Induction,identification and activity of human plasmacytoid dendritic cells

Objective To establish the method of inducing dendritic cells from monocytes from human peripheral blood, and evaluate the activity of induced dendritic cells. Methods Mononuclear cells were isolated from normal human peripheral blood, and cultured in medium with 100 ng/ml hGM-CSF. 30 ng/ml hIL-4 was added on the 4th day. During the culture, half of the media was replaced every two days. 1000 μ/ml INF was added on the 5th and 6th days respectively. The dendritic cells were harvested on the 7th day. The morphological characteristics were observed under inverted microscope everyday. The CD1a and CD83 of cell surface antigen of the dendritic cells were detected by flow cytometry (FCM) and immunofluorescence. T-lymphocytes proliferating activity stimulated by the dendritic cells were evaluated by MTT, in which dendritic cells were cultured with auto-mixed T-lymphocyte in the rate of 1:1,1:2 and 1:5 respectively. Results After 7 days in induction culture, dendritic cells with high purity were generated. The quintessential characteristics of dendritic cells, including large cell size and rapidly increasing capacity, irregular shape and dendrites on the cells surface, were observed under light-microscope. Immunofluorescence staining revealed high expression of CD1a and CD83, and FCM showed the percentage of CD1a+ cell was 80.80﹪, CD83+ cell was 19.50﹪ and CD1a+ /CD83+ cell was 16.9﹪. Mixed lymphocyte reaction revealed dendritic cells were potent stimulator of auto-T lymphocytes. Conclusion By induction with a cocktail of cytokines, large amount of mature dendritic cells can be induced from human palsmacytoid mononuclear cells in vitro.