Blood collection and processing.

Peripheral blood specimens were collected in K2EDTA Vacutainer tubes (Becton Dickinson) and processed within 1 h of phlebotomy. PBMC extraction was by either an ammonium chloride or Lymphoprep (STEMCELL Technologies) protocol. The Lymphoprep protocol was applied according to the manufacturer’s instructions. With the ammonium chloride protocol, 4–8 ml of blood was mixed with 20 ml of cold ammonium chloride lysing buffer (0.1 M of ammonium chloride, 0.01 M of Tris-HCl) and incubated for 5 min at room temperature. Cells were then centrifuged at 300g for 5 min and washed with 5 ml of cold PBS. PBMC samples were cryopreserved in 10% dimethyl sulfoxide/90% FBS. Cryovials were placed in Nalgene Mr. Frosty containers (Thermo Fisher Scientific) for 24 h, then stored in liquid nitrogen until cellular and RNA processing for expression analysis.