Journal: Science translational medicine

Article Title: Restoring oxidant signaling suppresses pro-arthritogenic T-cell effector functions in rheumatoid arthritis

doi: 10.1126/scitranslmed.aad7151

Figure Lengend Snippet: CD4+CD45RO− T-cells from patients with RA, patients with PsA and age-matched controls were stimulated for 72 hours. (A) Expression of G6PD and PFKFB3 in 31 RA patients, 14 PsA patients and 32 controls quantified by RT-PCR. (B) G6PD immunoblots from 4 control and 4 RA samples. Relative band densities from 8 RA-control pairs. (C) Correlation of G6PD and PFKFB3 mRNA expression in individual patients and controls. (D) Correlation of the disease activity DAS28 score with the ratio of G6PD and PFKFB3 transcripts. (E) G6PD enzyme activities quantified in 13 RA and 13 control samples. (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 T-cells. (H) Intracellular glutathione levels quantified by mBCI fluorescence. Data from 7 RA patient, 7 PsA patients and 9 controls. (I) Representative fluorescent imaging of mBCI staining in normal and RA T-cells. (J) Kinetics of intracellular ROS over 6 days following 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 mean±SEM.

Article Snippet: CD4 naïve (CD4 + CD45RO − ) T-cells were purified by negative selection with anti-human CD45RO microbeads followed by positive selection with anti-human CD4 microbeads (Miltenyi Biotec, Auburn, CA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Activity Assay, Staining, Fluorescence, Imaging

Journal: Science translational medicine

Article Title: Restoring oxidant signaling suppresses pro-arthritogenic T-cell effector functions in rheumatoid arthritis

doi: 10.1126/scitranslmed.aad7151

Figure Lengend Snippet: (A) Proliferation of CD4+CD45RO− T-cells with and without the G6PD inhibitor 6-AN measured by CFSE dilution 72h post stimulation. Representative histogram (left) and division indices from 7 experiments. (B) Intracellular ROS in RA T-cells cultured with and without 6-AN. Representative histogram (left) and MFI from 5 experiments (right). (C) T-cells from 4 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 72h later. (D) MFI of intracellular IL-2 in 4 patients and 4 controls. (E) Naïve-to-memory conversion of CD4 T-cells after TCR stimulation monitored by flow cytometry of CD45RA. Data from 4 patient-control pairs. (F) CFSE-labeled CD45RO− PBMC from RA patients and controls were injected i.v. 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) 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, 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 3 patient-control pairs. (J) CD4+CD45RO− T-cells cultured with and without the ROS scavenger Tempol. Assignment to the G1, S and G2/M phase 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 Ab staining. Percentages of phospho-histone H3+ cells in 7 patients and 7 controls. All results are mean±SEM.

Article Snippet: CD4 naïve (CD4 + CD45RO − ) T-cells were purified by negative selection with anti-human CD45RO microbeads followed by positive selection with anti-human CD4 microbeads (Miltenyi Biotec, Auburn, CA).

Techniques: Cell Culture, Transfection, Control, Flow Cytometry, Labeling, Injection, In Vivo, Activity Assay, Staining

Journal: Science translational medicine

Article Title: Restoring oxidant signaling suppresses pro-arthritogenic T-cell effector functions in rheumatoid arthritis

doi: 10.1126/scitranslmed.aad7151

Figure Lengend Snippet: (A) ATM gene expression in activated CD4+CD45RO− T-cells measured by RT-PCR in 7 controls and 6 patients. (B) Quantification of ATM monomers and dimers by Western blotting. Post stimulation dynamics of protein expression for a representative control and RA patient. (C) Relative band intensities for total ATM quantified at 72 h. Results from 8 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 4 controls and 4 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 1 of 4 experiments are shown. (F) Cells were cultured with the ATM inhibitor KU-55933 and proliferation assessed by CFSE dilution. Frequencies of proliferating T-cells in 5 experiments. (G) Effect of the ATM inhibitor KU-55933 on naïve-to-memory conversion. KU-55933-treated T-cells were phenotyped as CD45RA–CD62L+ central memory (CM), CD45RA–CD62L– effector memory (EM) and CD45RA+CD62L– end-differentiated effector T-cells (TEM) by flow cytometry. Results from 6 experiments. (H) Increasing cellular ROS levels restores ATM activation. T-cells were treated with menadione (3 μM) for 72 h. ROS were measured with the fluorogenic probe CellRox (left). ATM and pATM were quantified by Western blotting; 1 of 4 experiments is shown (right). All results are mean±SEM.

