The DC and lymphocyte populations were gated based on their forward-scatter and side-scatter profile (large MEK162 mouse or small granular cell population, respectively). The results are expressed as percentage of positive cells and for IL-12 and IL-10 expression,

the mean fluorescence intensity was also observed. CFSE Labeling PBMCs (1 × 107) were incubated at 37°C for 15 min in 1 mL of PBS containing CFSE (Molecular Probes Europe, Leiden, The Netherlands) at 0.6 μM, a concentration which was determined in preparatory experiments as useful. After one washing step in PBS containing 1% FCS, the cells were re-suspended at a density of 1 × 106 cells/mL and used to perform the lymphoproliferation assay. After 6 days of incubation, the CFSE-labeled cells were washed once in PBS and then either

immediately fixed in PBS containing 4% formaldehyde, and subjected to analysis by a FACSArea and CellQuest software (BD, Mountain View, CA, USA). The CFSE-fluorescence was plotted against forward scatter. The retained bright check details CFSE staining consistent with no proliferative response and the lost CFSE-fluorescence indicated an induced proliferation. The reduced level of CFSE staining in the stimulated lymphocyte in relation to the unstimulated was used to calculate a proliferation index. Immunization Protocol A prime vaccine and a single boost were given fifteen days apart. For each dose of vaccine, two aliquots were prepared in separated syringes with saline solution (500 μl/dose) containing 5 × 107 cells. First, a dose was subcutaneously administered in the arm and after 1 hour the second dose was given intravenously in the other arm. After the second dose, the patient remained under observation for 1 hour for evaluation of immediate unexpected adverse events. Clinical Evaluation The follow-up included routine history and physical exam, chest x-ray and computed tomography scans at regular intervals post immunization or as directed by signs or symptoms Montelukast Sodium of tumor recurrence. Immunologic Assessment A. Phenotypic characterization of immune cells from patients’ peripheral

blood The cellular composition of the immune system, LY2874455 order before and after vaccination with the dendritic cells, was assessed from peripheral blood samples using flow cytometry. The day of immunization was considered as “”Day 0″”. The peripheral blood samples were collected one week before vaccination (“”Day -7″”), two weeks after the first dose of vaccine (“”Day 14″”), two weeks after the second dose of vaccine (“”Day 28″”) and one month (“”Day 43″”) after the end of the vaccination protocol. Surface antigens labeled with specific fluorochromes for T lymphocytes (CD4 and CD8), NK cells (CD56), B lymphocytes (CD19) and mature dendritic cells (CD86, CD80, CD83, CD40 and HLA-DR) were used for immunophenotyping of the patients’ blood cells.