Anti il 17a apc

The following anti-human monoclonal antibodies were used for surface phenotype and intracellular cytokine staining: anti-CD4-fluorescein isothiocyanate (FITC); anti-CD4-phycoerythrin (PE); anti-CD161-PE; anti-CCR6-PE; anti-CD45RO-PE; anti-CD147-peridinin chlorophylla protein cyanine 5.5 dies (Percp–cy5.5); anti-interferon (IFN)-γ-FITC (all from BD Biosciences); and anti-IL-17A-allophycocyanin (APC; eBiosciences). Anti-mouse monoclonal antibodies included: anti-CD4-Percp; anti-IL-17A-APC; and anti-IFN-γ-FITC (all from BD Biosciences). Appropriately conjugated IgG antibodies were used as isotype controls. Cells were acquired on a FACSCalibur flow cytometer (BD Biosciences) and analyzed using Cell Quest software (BD Bioscience) and FlowJo 7.6.1 software (Tree Star).

Naïve CD4+ T cells from human and mouse were cocultured with human and mouse BMMSC at a ratio of 10:1, respectively. The cells were cultured in Th17 polarization conditions containing anti‐CD3/CD28 antibodies, IL‐2, 20 µg mL⁻¹ IL‐23 (PeproTech, #200‐23, R&D, #1887‐ML), 10 µg mL⁻¹ IL‐6 (PeproTech, #200‐06, R&D, #406‐ML) and 5 µg mL⁻¹ TGF‐β1 (PeproTech, #100‐21C, R&D, #7666‐MB). The Th17 polarization conditions were refreshed on the fourth day. After 6 days of culture, the Cell Stimulation Cocktail (eBioscience, #00‐4975‐03) was added to the cells at a dilution of 1:500 and incubated for 6 h. The cells were then collected and stained with live/dead violet dye (BioLegend, #423113) for 15 min. After fixation for 20 min and permeabilization for 30 min with solution A and solution B from the FIX&PERM Kit (Invitrogen, #FAS004), respectively, the cells were incubated with anti‐IL‐17A‐APC (BD, #560437, BioLegend, #506916) for 30 min. The cells were then washed and analyzed by flow cytometry.

PBMCs (2 × 10⁵ cells) were stimulated with M. avium bacilli and M. intracellulare bacilli individually for 48 h and incubated with GolgiPlug (1 μL/mL; BD Pharmingen, San Diego, CA, USA) for the final 5 h of culture. Cells were measured by flow cytometry using a fluorescence activated cell sorter (FACS) (FACSVerse, BD Biosciences, San Jose, CA, USA) and anti-CD3-APC-Cy7, anti-CD4-FITC, anti-IFN-γ-PE, anti-IL-4-PE, anti-IL-17A-APC, anti-CD25-PE, anti-Foxp3-APC, anti-T-bet-APC, anti-GATA3-APC, and anti-RORγT-APC antibodies (BD Pharmingen, San Diego, CA, USA). The PD-1, CTLA-4, and TIM-3 were also stained with anti-PD-1-PE, anti-CTLA-4-APC, and anti-TIM-3- APC (BD Biosciences, San Diego, CA, USA). Data were analyzed using BD FACSuite software (BD Biosciences, San Jose, CA, USA). A gate was set on the lymphocytes using characteristic forward scatter and side scatter parameters followed by CD3⁺CD4⁺ T cells (Supplementary Figure S1).