reversed immunophenotypes in NZB/W mice.

    (A and B) ECAR (A) and OCR (B) in splenic CD4+ T cells. (C) Change between the terminal and the initial serum anti-dsDNA IgG for each mouse. (D) Serum ANA intensity, with each linked symbol representing the initial and final values for each mouse. (E and F) Change between the terminal and initial total serum IgM (E) and IgG (F). (G to L) Frequency of total CD4+ T cells (G); CD69+ (H), Tem (I), Tfh (J), and Tfr (K) CD4+ T cells; and GC B cells (L). (M to P) Effect of treatment of mTORC1 targets: phosphorylation of S6 (M) and 4E-BP1 (N) and expression of CD98 (O) and CD71 (P) in total CD4+ T cells. (Q and R) Renal pathology assessed by severity rank (median and interquartile range) (Q) and distribution of GN mesangial (Mn 2–3 and 4) scores (R). n = 4 to 5.

  • Fig. 7. CD4+ T cells from SLE patients have an enhanced metabolism.

    (A and B) Representative ECAR (A) and OCR (B) graphs of human CD4+ T cells during a mitochondrial stress test. Anti-CD3/CD28 or isotope controls, oligomycin, FCCP, and antimycin A/rotenone (Rote + Anti) were added to the cells as indicated. (C to E) ECAR (C), OCR (D), and SRC (E) in HC and SLE CD4+ T cells, with and without anti-CD3/CD28 activation. n = 19 HCs and 20 SLE patients. (F to H) Correlations between Tn percentages and activated ECAR (F) or basal OCR (G) and between Treg percentages and activated ECAR (H). The significance and correlation coefficient of Pearson tests are shown. (I) IFN-γ production in TH1-polarized CD4+ T cells with or without Met. Two-tailed paired t tests compared the effect of treatment within each cohort. n = 6.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/274/274ra18/DC1

    Fig. S1. TC mice showed enhanced CD4+ T cell activation.

    Fig. S2. Representative FACS plots of ex vivo CD4+-gated T cells from B6 (black) and TC (red) mice for pS6, p4E-PB1, CD98, and CD71.

    Fig. S3. CD4+ T cells from TC mice showed an altered expression of metabolic genes.

    Fig. S4. IL-2 gating in CD4+ T cells.

    Fig. S5. The Met + 2DG treatment normalized the metabolism of effector CD4+ T cells.

    Fig. S6. A 3-month Met + 2DG in vivo treatment resulted in a global normalization of TC CD4+ T cell effector phenotypes.

    Fig. S7. The Met + 2DG treatment did not have adverse effects on body weight and blood sugar.

    Fig. S8. Met + 2DG treatment did not affect normal humoral response.

    Fig. S9. One-month Met + 2DG treatment reduced disease severity in TC mice.

    Fig. S10. Met + 2DG treatment prevented autoantibody production in the cGVHD model.

    Fig. S11. 2DG treatment failed to reverse immunophenotypes in TC mice.

    Fig. S12. Met treatment failed to reverse immunophenotypes in TC mice.

    Fig. S13. CD4+ T cell immunophenotypes in SLE patients.

    Table S1. Human subject demographics and disease parameters.

    Table S2. Primers for qPCR assays.

    Table S3. Antibodies used in this study.

    Table S4. Source data (Excel file).

  • Supplementary Material for:

    Normalization of CD4+ T cell metabolism reverses lupus

    Yiming Yin, Seung-Chul Choi, Zhiwei Xu, Daniel J. Perry, Howard Seay, Byron P. Croker, Eric S. Sobel, Todd M. Brusko, Laurence Morel*

    *Corresponding author. E-mail: morel{at}ufl.edu

    Published 11 February 2015, Sci. Transl. Med. 7, 274ra18 (2015)
    DOI: 10.1126/scitranslmed.aaa0835

    This PDF file includes:

    • Fig. S1. TC mice showed enhanced CD4+ T cell activation.
    • Fig. S2. Representative FACS plots of ex vivo CD4+-gated T cells from B6 (black) and TC (red) mice for pS6, p4E-PB1, CD98, and CD71.
    • Fig. S3. CD4+ T cells from TC mice showed an altered expression of metabolic genes.
    • Fig. S4. IL-2 gating in CD4+ T cells.
    • Fig. S5. The Met + 2DG treatment normalized the metabolism of effector CD4+ T cells.
    • Fig. S6. A 3-month Met + 2DG in vivo treatment resulted in a global normalization of TC CD4+ T cell effector phenotypes.
    • Fig. S7. The Met + 2DG treatment did not have adverse effects on body weight and blood sugar.
    • Fig. S8. Met + 2DG treatment did not affect normal humoral response.
    • Fig. S9. One-month Met + 2DG treatment reduced disease severity in TC mice.
    • Fig. S10. Met + 2DG treatment prevented autoantibody production in the cGVHD model.
    • Fig. S11. 2DG treatment failed to reverse immunophenotypes in TC mice.
    • Fig. S12. Met treatment failed to reverse immunophenotypes in TC mice.
    • Fig. S13. CD4+ T cell immunophenotypes in SLE patients.
    • Table S1. Human subject demographics and disease parameters.
    • Table S2. Primers for qPCR assays.
    • Table S3. Antibodies used in this study.
    • Legend for table S1

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S4. Source data (Excel file).

    [Download Table S4]

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