Journal: Cellular immunology

Article Title: Downregulating Galectin-3 Inhibits Proinflammatory Cytokine Production by Human Monocyte-Derived Dendritic Cells

doi: 10.1016/j.cellimm.2015.01.017

Figure Lengend Snippet: Effect of Gal-3 siRNA-treated MoDCs on CD4 T cell differentiation. CD45RO T cells were prepared with by positive selection with CD45RO magnetic beads of the CD4-T cells selected by the CD4 MACS multisort kit. Gal-3 siRNA-4 treated MoDCs were stimulated with LPS (250 ng/ml) and/or R848 (5 µg/ml), Pam3CSK4 (10 µg/ml) or MDP (2 µg/ml) for 48 hr in the presence of SEB (100 ng/ml). To assess IL-5 production, siRNA or scRNA treated MoDCs were also coincubated with CD40L (10 µg/ml) along with innate immunity ligand. MoDCs (3.3×10 5 ) were then washed and added to CD45RO (1×10 6 ) at a ratio of 1:3, and cocultures were incubated continually for seven days. Supernatants were then collected and evaluated for IFN-γ ( A ), IL-17A ( B ), and IL-5 ( C ) by ELISAs. The experiments were repeated five times with the optimal conditions obtained and the representative experiment is presented. Comparator between the two selected groups are specified in the arrowed legend placed within the respective drawing (Panel A to C). Thus a siRNA treatment group will be compared to the snRNA control group (e.g. siRNA vs. snRNA) as well as compared to another treatment group with added mAbs (e.g., siRNA vs. siRNA+ mAbs) to yield pertinent information. Experiments were repeated three times from different donors. The results of the representative experiments in triplicates as means+/−SE, and the p value computed.

Article Snippet: Other reagents are Ficoll-Hypaque (Amersham/GE, Piscataway, NJ); CYBR Green PCR Master Mix (AB Applied Biosystems/Invitrogen); anti-α-tubulin (Thermo/Fisher, Waltham, MA); human AB serum (VWR, Radnor, PA), staphylococcal superantigen B (SEB, Sigma), human CD4+ T cells and CD45RO separation kits (Miltenyi Biotec, Gladbach, Germany).

Techniques: Cell Differentiation, Selection, Magnetic Beads, Incubation

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