Research ArticleAutoimmunity

Restoring oxidant signaling suppresses proarthritogenic T cell effector functions in rheumatoid arthritis

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Science Translational Medicine  23 Mar 2016:
Vol. 8, Issue 331, pp. 331ra38
DOI: 10.1126/scitranslmed.aad7151
  • Fig. 1. Glucose shunting toward the PPP results in an accumulation of NADPH and reduced glutathione and loss of ROS.

    CD4+CD45RO T cells from patients with RA, patients with PsA, and age-matched controls (Con) were stimulated for 72 hours. (A) Expression of G6PD and PFKFB3 in 31 RA patients, 14 PsA patients, and 32 controls quantified by reverse transcription polymerase chain reaction (RT-PCR). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) G6PD immunoblots from four control and four RA samples. Relative band densities from eight RA-control pairs. (C) G6PD enzyme activities quantified in 13 RA and 13 control samples. (D) Correlation of G6PD and PFKFB3 mRNA expression in individual patients and controls. A.U., arbitrary units. (E) Correlation of the disease activity DAS28 score with the ratio of G6PD and PFKFB3 transcripts. (F) NADPH levels measured in T cell extracts of 11 RA patients, 8 PsA patients, and 14 controls. (G) Representative dot blots of monochlorobimane (mBCI) staining in control and RA Tcells. (H) Intracellular glutathione levels quantified by mBCI fluorescence. Data from seven RA patients, seven PsA patients, and nine controls. MFI, mean fluorescence intensity. (I) Representative fluorescent imaging of mBCI staining in normal and RA T cells. DAPI, 4′,6-diamidino-2-phenylindole. (J) Kinetics of intracellular ROS over 6 days after stimulation measured with the fluorogenic probe CellROX in 11 RA patients and 7 controls. (K) Intracellular ROS levels measured in T cell extracts of 15 RA patients, 8 PsA patients, and 14 controls. All data are means ± SEM.

  • Fig. 2. ROS-depleted T cells hyperproliferate and bypass the G2/M cell cycle checkpoint.

    (A) Proliferation of CD4+CD45RO Tcells with and without the G6PD inhibitor 6-AN measured by carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution 72 hours after stimulation. Representative histogram (left) and division indices from seven experiments. Max, maximum. (B) Intracellular ROS in RA Tcells cultured with and without 6-AN. Representative histogram (left) and MFI from five experiments (right). (C) T cells from four RA patients were transfected with control siRNA or two different G6PD-targeting siRNAs (si-1, si-2). NADPH levels, GSH, intracellular ROS, and division indices were measured 72 hours later. (D) MFI of intracellular IL-2 in four patients and four controls. (E) Naïve-to-memory conversion of CD4 T cells after TCR stimulation monitored by flow cytometry of CD45RA. Data from four patient-control pairs. d0, day 0. (F) CFSE-labeled CD45RO PBMCs from RA patients and controls were injected intravenously into NSG mice. Left: CFSE dilution in CD4 T cells as a measure of in vivo proliferative activity. Right: Division indices from 12 patients and 15 controls. (G) Fluorescence-activated cell sorting (FACS) analysis of NSG splenocytes to identify human CD4 and CD8 T cells converted to the CD4+CD95+ and CD8+CD95+ memory phenotype. Results from 12 experiments. (H and I) T cells were cultured with and without the ROS scavenger Tempol. Generational assignment was made by CFSE dilution. (H) Representative patient-control pairs. (I) Percentages of T cells that underwent >5 doublings from three patient-control pairs. (J) CD4+CD45RO T cells cultured with and without the ROS scavenger Tempol. Assignment to the G1, S, and G2/M phases of the cell cycle by propidium iodide staining. Percentages in each cell cycle phase for 6 patients and 12 controls. (K) Representative scatter blots of cells in the G2/M phase identified with anti–phospho-histone H3 antibody staining. Percentages of phospho-histone H3+ cells in seven patients and seven controls. All results are means ± SEM.

  • Fig. 3. Insufficient activation of the ROS-sensitive cell cycle regulator ATM results in T cell hyperproliferation.

