In Vitro Culture Of Immature Dendritic Cells And Experimental Study On The Bioimmunological Function

Objective:To establish the culture method of immature dendritic cells (imDCs) derived from bone marrow in vitro, followed by identification of its morphology, immunological phenotype, bioimmunological function. In order to study the mechanism of imDCs inducing allogeneic T lymphocyte tolerance in the therapy of allograft rejection or autoimmune diseases, the interaction between allogeneic T cells and DCs, both immature and mature, was investigated. Methods:1. Propagation of BM-DC: Bone marrow-derived dendritic cells (BM-DC) were generated from bone marrow precursors cultured with recombinant mouse granulocyte-macrophage colony-stimulating factor ( rmGM-CSF ) and interleukin-4 (IL-4). The cells were divided into two groups (mature group and immature group) . The DCs in mature group were stimulated with LPS for the last 18h of the culture, the DCs in immature group were not. The loosely adherent cells in both groups were collected for experiment at day 6.2. Morphological Observation : The cell morphological differences between two groups were examined under optical microscope and electromicroscope.3. Flow Cytometry: The differences on cell phenotype were analyzed by flow cytomety ( FCM ) : the purified rate of DC were measured by the positive rate of CD lie, the maturation state of DCs were detected through MHC-II and costimulatory molecules (CD40, CD86, etc.) .4. The Level of IL-12 Secreted by DC: To collect the culture medium of the two groups at different time during the culture, interleukin 12 (IL-12 ) was measured with enzyme linked immunosorbent assay (ELISA) kit to evaluate the relationship between the IL-12 secrection and the maturation state of DCs.5. The Influence of DC on T cell: The two groups of DCs were cocultured with the allogeneic T lymphocyte. The proliferation of T lymphocytes in mixed leukocyte reaction (MLR) , Thl cytokines (IL-2 and IFN- Y ) and Th2 cytokines (IL-4 and IL-10) in the coculture medium, CD4+CD25+ T lymphocytes were examinated through 3H-TdR method, liquchip technique and FCM respectively.Results:1. About 1-2 X 107 dendritic cells at above 70% purity (the percentage of CD 11 c+ ) were obtained from bone marrow of two mice after 6 days of culture with the method established in our lab. The cells possessed morphological characteristic of typical DCs.2. The DCs of immature group intermediately expressed MHC-II, lowly express co-stimulatory molecules, whereas the DCs of mature group reflected highly level of cell surface MHC class II and costimulatory molecules.3. DC secrected IL-12 in a time-dependent manner, the level of IL-12 in mature group is significantly higher than that in immature group (PO.01) .4. DCs of mature group induced strong allogeneic T lymphocytes proliferation, whereas DCs of immature group were poor stimulators in MLR (PO.O1) .5. In the medium of MLR, the level of Thl cytokines (IL-2 and IFN- Y ) inimmature group is lower than that in mature group (P<0.01). On the other hand,the level of Th2 cytokines (IL-4) in immature group is higher than that inmature group (P<0.05) . 6. The percentage of CD4+CD25+ T lymphocytes were increased by stimulation ofeither group of DCs. The DCs of immature group seem to be more powerfulinducer than the DCs of mature group (P<0.01) . Conclusion:1. Large quantities of pure dendritic cells can be generated from mouse bone marrow with this established method, which is valuable to clinical application.2. Evaluation system has been established to identify the mature state of DC, which involved morphology, phenotype, the production of IL-12 and the effects of DC on T cell including the stimulatary potential, the cytokines secretion and the percentage of CD4+CD25+ T cell, etc.3. Immature DCs show the following tolerogenic properties in relationship with immune tolerance, the low surface MHC-II and costimulatory molecule expression, the decrease of secretion of IL-12, the poorly T cells stimulatory capacity, promoting naive Th cell skew toward Th2 cell, induction of T cell anergy, hyporesponse or T regulatory cell activity. The above characteristics can provide the mechanism for immature DC inducing immunotolerance.4. DCs in different mature stages had diverse effects on allogeneic T lymphocytes. Mature DC can highly stimulate allogeneic T cells to proliferate, which associate with immune response. Immature DC weakly stimulated allogeneic T cells, but induced allogeneic T cells anergy and hypo-response which is related to immune tolerance. Therefore, further research of DCs with immature properties is thought to facilitate their use for the prevention and the treatment of immunopathogenic disease.