The isolated CD14+ population had >70% purity as determined by flow cytometry, contaminating cells being mostly CD19+. Monocytes were cultured in serum-free CellGro DC medium (CellGenix, Freiburg, Germany) for 5 days in 24-well plates at 3 × 105 cells per well with recombinant human GM-CSF and IL-4 (R&D Systems, Abingdon, UK; both at 1000 U/ml) to obtain immature DCs. Maturation of dendritic cells.  Maturation of immature DCs was induced by supplementing STI571 mw the culture media with the standard maturation cocktail consisting of TNF-α (50 ng/ml), IL-1β (25 ng/ml), IL-6 (10 ng/ml) (all from R&D Systems) and PGE2 (Sigma–Aldrich, Stenheim, Germany; 1 μg/ml).

Alternatively, DCs were matured by adding IFN-α (3000 U/ml), IFN-γ (1000 U/ml), TNF-α (50 ng/ml), IL-1β (25 ng/ml) (all from R&D Systems) and p-I:C (Sigma–Aldrich; 20 μg/ml) to obtain αDC1. DCs were cultivated for 24 h at 37 °C, and immature DCs cultivated without maturation cocktail were used MAPK inhibitor as controls. When indicated, DCs were pulsed with heat-stressed, necrotic CLL cells at a ratio of 1:1 at the same time as the maturation-inducing cytokines were added. Preparation of heat-stressed necrotic CLL cells as antigen source.  CLL cells were isolated from PBMCs using CD19+ magnetic beads (Miltenyi Biotec). The purity of isolated CLL cells (CD5+ CD19+) was >98%. These CLL cells were resuspended

at a density of 30 × 106/ml in CellGro medium and subjected to heat stress by incubation at 42 °C for 2 h. Heat-stressed cells were then incubated for a further 1 h in 56 °C

and stored in culture medium at −80 °C. When cells were thawed, trypan blue staining showed a cell viability of 0%. Necrotic CLL cells from one patient were used in all experiments. http://www.selleck.co.jp/products/lee011.html When indicated, a suspension of these heat-stressed necrotic cells was used for the pulsing of DCs. Chemokine determination and immunophenotyping.  For measurement of chemokines, 24 h culture supernatants from previously washed mature DCs were collected and stored at −80 °C until chemokine concentrations were determined by specific ELISAs. When indicated, DCs were pulsed with heat-stressed, necrotic CLL cells at a ratio of 1:1. The commercially available ELISAs for CXCL9/MIG, CXCL10/IP-10, CXCL11/I-TAC, CCL17/TARC and CCL22/MDC (R&D Systems) were performed according to the manufacturer’s instructions. The lower limits of detection were 125 pg/ml for CXCL9/MIG, 62.5 pg/ml for CXCL10/IP-10, 15.6 pg/ml for CXCL11/I-TAC, 15.6 pg/ml for CCL17/TARC and 15.6 pg/ml for CCL22/MDC. For immunophenotyping, fluorochrome-conjugated antibodies anti-CD45 FITC, anti-CCR7 FITC, anti-CD40 PE, anti-CD14 PerCP, anti-CD83 APC and anti-CD86 APC (all from BD Biosciences, San Jose, CA, USA) were added to DCs cultured for 24 h in indicated maturation conditions, incubated at 4 °C for 20 min and washed.