Cd14 and cd15 positive selection kits cd14 cd15

gMDSC were enriched or depleted using sequential magentic bead isolation: CD14 and CD15 positive selection kits (CD14⁻CD15⁺) (Miltenyi Biotec) as per the manufacturer’s instructions. In selected cases gMDSC were highly purified by flow cytometry on the basis of CD11b, CD14 and CD15 expression on a FACSAria (BD Bioscience). Where indicated PBMC or PBMC enriched/depleted of gMDSC were stimulated with either: 0.5 μg/ml of an HLA-A and HLA-B-restricted peptide pool spanning the immune-dominant proteins of CMV, EBV and influenza (CEF) (JPT Peptide Technologies), 1 μg/ml overlapping peptides (pool of 15-mer peptides overlapping by 10 residues) spanning the core of HBV genotype D (JPT Peptide Technologies), 1 μg/ml HLA-A2-restricted NLVPMVATV peptide from CMV pp65 (ProImmune) or 0.5 μg/ml plate-bound anti-CD3 and 0.5 μg/ml anti-CD28 (eBioscience). All cultures were carried out in the presence of 20 IU/ml recombinant human IL-2 (Miltenyi Biotec) in cRPMI (Life Technologies) for 5-7 days at 37 °C. Specific responses were detected by intracellular cytokine staining after re-stimulation on day 4/6 in the presence of 1 μg/ml brefeldin-A (BFA, Sigma-Aldrich). Where indicated, 0.5 mM N-hydroxyl-nor-L-arginine (nor-NOHA) (Calbiochem), an arginase I specific inhibitor, was added to the culture on day 0.