Cd4 fitc

Monensin (2 µM, Sony Biotechnology, USA) and anti-CD107a APC-Cy7 (Sony Biotechnology, USA) were added to the cells for the last 5 h of incubation. Cells were then harvested and stained for CD3 (Pacific Blue), CD4 (FITC), and CD8 (PE/Cy5) (all Sony Biotechnology, USA) and analyzed by flow cytometry. Since HSP90 inhibition may affect allogeneic mo-DCs, a similar experiment was performed using CD3/CD28 stimulation.

Human peripheral blood mononuclear cells (PBMCs) from healthy controls were isolated from whole blood by Ficoll-Hypaque density centrifugation (Amersham-Pharmacia-Biotech, Sweden). The cells were counted and plated at 2 × 10⁶ cells/well in 96-well V-bottom plates (Thermo-Fisher-Scientific), in 100 µL RPMI (GibcoBRL, Invitrogen), supplemented with 10% fetal bovine serum (FBS) (GibcoBRL, Invitrogen) or 100 µL RPMI supplemented with 10% plasma from patients or controls. PBMCs were left unstimulated or were stimulated with 5 to 80 ng/mL rhGM-CSF or 100 ng/mL rhIL-3 (Miltenyi-Biotec) for 30 min at 37°C, and the cells were then fixed permeabilized with a fixation/permeabilization kit (eBioscience). Extracellular labeling was performed with CD14-Pacific Blue and CD4-FITC (Sony-Biotechnology, clones M5E2 and RPA-T4, respectively). Cell viability was determined with the Aqua Dead Cell Stain Kit (Thermo-Fisher-Scientific), and STAT5 phosphorylation (p-STAT5 levels) was assessed by intracellular staining with Phospho-Flow PE Mouse Anti-p-STAT5 (pY694) antibody (BD-Biosciences). Data were collected with a Gallios flow cytometer (Beckman-Coulter) and analyzed with FlowJo software v.10.6.2 (Becton–Dickinson).