17a n  (ATCC)

Journal: Scientific Reports

Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

doi: 10.1038/s41598-019-52562-2

Figure Lengend Snippet: Schematic representation of the procedure used for the isolation and expansion of Th17 cells. CD4+ cells were isolated from PBMC by magnetic sorting, expanded in a culture medium without serum in the presence of polarizing cytokines and TCR-stimulation, activated, labelled for surface IL-17A and sorted by flow cytometry. The sorted cells were expanded and maintained in culture for several weeks. The indicated timeline is kept in all the figures to make it clear at which step cells were assayed. Concentrations of cytokines were 10 ng/mL (IL-2), 40 ng/mL (IL-6) and 2 ng/mL (TGF-β1). D: day; CSA: cytokine secretion assay; CSS: cytokine surface staining. α-: antibody anti-.

Article Snippet: Monoclonal antibodies to the N-terminal and C-terminal sequences of bovine IL-17A were developed (ProteoGenix SAS, Schiltigheim, France).

Techniques: Isolation, Flow Cytometry, Staining

Journal: Scientific Reports

Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

doi: 10.1038/s41598-019-52562-2

Figure Lengend Snippet: Identification of IL-17A and IFN-γ producing bovine lymphocytes. PBMC were stimulated with PMA/ionomycin, cytokine secretion blocked with Brefeldin A before flow cytometry analysis. Debris were excluded by gating according to FSC/SSC, and after gating on singlet cells, dead cells were excluded by gating on live cells. The CD4+ cells were gated by taking into account the isotype control, and the production of IL-17A and IFN-γ was measured by intracellular labelling with specific antibodies. Results shown are from a representative experiment. FSC: forward scatter: SSC: side scatter.

Article Snippet: Monoclonal antibodies to the N-terminal and C-terminal sequences of bovine IL-17A were developed (ProteoGenix SAS, Schiltigheim, France).

Techniques: Flow Cytometry

Journal: Scientific Reports

Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

doi: 10.1038/s41598-019-52562-2

Figure Lengend Snippet: Culture conditions for expansion of Th17 cells. ( a ) Number of cells after 6, 9 and 13 days of culture as a function of the concentration (µg/mL) of the coating antibodies to CD3 with or without 10 ng/ml recombinant human IL-2. Results are from a representative experiment. ( b ) Concentrations of IL-17A and IFN-γ under these culture conditions. ( c ) Proportion of cells producing IL-17A or IFN-γ (ICS) at day 6 of culture. Results are means from the cells of two cows. Percentages of IL-17A positive cells differed as a function of treatment (p = 0.034, one-way ANOVA), but not percentages of IFN-γ positive cells (p = 0.24, one-way ANOVA). Results with α-CD3 1 µg/mL are not shown because too few cells were available for staining.

Article Snippet: Monoclonal antibodies to the N-terminal and C-terminal sequences of bovine IL-17A were developed (ProteoGenix SAS, Schiltigheim, France).

Techniques: Concentration Assay, Recombinant, Staining

Journal: Scientific Reports

Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

doi: 10.1038/s41598-019-52562-2

Figure Lengend Snippet: Choice of culture medium for the expansion of Th17 cells. ( a ) Cell growth after 3, 6, 8 or 13 days in RPMI (+FCS and 2 ng/mL TGF-β1) or IMDM (+10% KnockOut Serum Replacement) with different concentrations of recombinant human TGF-β1. ( b ) Proportions of IL-17A, IFN-γ and double positive cells after 6 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( c ) Proportions of IL-17A+ cells after 6 and 13 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( d ) Comparison of cell growth in RPMI and X-VIVO™ 15 media with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1), after 3 and 6 days of culture. ( e ) Proportions of cells IL-17A+ and IFN-γ+ (ICS) with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1) after 6 days of culture. Results are means from two cows.

Article Snippet: Monoclonal antibodies to the N-terminal and C-terminal sequences of bovine IL-17A were developed (ProteoGenix SAS, Schiltigheim, France).

Techniques: Knock-Out, Recombinant, Concentration Assay

Journal: Scientific Reports

Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

doi: 10.1038/s41598-019-52562-2

Figure Lengend Snippet: Efficiency of the monoclonal antibodies in the cytokine secretion assay. ( a ) Percentages of CD4+ T cells labelled by the capture complex with either the α-Cter or α-Nter monoclonals, and comparison with the percentages of IL-17A+ cells identified by ICS. Data from two cows (C1 & C2) are shown, one per row. ( b ) Concentrations of IL-17A in culture supernatant before capture, at the end of the 3.5 h of stimulation. ( c ) Concentrations of IL-17A in culture supernatant at the end of the secretion step (after capture) in the presence of the complex capture with either one of the two monoclonal antibodies to IL-17A, an isotype control or the polyclonal antiserum to IL-17A, showing the efficiency of the capture of IL-17A as it is secreted. PI: PMA/ionomycin, PIB: PMA/ionomycin/brefeldin A. ICS: intracellular staining.

Article Snippet: Monoclonal antibodies to the N-terminal and C-terminal sequences of bovine IL-17A were developed (ProteoGenix SAS, Schiltigheim, France).

