S were also measured in IRX-2 mixed 1:1 with cell culture medium

S were also measured in IRX-2 mixed 1:1 with cell culture medium to determine background cytokine levels. Background cytokine levels were then subtracted from experimental values.In vitro Migration AssayDC migration was investigated as Autophagy previously described [21]. Briefly, the lower chamber of 24 trans-well plates (Corning Inc., Corning, NY) with polycarbonate membranes and 5 mm pore size was filled with 200 ml of RPMI1640 media containing 10 FBS and CCL21 (Peprotech, Rocky Hill, NJ) used at concentrations ranging from 0 to 100 ng/ml. Next, mDC (16105/100 ml medium) were seeded in the upper chamber, and plates were incubated for 2 h at 37uC. Cells in the lower chamber were counted using a Z1 Beckman Coulter particle counter.Cell LinesThe HLA-A2+ head and neck squamous cell carcinoma (HNSCC) cell line, PCI-13, generated in our laboratory as previously described [38], and the HLA-A2+ breast cancer cell line MCF-7 were cultured in plastic culture flasks (Costar, Cambridge, CA) under standard conditions (37uC, 5 CO2 in air) using RPMI1640 medium (Lonza, Walkersville, MD) supplemented with 10 (v/v) FBS (Gibco-Invitrogen, Carlbad, CA). Cell cultures were tested every 3 months for endotoxin and Mycoplasma and were found to be negative.In vitro Sensitization (IVS) of CD8+ T CellsCTLs were induced as previously described [37]. Briefly, PCI13 cell lysates of were generated by 5 cycles of rapid freezing and thawing. DCs from HLA-A2+ donors were pulsed with tumor cell lysates for the last 24 h of maturation. Based on the number of tumor cells before lysis, tumor cells were added to DC at a 3:1 ratio. TA-pulsed mDC were then irradiated (3000 rad) and washed with PBS. Autologous CD8+ T cells were isolated from cryopreserved PBMC by negative selection using magnetic bead separations (Miltenyi, Auburn, CA) and added to the mDC at the 10:1 ratio. Cells were cultured in an atmosphere of 5 CO2 in air at 37uC for 7 days in AIM-V media containing 10 ng/ml IL-7 and 10 ng/ml IL-21 (Peprotech) and 5 (v/v) FBS. On day 7, fresh, tumor cell lysate-pulsed and irradiated mDC were added and T cells were cultured for additional 7 days in AIM V containing 20 IU/ml IL-2 (Peprotech), 5 ng/ml IL-7, 10 ng/ml IL-21 and 5 (v/v) FBS. On day 1081537 14, cells were harvested and used for functional studies.Surface and Intracellular Staining for Flow CytometryCells were incubated for 20 min on ice with human Fc-Block (eBioscience, San Diego, CA) according to the manufacturer’s instructions. Without washing, cells were stained as described previously [23]. All antibodies were pre-titrated on freshly-harvested and activated PBMC to determine optimal working dilutions. Surface and intracellular staining of the various APM components was performed as previously described [10]. Samples were tested using a 4-color Beckman Coulter XL, and data were analyzed using the Expo32-Software.Phagocytosis AssayPhagocytosis of lysed tumor cells by DC was determined using flow cytometry as previously described [40]. Briefly, PCI13 cells were stained with 2 mMol carboxyfluorescein succinimidyl ester (CFSE, Invitrogen) and extensively washed. Necrosis was induced in PCI13 cells by 5 cycles of freeze/thawing. iDC were stained with 4 mM PKH26 (Sigma-Aldrich) for 5 min at RT and washed afterwards. inhibitor maturation was induced by adding IRX-2 and the conventional maturation cocktail, each diluted 1:1 with medium. Lysed, CFSE-labeled PCI-13 cells were added at the 1:3 ratio after the first 24 h of maturation. Co.S were also measured in IRX-2 mixed 1:1 with cell culture medium to determine background cytokine levels. Background cytokine levels were then subtracted from experimental values.In vitro Migration AssayDC migration was investigated as previously described [21]. Briefly, the lower chamber of 24 trans-well plates (Corning Inc., Corning, NY) with polycarbonate membranes and 5 mm pore size was filled with 200 ml of RPMI1640 media containing 10 FBS and CCL21 (Peprotech, Rocky Hill, NJ) used at concentrations ranging from 0 to 100 ng/ml. Next, mDC (16105/100 ml medium) were seeded in the upper chamber, and plates were incubated for 2 h at 37uC. Cells in the lower chamber were counted using a Z1 Beckman Coulter particle counter.Cell LinesThe HLA-A2+ head and neck squamous cell carcinoma (HNSCC) cell line, PCI-13, generated in our laboratory as previously described [38], and the HLA-A2+ breast cancer cell line MCF-7 were cultured in plastic culture flasks (Costar, Cambridge, CA) under standard conditions (37uC, 5 CO2 in air) using RPMI1640 medium (Lonza, Walkersville, MD) supplemented with 10 (v/v) FBS (Gibco-Invitrogen, Carlbad, CA). Cell cultures were tested every 3 months for endotoxin and Mycoplasma and were found to be negative.In vitro Sensitization (IVS) of CD8+ T CellsCTLs were induced as previously described [37]. Briefly, PCI13 cell lysates of were generated by 5 cycles of rapid freezing and thawing. DCs from HLA-A2+ donors were pulsed with tumor cell lysates for the last 24 h of maturation. Based on the number of tumor cells before lysis, tumor cells were added to DC at a 3:1 ratio. TA-pulsed mDC were then irradiated (3000 rad) and washed with PBS. Autologous CD8+ T cells were isolated from cryopreserved PBMC by negative selection using magnetic bead separations (Miltenyi, Auburn, CA) and added to the mDC at the 10:1 ratio. Cells were cultured in an atmosphere of 5 CO2 in air at 37uC for 7 days in AIM-V media containing 10 ng/ml IL-7 and 10 ng/ml IL-21 (Peprotech) and 5 (v/v) FBS. On day 7, fresh, tumor cell lysate-pulsed and irradiated mDC were added and T cells were cultured for additional 7 days in AIM V containing 20 IU/ml IL-2 (Peprotech), 5 ng/ml IL-7, 10 ng/ml IL-21 and 5 (v/v) FBS. On day 1081537 14, cells were harvested and used for functional studies.Surface and Intracellular Staining for Flow CytometryCells were incubated for 20 min on ice with human Fc-Block (eBioscience, San Diego, CA) according to the manufacturer’s instructions. Without washing, cells were stained as described previously [23]. All antibodies were pre-titrated on freshly-harvested and activated PBMC to determine optimal working dilutions. Surface and intracellular staining of the various APM components was performed as previously described [10]. Samples were tested using a 4-color Beckman Coulter XL, and data were analyzed using the Expo32-Software.Phagocytosis AssayPhagocytosis of lysed tumor cells by DC was determined using flow cytometry as previously described [40]. Briefly, PCI13 cells were stained with 2 mMol carboxyfluorescein succinimidyl ester (CFSE, Invitrogen) and extensively washed. Necrosis was induced in PCI13 cells by 5 cycles of freeze/thawing. iDC were stained with 4 mM PKH26 (Sigma-Aldrich) for 5 min at RT and washed afterwards. Maturation was induced by adding IRX-2 and the conventional maturation cocktail, each diluted 1:1 with medium. Lysed, CFSE-labeled PCI-13 cells were added at the 1:3 ratio after the first 24 h of maturation. Co.