Phytohemagglutinin (pha)

To evaluate the proliferative responses of lymphocytes to C-Vx, carboxyfluorescein succinimidyl ester (CFSE) dilution method was used which is based on labelling cells with CFSE and evaluating the fluorescence halved by each cell division. PBMCs (up to 2 × 10⁷), freshly isolated and suspended in RPMI-1640 medium (Gibco, Paisley, UK), were stained with 1 μl of 5 mM CFSE solution (Thermo Fisher Scientific, USA) and incubated for 6 min at 4°C. Following washing with PBS, PBMCs were cultured for 120 h at 37°C with or without C-Vx (Miracle Labs PHArmaceutical Industry-Turkey) together with the absence or presence of 5 µl/mL PHA (Thermo Fisher, USA). Following cell culture, supernatants were collected and stored at −20°C for further analysis. PBMCs were harvested from the respective wells (US: unstimulated, PHA: phytohemagglutinin-stimulated, C-Vx, and PHA + C-Vx) into separate tubes for cell surface staining with anti-human-CD3-BV785, -CD4-PE-Cy7, -CD8-APC/Cy7, -CD16-BV570, and -CD56-BV711 (all from Biolegend, USA) mAbs. After the incubation, stained cells were washed with PBS and analyzed by flow cytometry.

To determine the effects of blocking co-stimulation through CD137 on T cells, NKT-like and NK cells, PBMC from COPD patients and control subjects were stimulated with 1 μg/mL PHA (Sigma, Sydney, Australia). For co-stimulation blockade, PBMC were activated with 1 μg/mL PHA alone or in the presence of 10 μg/mL purified blocking antibodies for CD137 (Biolegend, clone 4B4-1) and isotype-matched control antibodies (eBioscience, Sydney, Australia). Stimulation of T cells, NKT-like and NK cells through CD137 was performed with 10 μg/mL stimulatory antibodies for CD137 (R&D Systems, AF838).