Phorbol myristate

Splenocytes were washed, resuspended in complete RPMI-1640 medium, and transferred to 6-well plates. Cultures were stimulated with 50 ng/ml phorbol myristate acetate (PMA; Sigma-Aldrich) and 1 μg/ml ionomycin (Sigma-Aldrich) for 5 h in the presence of Golgi-stop (BD Biosciences). After 5 h of culture, the contents of the wells were transferred and centrifuged at 300 g for 5 min. Cells were aliquoted into tubes and washed once in stain buffer (BD Bioscience). Cells were incubated with FITC-labeled anti-CD4 Ab for surface staining. After surface staining, cells were fixed and permeabilized with Perm/Fix solution (eBioscience) and stained with PE-conjugated anti-IFN-γ, IL-4, and IL-17A Abs (eBioscience). Stained cells were acquired by BD FACSCalibur flow cytometer, and the data were analyzed using FLOWJO software (BD Biosciences).

On the 4^th day post-activation with PHA ± blocking anti-CD137, cultures were stimulated with phorbol myristate (25 ng/mL) (Sigma, Sydney, Australia) and ionomycin (1 μg/mL) (Sigma). Brefeldin A (10 μg/mL) was added as a “Golgi block” (Sigma) and the tubes re-incubated in a humidified 5% CO₂/95% air atmosphere at 37°C for 16 h. Cytokine and granzyme B determination was performed as previously reported [8 ]. Five μL of appropriately diluted anti- IFNγ FITC (BD), granzyme B V450 (BD), CD137 PE (BD), CD3 perCP.CY5.5 (BD), CD28 PE.CY7 (BD), CD56 APC (Beckman Coulter, Sydney, Australia), CD8 APC.CY7 (BD), TNFα V450 (BD) and CD45 V500 (BD) monoclonal antibodies were added for 15 min in the dark at room temperature. Two mL of 0.5% bovine serum albumin (Sigma)/Isoton II (Beckman Coulter) was then added and the tubes centrifuged at 300 × g for 5 min. After decanting, cells were analyzed as above.

Phorbol myristate acetate (PMA), dimethyl sulfoxide (DMSO) and methotrexate (MTX) were obtained from Sigma (St Louis, MO). CCK-8 cell proliferation and cytotoxicity assay kits were supplied by KeyGEN (Nanjing, China). Cell stimulation and protein inhibition cocktails were purchased from eBioscience Biotech (CA, USA). APC-conjugated anti-rat CD4 and FITC-conjugated anti-rat CD25 monoclonal antibodies were obtained from BioLegend Biotech (CA, USA). PE-conjugated anti-rat CD127 and IL-17A monoclonal antibodies were purchased from R&D Biotech (MN, USA). Tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β), interleukin-6 (IL-6) and IL-17 ELISA kits were purchased from Elabscience Biotech (GA, USA).

To determine possible association between pro-inflammatory cytokines and BLTR1 expression, CD28±, CD8+, and CD8− T and NKT-like cells; whole blood; BAL; and proximal and distal brushings were treated as previously described, as leucocyte stimulation was required for both intracellular cytokine and BLTR1 expression by T and NKT-like cells. One-mL aliquots of blood (diluted 1:2 with RPMI 1640 medium), BAL, and proximal and distal brushings were treated as described above and placed in sterile 10 mL conical PVC tubes (Johns Professional Products, Sydney, Australia). Phorbol myristate (25 ng/mL; Sigma) and ionomycin (1 μg/mL; Sigma) were added. Brefeldin A (10 μg/mL) was added as a “Golgi block” (Sigma, Sydney, Australia) and the tubes re-incubated in a humidified 5% CO₂/95% air atmosphere at 37 °C for 16 h. Preliminary experiments showed that addition of brefeldin A had no effect on expression of BLTR1.
Following stimulation and processing, 5 μL of appropriately diluted IFNγ FITC, BLTR1 PE, CD3 perCP.Cy5.5, CD28 PE.CY7, CD56 APC, CD8 APC.CY7, TNFα V450, and CD45 V500 (all BD Biosciences) were added for 15 min in the dark at room temperature. Cells were washed and events acquired and analysed as described above.

Fresh PBMCs or CD4⁺ T cells were resuspended in RPMI-1640 medium supplemented with 10% foetal bovine serum and stimulated with 50 ng/ml phorbol myristate acetate (PMA; Sigma–Aldrich) and 1 µg/ml ionomycin (Sigma–Aldrich) for 2 hours. Subsequently, the cells were incubated for an additional 4 hours in the presence of 1 µg/ml brefeldin-A (eBioscience, San Diego, USA) at 37°C with 5% CO₂. The suspended cells were subsequently stained with relevant mAbs, including phycoerythrin-cyanin 5 (PE-Cy5)-conjugated anti-human CD3 mAb, fluorescein isothiocyanate (FITC)-conjugated anti-human CD8 mAb (eBioscience), PE-conjugated anti-human IL-17A mAb, and PE-conjugated anti-human IL-23R mAb (R&D Systems, Minnesota, USA). FlowJo 10 (Stanford University, San Francisco, USA) was used to analyse the data. In this study, we defined CD3⁺ CD8^- IL-17A⁺ cells as Th17 cells.

Heparinized whole blood cultured with complete RPMI medium and 10% FCS in the ratio of 1 : 1 was activated with 50 ng/mL phorbol myristate acetate (PMA; Sigma, St. Louis, USA) and 1 μg/mL ionomycin (Sigma, St. Louis, USA) for 6 hours and Golgi Plug 2 μM monensin (Sigma, St. Louis, USA) was added for the last 4 four hours of activation. Cells were surface-stained with PECy7-labeled monoclonal anti-CD4 and APC-labeled monoclonal anti-CD3 antibodies (BD Biosciences, Franklin Lakes, New Jersey, USA) followed by red blood cells (RBC) lysis using FACS lysing solution (BD Biosciences, Franklin Lakes, New Jersey, USA). After washing with phosphate buffered saline (PBS), the cells were fixed using a fixation buffer (Leucoperm, ABD Serotech, San Diego, CA, USA) and washed twice with PBS (pH7.2, 0.15 M). The cells were then permeabilized with permeabilization buffer (Leucoperm, ABD Serotech, San Diego, CA, USA) and intracellularly stained with phycoerythrin- (PE-) labeled monoclonal antibodies against IL-17A. Cells were washed and resuspended in PBS.