Journal: Frontiers in Immunology

Article Title: Partial CD25 Antagonism Enables Dominance of Antigen-Inducible CD25 high FOXP3 + Regulatory T Cells As a Basis for a Regulatory T Cell-Based Adoptive Immunotherapy

doi: 10.3389/fimmu.2017.01782

Figure Lengend Snippet: A 2D2-FIG, MOG-specific FOXP3 + regulatory T cell (Treg) line exhibited in vitro and in vivo suppressive activities. (A,B) To prepare Tregs and a control line, CD45.2 2D2-FIG FOXP3 + Tregs were cultured in PC61 and IL-2 for either 13 or 40 days, and CD45.2 2D2-FIG conventional T cells (Tcons) were cultured in IL-2 for 13 days. To test the ability of Tregs to suppress naïve T cell activation, 25,000 CD45.2 2D2-FIG Tregs or 25,000 CD45.2 2D2-FIG Tcons were co-cultured with 150,000 CTV-stained CD45.1 2D2-FIG SPL responders for 5 days with 1 µM MOG35-55 and IL-2 (200 µl cRPMI). (A) CTV dye dilution and (B) percent suppression of CD45.1-gated responder T cells were analyzed by one-way ANOVA ( n = 3). (C–G) A continuous line of FOXP3 + Tregs was activated with irradiated splenic APC, 1 µM MOG35-55, IL-2, and 100 pM TGF-β for 3 days. The PC61 monoclonal antibody was not added to these cultures to avoid adoptive transfer of antibody-coated T cells. Activated Tregs were cultured one additional day in IL-2 and were analyzed for expression of CD25 (clone 3C7) (C) , surface-bound PC61 (anti-rat IgG) (D) , and CD44 and CD62L (E) . Tregs were extensively washed and injected (3 × 10 6 /mouse) into naïve recipients on day −1. Recipients were then challenged with 100 µg MOG35-55 in CFA on day 0 and were given Pertussis toxin on days 0 and 2 to elicit experimental autoimmune encephalomyelitis (EAE). Significant differences ( p < 0.05) were noted for the mean daily EAE scores on days 9–13 and 15–21 (F) and for daily weight loss on days 11–20. (H,I) EAE was elicited by administering 200 µg MOG35-55 in CFA on day 0 and Pertussis toxin on days 0 and 2. At peak disease (day 15, indicated by black arrow), activated 2D2 Tregs (3 × 10 6 /mouse) were administered i.v. by retro-orbital injection. Significant differences ( p < 0.05) were noted for mean daily EAE scores on days 18–28 (H) and for daily weight loss on days 21–28 (I) .

Article Snippet: The PC61-5.3 anti-CD25 rat IgG1(λ) hybridoma , the 7D4 anti-CD25 rat IgM(κ) hybridoma, and the 1D11.16.8 anti-mouse-TGF-β1/2/3 mouse IgG1 hybridoma ( , ) were obtained from ATCC.

Techniques: In Vitro, In Vivo, Control, Cell Culture, Activation Assay, Staining, Irradiation, Adoptive Transfer Assay, Expressing, Injection

Journal: Journal of Veterinary Science

Article Title: In vitro effects of meloxicam on the number, Foxp3 expression, production of selected cytokines, and apoptosis of bovine CD25+CD4+ and CD25-CD4+ cells

doi: 10.4142/jvs.2013.14.2.125

Figure Lengend Snippet: The effect of MEL on IL-10 (A), TGF-β (B), and IFN-γ (C) production. The results are expressed as a percentage of CD25 high CD4+, CD25 low CD4+, and CD25-CD4+ cells expressing IL-10, TGF-β, or IFN-γ. Typical cytograms (D) illustrating IFN-γ expression in particular lymphocyte subpopulations are shown. Results are presented as the mean ± SE (n = 18) of three independent experiments (each one using six animals). * p < 0.01, MEL-treated cells versus the control cells.

Article Snippet: After extracellular staining (as described above) and fixation (200 μL 2% paraformaldehyde in Dulbecco's PBS per sample for 15 min on ice; both reagents were purchased from Sigma-Aldrich, USA), the cells were permeabilized with 2 mL SAP buffer [0.1% (w/v) saponin and 0.05% (w/v) NaN 3 in Hanks' balanced salt solution (HBSS; all from Sigma-Aldrich, USA)] and stained with APC (allophycocyanin) -conjugated mouse anti-TGF-β mAb (1 : 20, 1D11, IgG1, this antibody reacts with human, mouse, and bovine TGF-β1 and TGF-β2; R&D Systems, USA) for 45 min at RT in the dark.

