| It is well known dendritic cells are the most important antigen presenting cells(APCs).They are highly efficient for antigen uptake and processing and for the initiation and regulation of innate and adapati ve immune responses.In recent years ,it has become increasingly clear that DCs are a heterogenous population composed of several subsets that share common features but that also exhibit distinct biologic properties. It is generally accepted that dendeitic cells can be divided into two subsets according to their generators, DC1s (myeloid dendritic cells, MDCs) , who are originate from myeloid derived progenitors, and DC2s (lymphoid dendritic cells, LDCs), who are derived from lymphoid progenitors. Ample information has been collected on the development nature function and culture characteristics in vitro of DC1s. In contrast, our current understanding on the origin function and culture characteristics in vitro of DC2s is still limited, but is increasing rapidly.Molecule CD40 is highly expressed on DCs. We have demonstrated that mouse anti-human CD40 monoclonal antibody(McAb) can induce the differentiation and maturation of DC1 and it is an effective stimultor in up-regulating the expression of CD80 and CD86 on DC1 .Whether anti-CD40McAb can also induce the differentiation and maturation of DC2 is unclear. Our goal is to develop a method of inducing DC2 from human cord blood CD34+ hemopoietic cell with cytokines, and to investigate the effect of mouse anti-human CD40 McAb on activation and maturation of DC2 in vitro for future research in biologic properties and clinical trials of DC2.In this study, CD34+ hemopoietic cells were isolated from human cord blood with magnetic microbeads, and cultured with rhIL-3 (10ng/ml). rhFlt-3L(FL, lOOng/ml) and rhGM-CSF(100ng/ml).On the 13th day, rhIL-3 (lOng/ml) and anti-human CD40 McAb (5ug/ml) or rhIL-3 (lOng/ml) and TNFa (lOng/ml) were added to induce DC2 maturation respectively and two days later the surface membrane DC2 related antigens, such as CD 123, HLA-DR, CD83, CD86, CD80, were analyzed by FCM. Our results found that the percentage of lineage negative cells was 81.78% and84.73% in two groups respectively, The expression rate of surface antigens, such as CD 123. HLA-DR. CD83> CD86> CD80, was 24.54%> 88.74%. 37.40%. 32.79% and 99.13% respectively after induced by rhIL-3 (lOng/ml) and anti-human CD40; Otherwise the expression rate of surface antigens was 21.01%. 78.85%. 32.23%. 29.53% and 98.89% respectively after induced by rhIL-3 (lOng/ml) and TNFa (lOng/ml) .It is clear that we have effectively induced rich DC2s from cord blood CD34+ hemopoietic cells and the quantity of DC2 induced by anti-human CD40 is slightly more than that of TNF-a. In the meanwhile, the supematants of the two experimental group and control group culture systemswere collected. IL-12, IFN-y, IL-4 and IL-10 were measured by ELISA respectively. There was no significant difference between the two experimental groups (P>0.05).DCs induced and maturated by FL+GM-CSF+IL-3+TNF-a and FL+GM-CSF+IL-3+anti-human CD40 were cultured with allogeneic cord blood lymphocyte from another healthy person, respectively. IL-1CK IFN-y^ IL-4 and IL-2 levels in supernatants were measured respectively, our results indicated that the induced DCs can induce allogeneic T lymphocytes to secret high level of IL-l(h IFN-y^ IL-4 and IL-2, demonstrating that the induced DCs have the function of DC2s.In conclusion, CD123+DC2 can be successfully induced from CD34+ hemopoietic cells with rhIL-3 ( lOng/ml ) > rhFL(100ng/ml) and rhGM-CSF(100ng/ml) mixture in vitro. The agonist anti-human CD40 monoclonal antibody can differentiate and maturate DC2s. DCs induced by the methods can induce allogeneic T lymphocytes to secret high level of IL-10 indicating that the induced DCs have the function of DC2s.
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