    (A) ATM gene expression in activated CD4+CD45RO T cells measured by RT-PCR in seven controls and six patients. (B) Quantification of ATM monomers (mATMs) and dimers (dATMs) by Western blotting. Poststimulation dynamics of protein expression for a representative control and RA patient. (C) Relative band intensities for total ATM quantified at 72 hours. Results from eight patient-control pairs. (D) Kinetics of ATM phosphorylation on days 0, 1, 3, and 6 after T cell stimulation. Representative immunoblots (left) and results from four controls and four patients (right). (E) Healthy stimulated T cells were treated with H2O2 on day 3. Cell extracts were immunoblotted with anti-ATM and pATM (Ser1981). Results from one of four experiments are shown. (F) Cells were cultured with the ATM inhibitor KU-55933, and proliferation was assessed by CFSE dilution. Frequencies of proliferating T cells in five experiments. (G) Effect of the ATM inhibitor KU-55933 on naïve-to-memory conversion. KU-55933–treated T cells were phenotyped as CD45RA+CD62L+ naïve, CD45RACD62L effector memory (EM), and CD45RA+CD62L end-differentiated effector T cells (TEM) by flow cytometry. Results from six experiments. (H) Increasing cellular ROS levels restore ATM activation. T cells were treated with menadione (Me) (3 μM) for 72 hours. ROS were measured with the fluorogenic probe CellROX (left). ATM and pATM were quantified by Western blotting; one of four experiments is shown (right). All results are means ± SEM. KU, KU-55933.

  • Fig. 4. ROS scavenging mimics the maldifferentiation of RA T cells.

    (A and B) CD4+CD45RO T cells were cultured under TH1- and TH17-skewing conditions with or without the ROS scavenger Tempol, restimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin, and stained for intracellular cytokines. (A) Representative dot plots. (B) Percentages of IFN-γ–producing (left) and IL-17–producing (right) cells from four experiments. FoxP3, forkhead box P3. (C) Healthy PBMCs depleted of CD45RO+ cells were adoptively transferred into NSG mice. On day 7, splenocytes were analyzed for human CD45+CD4+IFN-γ+ cells by flow cytometry. Representative dot plots from one control-patient pair (left) and results from four independent experiments (right).

  • Fig. 5. Arthritogenic effector functions in RA T cells.

    (A) CD4+CD45RO T cells were stimulated for 6 hours. IFN-γ, IL-4, IL-17, and FoxP3 were detected by intracellular staining in six patients and six controls. All results are means ± SEM. (B) NSG mice were engrafted with human synovium, and CD45RO-depeleted PBMCs from healthy controls or RA patients were adoptively transferred into the chimeras. Synovial inflammation was assessed by RT-PCR analysis of 17 inflammation-related genes. Results from 8 to 16 tissue grafts are shown as a heat map. RANKL, receptor activator of nuclear factor κB ligand; MMP3, matrix metalloproteinase-3; TNF-α, tumor necrosis factor–α. (C) Densities of synovial T cell infiltrates were analyzed by immunostaining for human CD3. (D) T cells migrated into synovial tissue were quantified by RT-PCR for TCR transcripts, and tissue-infiltrating T cells were enumerated by anti-CD3 staining per high-power field (HPF). (E) T cell mobility was measured in Transwell migration assays. Means ± SEM from nine patient-control pairs.

  • Fig. 6. The cell cycle kinase ATM regulates the lineage commitment and the arthritogenic potential of T cells.

    (A) CD4+CD45RO T cells were cultured with the ATM inhibitor KU-55933. Cytokine production patterns after nonpolarizing conditions from six experiments. (B) Cytokine production patterns after culture under TH1- and TH2-skewing conditions with and without KU-55933. (C) T cells transfected with control or shATM (shRNA targeting ATM) plasmids were cultured under TH0-, TH1-, and TH2-polarizing conditions. Intracellular cytokine stains from a representative experiment. (D) Frequencies of cytokine-producing cells from five experiments with ATM-silenced cells. (E) NSG mice were reconstituted with CD45RO-depleted PBMCs and injected with KU-55933 (0.5 mg/kg intraperitoneally) or vehicle daily. Cytokine production in splenocytes was measured by intracellular cytokine staining in human cells. Left: Representative dot plots. Right: Percentages of IFN-γ, IL-4, IL-17, and FoxP3+ cells from four independent experiments. (F) Flow cytometric analysis of lineage-defining transcription factors in T cells cultured under TH1-, TH2-, TH17-, and Treg-skewing conditions with or without KU-55933. Means ± SEM of MFI from three experiments. (G) CD45RO+-depleted PBMCs from healthy individuals or RA patients were adoptively transferred into synovium-engrafted NSG mice. Mice were treated with the ATM inhibitor KU-55933 for 9 days. Gene expression was quantified in explanted synovial tissues by RT-PCR. Means ± SEM from eight tissues. *P < 0.05; **P < 0.01; ***P < 0.001. (H) Immunohistochemistry of synovial tissue sections. The osteoclastogenic ligand RANKL is visualized by brown staining.

