Expression of CD14, HLA-DR, CD83, and DC-LAMP by DCs on maturation with CD154 trimer or TNF-α of HIGM3 patients. (A) FACS analysis of CD14, HLA-DR, and CD83 expression by DCs on maturation with CD154 trimer or TNF-α from a healthy subject and from HIGM3 patients. Monocyte-derived DCs were cultured and matured with coated CD154 trimer or TNF-α, as described in “Patients, materials, and methods,” and were incubated with the appropriate antibody, washed twice, and analyzed using a flow cytometer. Expression of CD14 and HLA-DR was analyzed by CD14-FITC/HLA-DR-PE double staining. Extent of CD83 expression was analyzed by CD83-PE single staining. HLA-DR, CD14, and CD83 antibody stainings are presented (thick line) in comparison with mouse IgG antibody (thin line). The x-axis represents the intensity of green or red fluorescence expressed in a log scale as mean channel, and the y-axis represents the number of cells per channel. Data shown are representative of 3 independent experiments. (B) HIGM3 patient DCs fail to express DC-LAMP on CD154 stimulation. Monocyte-derived immature and CD154 trimer or TNF-α matured DCs were cultured as described in “Patients, materials, and methods.” After cytospin preparation and immunocytochemical staining with DC-LAMP monoclonal antibody, cells were photographed using an optic microscope. Original magnification, × 200. Data are from 1 of 2 representative experiments.