Interleukin 4 (il 4)

Human monocytes from nine human leukocyte antigen (HLA) -A2, and two non-HLA-A2 healthy donors were obtained from the NIH Blood Bank. The monocytes were cryopreserved in liquid N2 until use. To generate dendritic cells, the cryopreserved monocytes were thawed, put in tissue culture plate for 4 hrs at 37°C. The non-adherent cells were removed by washing with phosphate buffered saline (PBS). The adherent cells were cultured in complete RPMI 1640 medium (Mediatech, Inc. Manassas, VA) supplemented with human GM-CSF (1000 U/ml, BD Biosciences, San Jose, CA) for two days to generate pre-immature dendritic cells (piDC) or with human GM-CSF (1000 U/ml) and IL-4 (50 ng/ml, BD Biosciences) for 4 days to generate immature dendritic cells (iDC), or with human GM-CSF (1000 U/ml) and IL-4 (50 ng/ml) for 4 days and additional 2 days with TNFα (20 ng/ml, BD Biosciences) and CD40 ligand (200 ng/ml, InvivoGen, San Diego, CA) to generate mature dendritic cells (mDC) (Table 1). Two donors (D1 and D2) were also matured by culturing 2 days with a cytokine cocktail, which contained IL-1β (10 ng/ml, BD Biosciences), IL6 (10 ng/ml, InvivoGen), TNFα (10 ng/ml) and PGE₂ (10⁻⁷ M, Sigma-Aldrich, St. Louis, MO).