Techniques: Expressing, Control

Journal: Infection and Immunity

Article Title: Expression and Function of Transforming Growth Factor ? in Melioidosis

doi: 10.1128/IAI.05534-11

Figure Lengend Snippet: TGF-β levels are elevated in melioidosis patients. (A) On admission, TGF-β levels in plasma of patients with culture-proven melioidosis (n = 33) were elevated compared to healthy controls (n = 30). (B) Plasma levels of TGF-β decreased in melioidosis patients 2 weeks after successful antibiotic treatment (n = 6). *, P < 0.05.

Article Snippet: In depletion experiments, mice were injected intraperitoneally with 200 μg of a monoclonal anti-TGF-β (1D11, anti-TGF-β1, -β2, and -β3; Bioceros) or an isotype-matched control IgG antibody (anti-β-galactosidase, G113; Bioceros) 2 h before and 48 h after bacterial inoculation ( 16 , 19 ).

Techniques:

Journal: Infection and Immunity

Article Title: Expression and Function of Transforming Growth Factor ? in Melioidosis

doi: 10.1128/IAI.05534-11

Figure Lengend Snippet: Increased expression of TGF-β in lung tissue of wild-type mice infected with B. pseudomallei. Elevated pulmonary TGF-β levels were observed in mice (n = 8) 72 h after intranasal infection with 7.5 × 102 CFU of B. pseudomallei compared to 24 h after infection. ***, P < 0.001.

Article Snippet: In depletion experiments, mice were injected intraperitoneally with 200 μg of a monoclonal anti-TGF-β (1D11, anti-TGF-β1, -β2, and -β3; Bioceros) or an isotype-matched control IgG antibody (anti-β-galactosidase, G113; Bioceros) 2 h before and 48 h after bacterial inoculation ( 16 , 19 ).

Techniques: Expressing, Infection

Journal: Infection and Immunity

Article Title: Expression and Function of Transforming Growth Factor ? in Melioidosis

doi: 10.1128/IAI.05534-11

Figure Lengend Snippet: Decreased expression of phosphorylated Smad2 in mice receiving anti-TGF-β treatment. TGF-β signaling was evaluated by quantifying the total levels of Smad2 and Smad3 and their phosphorylation in lung tissue by Western blot 48 h after intranasal infection with B. pseudomallei. The left 4 lanes show the expression of Smad2 (A) and Smad3 (B) in lung tissue of control mice and the right 4 lanes of mice that received anti-TGF-β treatment (2 × 200 μg TGFβ antibody). The bar graph shows the ratio of expressed phosphorylated Smad/total Smad corrected for actin loading levels. (C) A significant decrease in p-Smad2 is demonstrated in mice that were treated with anti-TGF-β antibody. *, P < 0.05.

Article Snippet: In depletion experiments, mice were injected intraperitoneally with 200 μg of a monoclonal anti-TGF-β (1D11, anti-TGF-β1, -β2, and -β3; Bioceros) or an isotype-matched control IgG antibody (anti-β-galactosidase, G113; Bioceros) 2 h before and 48 h after bacterial inoculation ( 16 , 19 ).

Techniques: Expressing, Western Blot, Infection

Journal: Infection and Immunity

Article Title: Expression and Function of Transforming Growth Factor ? in Melioidosis

doi: 10.1128/IAI.05534-11

Figure Lengend Snippet: Cytokine profile during experimental melioidosis in mice treated with control antibody or anti-TGF-β a

Article Snippet: In depletion experiments, mice were injected intraperitoneally with 200 μg of a monoclonal anti-TGF-β (1D11, anti-TGF-β1, -β2, and -β3; Bioceros) or an isotype-matched control IgG antibody (anti-β-galactosidase, G113; Bioceros) 2 h before and 48 h after bacterial inoculation ( 16 , 19 ).

