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Methods and compositions for obtaining mature dendritic cells
| Details |
Inventors: Steinman, Ralph M.; Bhardwaj, Nina; Schuler, Gerold;
Assignee: The Rockefeller University (New York, NY)
Primary Examiner: Lankford, Jr.; Leon B.
Assistant Examiner:
Attorney, Agent or Firm: Morgan & Finnegan, LLP
We describe an improved method for generating sizable numbers of mature dendritic cells from nonproliferating progenitors in human blood. The first step or "priming" phase is a culture of T cell depleted mononuclear cells in medium supplemented with GM-CSF and IL-4 to produce immature dendritic cells. The second step or "differentiation" phase requires the exposure to dendritic cell maturation factor such as monocyte conditioned medium. Using this two-step approach, substantial yields are obtained. The dendritic cells derive from this method have all the features of mature cells. They include a stellate cell shape, nonadherence to plastic, and very strong T cell stimulatory activity. The mature dendritic cells produced according to this invention are useful for activating T cells. |
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DETAILED DESCRIPTION OF THE INVENTION This invention relates to methods and compositions useful for promoting the maturation of immature dendritic cells to a mature and stable phenotype as well as to the stable mature dendritic cells.
The mature dendritic cells are useful as antigen presenting cells (APCs) which activate other immune cells including antigen specific helper and killer T cells.
Thus, this invention also provides methods and compositions useful for the treatment of disease.
The method of producing mature dendritic cells according to this invention comprises contacting immature dendritic cells with a dendritic cell maturation factor.
We have found such a dendritic cell maturation factor in conditioned medium obtained from cultures of peripheral blood mononuclear cells (PBMCs).
Immature dendritic cells may be derived from PBMCs by culturing PBMCs with cytokines, such as, for example, a combination of GM-CSF and IL-4, which promote their differentiation to immature dendritic cells.
Surprisingly, we have determined that unless they are exposed to a dendritic cell maturation factor, removal of the cytokines from contact with the cells causes the cells to revert back to a pluripotential cell having characteristics similar to macrophages.
The response of immature dendritic cells to contact with the maturation factor is an increased expression of CD83 and p55 dendritic cell markers; strong expression of antigen presenting MHC class I and II products; and expression of several accessory (adhesion and co-stimulatory) molecules including CD40, CD54, CD58, CD80 and CD86.
A decrease in expression of CD115 as well as CD14, CD68 and CD32, which are markers associated with immature dendritic cells, is also observed as a result of carrying out the method of this invention.
In addition, this phenotype remains stable for up to three days even after removal of cytokines used to promote cell maturation.
We have developed an in vitro culture system whereby dendritic cells are induced to undergo irreversible maturation
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