Histopaque

Patients with lung cancer and healthy donors were bled (20–60 mL) at Nottingham City Hospital. Following the separation using Histopaque, Peripheral blood mononuclear cells (PBMCs) were depleted of CD25 +T cells and/ or CD45RO+T cells using CD25 depletion kit (Miltenyi) and CD45RO depletion kit (Miltenyi), respectively. CD14 + cells were positively isolated (Miltenyi) from PBMCs and then reintroduced to the PBMCs after CD45RO depletion as CD14 +cells express CD45RO. PBMCs were carboxyfluorescein succinimidyl ester (CFSE) loaded and plated out as described previously.16 (link) PBMCs were stimulated with NPM266-285cit peptide at 10 µg/mL. PHA and medium were used as positive and negative controls, respectively. Proliferation responses which were double background control were considered to be significant. Buffy coats were purchased from NBT NHS Sheffield and PBMCs were isolated as described earlier. Following depletion of CD25 +T cells, PBMCs were stimulated with NPM266-285cit peptide at 10 µg/mL for 11 days. PBMCs were then restimulated with 10 µg/mL of NPM266-285cit or NPM266-285wt peptides to assess citrulline-specific response. The responses were measured using ELISpot assays with human IFNγ capture and detection reagents according to the manufacturer’s instructions (Mabtech, Sweden).