Article Snippet: CD4 naïve (CD4 + CD45RO − ) T-cells were purified by negative selection with anti-human CD45RO microbeads followed by positive selection with anti-human CD4 microbeads (Miltenyi Biotec, Auburn, CA).

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Control, Phospho-proteomics, Cell Stimulation, Cell Culture, Flow Cytometry, Activation Assay

Journal: Science translational medicine

Article Title: Restoring oxidant signaling suppresses pro-arthritogenic T-cell effector functions in rheumatoid arthritis

doi: 10.1126/scitranslmed.aad7151

Figure Lengend Snippet: (A, B) CD4+ CD45RO− T-cells were cultured under Th1- and Th17-skewing conditions with or without the ROS scavenger Tempol, restimulated with PMA/ionomycin and stained for intracellular cytokines. (A) Representative dot plots. (B) Percentages of IFN-γ- (left) and IL-17-producing (right) cells from 4 experiments. (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 1 control-patient pair (left) and results from 4 independent experiments (right).

Article Snippet: CD4 naïve (CD4 + CD45RO − ) T-cells were purified by negative selection with anti-human CD45RO microbeads followed by positive selection with anti-human CD4 microbeads (Miltenyi Biotec, Auburn, CA).

Techniques: Cell Culture, Staining, Flow Cytometry, Control

Journal: Science translational medicine

Article Title: Restoring oxidant signaling suppresses pro-arthritogenic T-cell effector functions in rheumatoid arthritis

doi: 10.1126/scitranslmed.aad7151

Figure Lengend Snippet: (A) CD4+ CD45RO− T-cells were stimulated for 6 hours. IFN-γ, IL-4, IL-17 and FoxP3 were detected by intracellular staining in 6 patients and 6 controls. All results are mean±SEM. (B) NSG mice were engrafted with human synovium and CD45RO-depeleted PBMC 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 8-16 tissue grafts are shown as a heat map. (C) Densities of synovial T-cell infiltrates analyzed by immunostaining for human CD3. (D) T-cells migrated into synovial tissue were quantified by RT-PCR 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. Mean±SEM from 9 patient-control pairs.

Article Snippet: CD4 naïve (CD4 + CD45RO − ) T-cells were purified by negative selection with anti-human CD45RO microbeads followed by positive selection with anti-human CD4 microbeads (Miltenyi Biotec, Auburn, CA).

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Immunostaining, Migration, Control

Journal: Science translational medicine

Article Title: Restoring oxidant signaling suppresses pro-arthritogenic T-cell effector functions in rheumatoid arthritis

doi: 10.1126/scitranslmed.aad7151

Figure Lengend Snippet: (A) CD4+CD45RO− T-cells were cultured with the ATM inhibitor KU-55933. Cytokine production patterns after non-polarizing conditions from 6 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 plasmids were cultured under Th0, Th1 and Th2-polarizing conditions. Intracellular cytokine stains from a representative experiment. (D) Frequencies of cytokine-producing cells from 5 experiments with ATM-silenced cells. (E) NSG mice were reconstituted with CD45RO-depleted PBMC and injected with KU-55933 (0.5 mg/kg i.p.) 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 4 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. Mean±SEM of MFI from 3 experiments. (G) CD45RO+ depleted PBMC 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. Mean±SEM from 8 tissues. (H) Immunohistochemistry of synovial tissue sections. The osteoclastogenic ligand RANKL is visualized by brown staining.

Article Snippet: CD4 naïve (CD4 + CD45RO − ) T-cells were purified by negative selection with anti-human CD45RO microbeads followed by positive selection with anti-human CD4 microbeads (Miltenyi Biotec, Auburn, CA).