Techniques:

Journal: Infection and Immunity

Article Title: Expression and Function of Transforming Growth Factor ? in Melioidosis

doi: 10.1128/IAI.05534-11

Figure Lengend Snippet: Less organ damage in mice receiving anti-TGF-β treatment. Levels of (A) BUN, (B) creatinine, (C) AST, and (D) ALT were measured in B. pseudomallei-infected mice (n = 8/group) that were treated with anti-TGF-β antibody and their controls at 72 h as parameters for organ damage. Expression of AST and BUN were decreased, demonstrating less organ damage. *, P < 0.05. Data shown from single independent experiments.

Article Snippet: In depletion experiments, mice were injected intraperitoneally with 200 μg of a monoclonal anti-TGF-β (1D11, anti-TGF-β1, -β2, and -β3; Bioceros) or an isotype-matched control IgG antibody (anti-β-galactosidase, G113; Bioceros) 2 h before and 48 h after bacterial inoculation ( 16 , 19 ).

Techniques: Infection, Expressing

Journal: Infection and Immunity

Article Title: Expression and Function of Transforming Growth Factor ? in Melioidosis

doi: 10.1128/IAI.05534-11

Figure Lengend Snippet: Anti-TGF-β treatment results in diminished bacterial growth during experimental melioidosis. Mice (n = 8/group) were treated with anti-TGF-β antibody or control IgG antibody and inoculated with 7.5 × 102 CFU of B. pseudomallei intranasally. Bacterial numbers were quantified 72 h postinfection. Less growth was seen in the lungs (A) and spleens (B) of mice treated with anti-TGF-β antibody. *, P < 0.05. Data shown from single independent experiments.

Article Snippet: In depletion experiments, mice were injected intraperitoneally with 200 μg of a monoclonal anti-TGF-β (1D11, anti-TGF-β1, -β2, and -β3; Bioceros) or an isotype-matched control IgG antibody (anti-β-galactosidase, G113; Bioceros) 2 h before and 48 h after bacterial inoculation ( 16 , 19 ).

Techniques:

Journal: Infection and Immunity

Article Title: Expression and Function of Transforming Growth Factor ? in Melioidosis

doi: 10.1128/IAI.05534-11

Figure Lengend Snippet: No influence on survival of anti-TGF-β treatment during murine melioidosis. Mice (n = 12/group) received either 2× 200 μg control antibody (black squares) or 2× 200 μg anti-TGF-β antibody (white squares) and were infected with 7.5 × 102 CFU, a lethal dose, of B. pseudomallei intranasally, after which they were followed until death. Mortality was assessed every 4 h. No difference in survival was seen.

Article Snippet: In depletion experiments, mice were injected intraperitoneally with 200 μg of a monoclonal anti-TGF-β (1D11, anti-TGF-β1, -β2, and -β3; Bioceros) or an isotype-matched control IgG antibody (anti-β-galactosidase, G113; Bioceros) 2 h before and 48 h after bacterial inoculation ( 16 , 19 ).

Techniques: Infection

Journal: bioRxiv

Article Title: Identification of Chlorophyll a-b Binding Protein AB96 as a novel TGFβ1 binding agent

doi: 10.1101/2020.08.20.258590

Figure Lengend Snippet: The effects of VA extract on endogenous (a) and exogenous (b) TGFβ1 levels in the supernatant of monocyte derived dendritic cells. n=1 for A and n=3 for B. (c) Unbound and bound active TGFβ1 concentrations (starting: 2ng/ml) after incubation with VA extract for 2h at RT. Representative of 3 independent assays. Technical triplicates. Mean +/− SEM shown. (d) The unbound and bound IL-10, IL12, latent TGFβ1 and active TGFβ1 concentrations (starting: 4ng/ml) after incubation with VA extract for 2h at room temperature (RT). Technical triplicates. Mean +/− SEM shown. (e) The unbound and bound active TGFβ1 concentrations (starting: 2ng/ml) after incubation with sesquiterpene lactones for 2h at RT. Technical triplicates. Mean +/− SEM shown. (f) The levels of active TGFβ1 bound to aqueous extracts (1mg/ml) from different species of plants (normalized to VA extract) when incubated with 2ng/ml active TGFβ1. Species in order of x axis: 1. Vernonia amygdalina Del. (Compositae). 2. Vernonia arkansana DC. (Compositae). 3. Helianthus annuus L. (Compositae). 4. Aster dumosus Hoffm. (Compositae). 5. Campanula lactiflora M.Bieb. (Campanulaceae). 6. Petroselinum crispum (Mill.) Fuss (Apiaceae). 7. Ocimum basilicum L. (Lamiaceae). 8. Solanum lycopersicum L. (Solanaceae). 9. Spinacia oleracea L. (Amaranthaceae). 10. Rosa sericea Wall. Ex Lindl. (Rosaceae) 11. Vitis vinifera L. (Vitaceae). 12. Dieffenbachia amoena Bull. (unresolved, Araceae) 13. Dracaena marginata Hort. (Asparagaceae). 14. Lilium candidum L. (Liliaceae). Technical triplicates. Mean +/− SEM shown. (g) The effect of VA extract on luciferase activity in a functional active TGFβ1 reporter cell line. Biological triplicates. Mean +/− SEM shown. Representative of 2 independent assays. (h) The levels of TGFβ1 in different fractions eluted from an anion exchange column after loading with indicated factors, using indicated % of 2M NaCl. Unpaired student’s t-test: * p<0.05 **** p<0.0001.

