Anti il 17a

A flow cytometry analysis was performed on the lung cells. To analyze the Th17/Th22 subsets, 1 × 10⁶ cells/mL were incubated with TruStain FcX anti-mouse CD16/32 (San Diego, CA) for 10 min at room temperature, and the cells were subsequently incubated with the fluorescently labeled monoclonal antibodies (MAbs) anti-CD45 (APC/Cy7), anti-CD3 (BV7856, PE, or FITC), anti-CD4 (APC/Cy7, PE, or BV786), anti-LIN (AF700), and NKP46 (BV510), purchased from Biolegend, for 30 min on ice. BD Cytofix/Cytoperm (BD Biosciences, Franklin Lakes, NJ) was used to fix and permeabilize the cells for intracellular staining using anti-IL-22 (AF647), anti-IL-17a (BV510 or FITC), and RORγt (PE, BV650, or APC). To determine the cell percentages of the Th17/Th22 cells, BD FACS Celesta and FlowJo v10 software were used. The percentages of Th17/Th22 were multiplied by the total number of cells isolated from the lungs to determine the absolute cell counts of the populations.

The following antibodies were used: anti-IFN-γ, anti-IL17A, anti-CD3, anti-CD27, anti-CD45RA and isotype-matched control mAbs, labelled with different fluorochromes, all purchased from BD Bioscience, and used according to the manufacturer's recommendations. Vγ9Vδ2 T cell proliferation was assessed after 6 days of co-culture according to loss of CFSE labelling in PI⁻ cells. To study intracellular IFN-γ and IL-17, Vγ9Vδ2 T cells were co-cultured with activated pDCs, Ionomycin and PMA or with BrHPP in the presence of monensin for the last 5 hrs at 37°C in 5% CO₂. The cells were harvested, washed twice in PBS with 1% FCS and fixed with PBS containing 4% paraformaldehyde overnight at 4°C. Fixation was followed by permeabilization with PBS containing 1% FCS, 0.3% saponin, and 0.1% Na azide for 15 min at 4°C. Staining of intracellular cytokines were performed by incubation of fixed permeabilized cells with FITC-labelled anti-IFN-γ and APC-labelled anti-IL17A mAbs. After two more washes in PBS containing 1% FCS, the cells were analyzed by FACS CANTO II flow cytometer (BD Bioscience). Viable lymphocytes were gated by forward and side scatter, and analysis was performed on 100,000 acquired events for each sample by using FlowJo and the following gating strategy to detect lymphocytes from FSC/SSC, live cells, single cells, double positive CD3, and TCR Vγ9Vδ2 cells.

Primary mouse splenic CD4 T cell culture supernatants were collected at designated time points and analyzed for cytokine secretion. 96-well Maxisorp plates were coated overnight at 4°C with the appropriate capture antibody (anti-mouse IFNγ, anti-mouse IL-2, anti-IL-4, anti-IL-17A, and anti-IL-10; BD Biosciences). Non-specific protein binding was prevented by blocking wells with 5% BSA in PBS for 3 h at room temperature. Culture supernatants and standards (recombinant IFNγ and IL-2, carrier-free) were diluted appropriately and added to wells. The plate was incubated overnight at 4°C, with continuous rocking. Biotinylated detection antibodies were added to wells followed by TMB substrate reagents (BD Biosciences) at a 1:1 ratio. Color development was monitored, and the reaction was terminated by the addition of stop solution (2N H₂SO₄). Absorbance was read at 450 nm using a microplate reader. Cytokine concentrations were determined relative to the standard curves generated.

Cells from the cervical lymph nodes were incubated in culture medium for 4 h in the presence of monensin and phorbol 12-myristate 13-acetate (Sapphire Bioscience Pty. Ltd. Redfern NSW, Australia)/ionomycin (Wako). Cells were first stained with IL-18 receptor (IL-18R)-FITC (Miltenyi Biotec, Gaithersburg, MD, USA), CD8s-PerCp-Cy5.5, gamma delta TCR-APC, and BioLegend Brilliant Violet 421 anti-mouse CD4 antibody (BioLegend, San Diego, CA, USA). Dead cells were eliminated using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific). Cells were fixed with 4% (w/v) paraformaldehyde (nacalai tesque), permeabilized with 0.1% saponin buffer (phosphate-buffered saline with 0.1% saponin, 1 mM HEPES; Thermo Fisher Scientific, and 0.1% bovine serum albumin; Sigma-Aldrich, St. Louis, MO, USA), and then stained with anti-IL-17A or a control rat IgG1 antibody (BD Biosciences), as previously described [10 (link)]. The cells were examined using a FACS Canto II (BD Biosciences), and the data were analyzed using FlowJo software (v10.5) (Tree Star, Ashland, OR, USA).

As a longer incubation period was needed to study the influence of IFN-α on the IL-17 production, we first compared the result of 20-h and 6-h incubation with phorbol myristate acetate (PMA) and ionomycin in five samples. Our result showed that there was no significant difference concerning both IL-17 expression and the frequency of live PBMCs between 20-h and 6-h incubation. Therefore, a 20-h incubation was used in the following experiments. Briefly, PBMCs from patients and controls were cultured with or without rhIFN-α 2a (PBL Biomedical Lab, Piscataway, NJ, USA), at a concentration of 2 × 10⁶ cells/ml in combination with 20 ng/ml PMA and 1 μg/ml ionomycin (Sigma, Saint Louis, MO, USA), for 20 h and then blocked with 10 μg/ml brefeldin A (Sigma, St Louis, MO, USA) for 4 h. The cultured cells were stained with anti-CD3, anti-CD4 antibodies (BD Company, USA), washed, fixed, permeabilized, and subsequently stained with anti-IL-17A (BD Company, USA) or appropriate isotypes (eBioscience, San Diego, CA). FACS analysis was performed using FACS Canton II and FACS Diva software (BD Company, USA) to determine the in vitro effect of IFN-α on the expression of IL-17.