Activation of APCs by rCysO in terms of phenotypic changes and cytokine production. a. Up-regulated expression of APC-associated molecules on DC by stimulation with rCysO protein. Monocyte-derived immature DC were stimulated with either rMMP-II or rCysO protein at 10 μg/ml, and cultured for another 2 days in the presence of rGM-CSF and rIL-4. The DC from day 6 were gated and analyzed. Dotted lines, isotype-matched control IgG; solid lines, the indicated test mAb. Representative results of three separate experiments are shown. The number at the top right-hand corner of each panel represents the difference in mean fluorescence intensity between the control IgG and the test mAb. b. Cytokine production from APCs by stimulation with rCysO protein. DC produced using rGM-CSF and rIL-4 and macrophages differentiated from monocytes by using M-CSF were stimulated with either rMMP-II or rCysO protein for 24 h. c. IFN-γ production from CD4+ T cells by stimulation with rCysO protein. Monocyte-derived DC were stimulated with either rMMP-II or rCysO protein, and were used as a stimulator of memory type or naïve CD4+ T cells in a 4-day culture. 105 responder T cells were stimulated with the protein-pulsed DC at T: DC = 10: 1. In case of the stimulation of naïve CD4+ T cells, the protein-pulsed DC were further treated with 1.0 μg/ml of rCD40 ligand for 24 h. The concentration of the indicated cytokine was determined by the ELISA method. A representative of three separate experiments conducted by using 3 different PBMCs-donors is shown. Assays were performed in triplicate and the results are expressed as the mean ± SD. Titers were statistically compared using Student’s t-test.