Article Snippet: Leaf extract (1mg/ml in PBS), TGFβ1 antibody (5μg/ml in PBS; clone 1D11 BioTechne), parthenium (1mg/ml in chloroform; Sigma), artemisinin (1mg/ml in chloroform; Sigma), full length Pisum sativum L. (Leguminosae) chlorophyll a-b binding protein AB96 (at indicated concentrations; mybiosource TTKKVASSSSPWHGPDGVKYLGPFSGESPSYLTGEFPGDYGWDTAGLSADPETFAKNRELEVIHSRWAMLGALG CVFPELLSRNGVKFGEAVWFKAGSQIFSEGGLDYLGNPSLVHAQSILAIWATQVILM GAVEGYRIAGGPLGEVVDPLYPGGSFDPLGLAEVPEAFAELKVKELKNGRLAMFSM FGFFVPAIVTGKGPLENLADHLADPVNNNAWSYATNFVPGK) or vehicle (PBS or chloroform) were plated on a high binding 96 well flat-bottomed plate (Greiner) and left at RT overnight.

Techniques: Derivative Assay, Incubation, Luciferase, Activity Assay, Functional Assay

Journal: bioRxiv

Article Title: Identification of Chlorophyll a-b Binding Protein AB96 as a novel TGFβ1 binding agent

doi: 10.1101/2020.08.20.258590

Figure Lengend Snippet: (a) Mass spectrometry data for pulled out proteins when using indicated bait proteins when a threshold of min number of peptides =2, protein threshold =20% and peptide threshold =95%. (b) The chlorophyll a-b binding protein AB96 peptides observed by mass spectrometry are highlighted in yellow. Green represents amino acid modifications: M, oxidation and C, carbamidomethyl. Pink represents Magnesium binding sites. (c) Quantitation of chlorophyll a-b binding protein AB96 pulled from VA extract using indicated bait protein. (d) Concentration of active TGFβ1 eluted from different concentrations of chlorophyll a-b binding protein AB96 after incubation for 2h at RT.

Article Snippet: Leaf extract (1mg/ml in PBS), TGFβ1 antibody (5μg/ml in PBS; clone 1D11 BioTechne), parthenium (1mg/ml in chloroform; Sigma), artemisinin (1mg/ml in chloroform; Sigma), full length Pisum sativum L. (Leguminosae) chlorophyll a-b binding protein AB96 (at indicated concentrations; mybiosource TTKKVASSSSPWHGPDGVKYLGPFSGESPSYLTGEFPGDYGWDTAGLSADPETFAKNRELEVIHSRWAMLGALG CVFPELLSRNGVKFGEAVWFKAGSQIFSEGGLDYLGNPSLVHAQSILAIWATQVILM GAVEGYRIAGGPLGEVVDPLYPGGSFDPLGLAEVPEAFAELKVKELKNGRLAMFSM FGFFVPAIVTGKGPLENLADHLADPVNNNAWSYATNFVPGK) or vehicle (PBS or chloroform) were plated on a high binding 96 well flat-bottomed plate (Greiner) and left at RT overnight.

Techniques: Mass Spectrometry, Binding Assay, Quantitation Assay, Concentration Assay, Incubation