Techniques: Cell Culture, Transfection, Control, Injection, Staining, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry

Journal: Science translational medicine

Article Title: Restoring oxidant signaling suppresses pro-arthritogenic T-cell effector functions in rheumatoid arthritis

doi: 10.1126/scitranslmed.aad7151

Figure Lengend Snippet: 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 and pChk2. (B) Amounts of dATM, mATM, p-dATM, p-mATM, Chk2 and pChk2 were quantified in 5 experiments. (C) Effect of menadione and 6-AN treatment on IFN-γ production under Th1-polarizing conditions. Representative dot plots (left) and results from 5 experiments (right). (D) CD45RO-depleted PBMC from RA patients were adoptively transferred into NSG mice engrafted with human synovium. To increase intracellular ROS levels, mice were treated with daily i.p. injections of menadione or BSO for 9 days. T-cell polarization and intensity of synovitis were analyzed as in Figure 5 and ​and6.6. Mean±SEM from 8-13 synovial tissues. (E) Immunohistochemically analysis of synovial tissues for human CD3 (pink) and RANKL (brown). Double positive cells are marked by a white arrow head, CD3+RANKL− T-cells by a black star. (F) Effects of menadione and BSO on T-cell mobility measured in Transwell migration assays. Mean ± SEM from 9 experiments.

Article Snippet: CD4 naïve (CD4 + CD45RO − ) T-cells were purified by negative selection with anti-human CD45RO microbeads followed by positive selection with anti-human CD4 microbeads (Miltenyi Biotec, Auburn, CA).

Techniques: Migration

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Telomerase insufficiency in rheumatoid arthritis

doi: 10.1073/pnas.0811332106

Figure Lengend Snippet: Impaired induction of telomerase activity in naïve CD4 T cells in RA. (A) Naïve (CD45RO−) and memory (CD45RA−) CD4 T cells were isolated from RA patients (n = 38, black bars) and age-matched healthy controls (n = 26, gray bars) and stimulated with anti-CD3/CD28-coated beads. Telomerase activity was measured by TRAP assay on days 0, 3, and 6 following stimulation. Data are presented as means ± SEM. (B) CD34+ cells were purified from the blood of RA patients (n = 10) and age-matched healthy controls (n = 10), and telomerase activity was determined by TRAP assay. (C) hTERT transcripts were quantified by qPCR on day 3 after T cell stimulation, normalized to 18S ribosomal RNA, and correlated with enzyme activity (r = 0.87, P < 0.001). (D) Naïve (CD45RO−) CD4 T cells were isolated from 6 RA patients (dashed lines) and 6 controls (solid lines) and stimulated with anti-CD3/CD28-coated beads. Cell surface expression of CD69, CD71, and CD25 was analyzed by flow cytometry 0, 4, 12, 24, 48, and 72 h after stimulation. Frequencies of positive cells are given as means ± SEM.

Article Snippet: T cell subsets were purified from PBMC using CD45RO, CD45RA, CD4, and CD71 (Miltenyi) microbeads.

Techniques: Activity Assay, Isolation, TRAP Assay, Purification, Cell Stimulation, Expressing, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Telomerase insufficiency in rheumatoid arthritis

doi: 10.1073/pnas.0811332106

Figure Lengend Snippet: Telomerase insufficiency is independent from disease activity and treatment and present in vivo. (A) Patients described in Fig. 1 were assigned to clinical categories [active vs. inactive disease, treated vs. untreated disease, early (≤ 2 years duration) vs. established disease (>2 years)], and telomerase activity was measured in naïve CD4 T cells 3 days after TCR-mediated stimulation by TRAP assay. Activities are expressed as arbitrary units (A.U.) per 1,000 cells after normalization to an internal standard. Results are shown as means ± SEM. (B) Telomerase-expressing T cell populations in freshly harvested blood samples were identified by isolating CD3+CD4+CD71− and CD3+CD4+CD71+ subsets. Results are shown as means ± SEM per 1000 cells. (C) Blood samples were collected from RA patients (n = 11) and controls (n = 9), and naïve and memory CD4 cells were purified. Telomerase activity measurements in each T cell subset were normalized to the frequency of CD71+ cells and are given as means ± SEM per 20,000 cells.

Article Snippet: T cell subsets were purified from PBMC using CD45RO, CD45RA, CD4, and CD71 (Miltenyi) microbeads.

Techniques: Activity Assay, In Vivo, TRAP Assay, Expressing, Purification