Techniques: Staining

Journal: Scientific Reports

Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

doi: 10.1038/s41598-019-52562-2

Figure Lengend Snippet: Schematic representation of the assays used for surface labelling of IL-17A secreting cells. In the cytokine secretion assay (CSA), after stimulation with PMA/ionomycin, the cells start to secrete cytokines for 3.5 h. Then the cells are allowed to bind the capture complex (Capture complex antibody staining) for 15 min before dilution and incubation for 1.5 h under agitation (New cytokine secretion step). The secreted cytokine is captured by antibodies to IL-17A that are maintained at the surface of the cell by antibodies to CD45. The capture complex comprises the two types of biotinylated antibodies linked by a streptavidin molecule. After washing, rabbit anti-bovine IL-17A antibodies are added that bind to the captured IL-17A (IL-17A detection labelling and revelation). Then the cells are washed and the binding of rabbit antibodies revealed with a secondary antibody conjugated to phycoerythrin. Alternatively, in the cytokine surface staining assay (CSS), after washing at the end of the 3.5 h stimulation, cells are incubated with rabbit antibodies to IL-17A, washed, and the binding of rabbit antibodies to surface-associated IL-17A is revealed with a secondary antibody conjugated to phycoerythrin.

Article Snippet: Monoclonal antibodies to the N-terminal and C-terminal sequences of bovine IL-17A were developed (ProteoGenix SAS, Schiltigheim, France).

Techniques: Staining, Incubation, Binding Assay

Journal: Scientific Reports

Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

doi: 10.1038/s41598-019-52562-2

Figure Lengend Snippet: Comparison of the Cytokine Secretion Assay with the cytokine surface Staining. ( a ) Gating strategy. At the end of the culture expansion step, the polarized cell populations were split and analyzed side-by-side by flow cytometry with Cytokine Secretion (left) and surface staining (right) assays for IL-17A secretion. The squares indicate the IL-17A+ cells sorting windows, the circles the IL-17A- sorting windows. ( b ) Proportions of cytokine-positive cells 8 days after sorting. Median values from two sorting experiments with cells of two cows are shown. ( c ) Numbers of IL-17A+ and IL-17A- cells at different times after sorting. Stars indicate stimulations with anti-CD3/CD28 antibodies, arrows the sampling of cells for freezing or RNA preparation. Values are from one sorting experiment with two cows. ( d ) Proportions of sorted IL-17A+ cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. Values are from one sorting experiment with two cows. e) Proportions of sorted IL-17A- cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. CSA: cytokine secretion assay; CSS: cytokine surface staining.

Article Snippet: Monoclonal antibodies to the N-terminal and C-terminal sequences of bovine IL-17A were developed (ProteoGenix SAS, Schiltigheim, France).

Techniques: Staining, Flow Cytometry, Sampling

Journal: Scientific Reports

Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

doi: 10.1038/s41598-019-52562-2

Figure Lengend Snippet: Maintenance of the IL-17A and IFN-γ phenotype of sorted and frozen-thawed cells upon subculture. ( a ) Sorted IL-17A+ cells stimulated with α-CD3/α-CD28 were cultured with IL-2 with or without the polarizing cytokines TGF-β1 and IL-6 for 9 days and analyzed by flow cytometry (ICS). Percentages of IL-17A+/IFN-γ+ double positive cells and of IL-17A-/IFN-g+ cells are shown. ( b ) Thawed IL-17A+ cells were cultured for 22 days and analyzed by ICS at days 12 and 22. Percentages of IL-17A+ cells (all: both IL-17A+/IFN-γ- and double positive cells) and IL-17A-/IFN-γ+ cells are shown. Results from two cows (C1 & C2) are shown.

Article Snippet: Monoclonal antibodies to the N-terminal and C-terminal sequences of bovine IL-17A were developed (ProteoGenix SAS, Schiltigheim, France).

Techniques: Cell Culture, Flow Cytometry

Journal: Scientific Reports

Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

doi: 10.1038/s41598-019-52562-2

Figure Lengend Snippet: Unambiguous identification of bovine Th17. ( a ) Secretion of cytokines by sorted Th17 cells. After sorting, the cells were stimulated with α-CD3 and α-CD28 and cultured for 6 days. Supernatant contents were analysed by ELISA. Median values (10 to 90 percentiles) from five cell preparations from three cows are shown. ( b ) Comparison of Th17 signature gene expression between IL-17A+ and IL-17A- cells. Heatmap shows expression intensity expressed as log2(fold-change) normalized relative to three reference genes (ACTB, PPIA and GAPDH). Expression is relative to the CD4+ subset of the corresponding cow at day 6 after positive selection using MACS® beads. Cells were cultured in X-VIVO™ 15 medium without polarizing cytokines for 6 days after an initial TCR stimulation and a partial (half) renewal of medium with addition of IL-2 on the third day. Results are from three cows (C1, C2, C3).

Article Snippet: Monoclonal antibodies to the N-terminal and C-terminal sequences of bovine IL-17A were developed (ProteoGenix SAS, Schiltigheim, France).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Selection