  • Fig. 7. Replenishing intracellular ROS in RA T cells corrects ATM insufficiency, T cell maldifferentiation, and arthritogenic effector functions.

    CD4+CD45RO T cells from RA patients were stimulated as above. (A) On day 3, T cells were treated with menadione or menadione plus KU-55933. Cell extracts were immunoblotted with anti-ATM, pATM, Chk2 (checkpoint kinase 2), and pChk2 (phosphorylated Chk2). (B) Amounts of dATM, mATM, pdATM (phosphorylated dATM), pmATM (phosphorylated mATM), Chk2, and pChk2 were quantified in five experiments. (C) Effect of menadione and 6-AN treatment on IFN-γ production under TH1-polarizing conditions. Representative dot plots (left) and results from five experiments (right). (D) CD45RO-depleted PBMCs from RA patients were adoptively transferred into NSG mice engrafted with human synovium. To increase intracellular ROS levels, mice were treated with daily intraperitoneal injections of menadione or BSO for 9 days. T cell polarization and intensity of synovitis were analyzed as in Figs. 5 and 6. Means ± SEM from 8 to 13 synovial tissues. *P < 0.05; **P < 0.01; ***P < 0.001. (E) Immunohistochemical analysis of synovial tissues for human CD3 (pink) and RANKL (brown). Double-positive cells are marked by a white arrow head, and CD3+RANKL T cells by a black star. (F) Effects of menadione and BSO on T cell mobility measured in Transwell migration assays. Means ± SEM from nine experiments.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/331/331ra38/DC1

    Fig. S1. G6PD is regulated by T cell stimulation.

    Fig. S2. The impact of therapy on G6PD expression in RA T cells.

    Fig. S3. Intracellular GSH in resting naïve and memory T cells.

    Fig. S4. G6PD protein expression in RA T cells transfected with gene-specific siRNA.

    Fig. S5. Reconstitution of NSG mice with human T cells.

    Fig. S6. Autologous monocytes are required for the expansion of human T cells in NSG hosts.

    Fig. S7. BSO increases intracellular ROS in RA T cells.

    Table S1. Demographic and clinical characteristics of the study population.

    Table S2. List of primers.

  • Supplementary Material for:

    Restoring oxidant signaling suppresses proarthritogenic T cell effector functions in rheumatoid arthritis

    Zhen Yang, Yi Shen, Hisashi Oishi, Eric L. Matteson, Lu Tian, Jörg J. Goronzy, Cornelia M. Weyand*

    *Corresponding author. E-mail: cweyand{at}stanford.edu

    Published 23 March 2016, Sci. Transl. Med. 8, 331ra38 (2016)
    DOI: 10.1126/scitranslmed.aad7151

    This PDF file includes:

    • Fig. S1. G6PD is regulated by T cell stimulation.
    • Fig. S2. The impact of therapy on G6PD expression in RA T cells.
    • Fig. S3. Intracellular GSH in resting naïve and memory T cells.
    • Fig. S4. G6PD protein expression in RA T cells transfected with gene-specific siRNA.
    • Fig. S5. Reconstitution of NSG mice with human T cells.
    • Fig. S6. Autologous monocytes are required for the expansion of human T cells in NSG hosts.
    • Fig. S7. BSO increases intracellular ROS in RA T cells.
    • Table S1. Demographic and clinical characteristics of the study population.
    • Table S2. List of primers.

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