Golgi block

Production of IFNγ and TNFα by T, NKT-like and NK cells was determined on blood and BAL samples from all subjects as previously reported [11 (link)–13 (link)].
Briefly, one mL blood diluted 1:2 in RPMI and prepared BAL cells 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. Aliquots of blood and BAL were added to FACS tubes (BD) and treated with FACSLyse and FACSPerm as above and five μL of appropriately diluted anti- IFNγ FITC (BD), CD3 perCP.CY5.5 (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) / Isoflow (Beckman Coulter) was then added and the tubes centrifuged at 300 ×g for 5 min. After decanting, cells were analyzed as above.

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.

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.