This invention provides methods to prepare and use immunostimulatory cells for enhancing an immune response. The invention provides a method for preparing mature dendritic cells (DCs), comprising the sequential steps of: (a) signaling isolated immature dendritic cells (iDCs) with a first signal comprising an interferon gamma receptor (IFN-³R) agonist and/or a tumor necrosis factor alpha receptor (TNF-±R) agonist to produce signaled dendritic cells and (b) signaling said signaled dendritic cells with a second transient signal comprising an effective amount of a CD40 agonist to produce CCR7+ mature dendritic cells. Also provided by this invention are enriched populations of dendritic cells prepared by the methods of the invention. Such dendritic cells have enhanced immunostimulatory properties and increased IL-12 secretion and/or decreased IL-10 secretion. CD40 signaling can be initiated by one or more of polypeptide translated from an exogenous polynucleotide encoding CD40L (e.g. mRNA or DNA), an agonistic antibody to CD40 receptor or by CD40 ligand polypeptide. The enriched populations can be further modified by the administration of an immunogen to the DC. The DC will take up and process the immunogen on its cell surface.本發明提供製備及使用免疫刺激細胞來增強免疫反應之方法。本發明提供一種用於製備成熟樹突細胞(dendritic cells;DCs)之方法,其包括以下依序步驟:(a)用一包含干擾素γ受體(interferon gamma receptor;IFN-γR)激動劑或腫瘤壞死因子α受體(tumor necrosis factor alpha receptor;TNF-αR)激動劑之第一信號向經分離之不成熟樹突細胞(immature dendritic cells;iDCs)傳導信號,以產生經信號標誌之樹突細胞;及(b)用一包含有效量CD40激動劑之第二瞬時信號向該等經信號標誌樹突細胞發出信號,以產生CCR7+成熟樹突細胞。本發明亦提供藉由本發明方法製備的樹突細胞富集群。此等樹突細胞具有增強的免疫刺激性及增加的IL-12分泌及/或降低的IL-10分泌。CD40信號傳導可藉由一或多種轉譯自編碼CD40L之外源多核苷酸(例如,mRNA或DNA)之多肽、CD40受體之激動抗體或藉由CD40配體多肽引發。藉由將免疫原施予該DC可進一步修飾該等富集群。該DC將於其細胞表面上捕獲並處理該免疫原。
