Mature Dendritic Cell Compositions and Methods of Culturing Same

Abstract

This invention provides novel CD40L polypeptides and nucleic acids, as well as antigen presenting cells and vaccines comprising such CD40L polypeptides and/or nucleic acids, and related methods for preparing the antigen presenting cells and vaccines. The antigen presenting cells and vaccines are useful 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-?R) 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.

1 - 9 . (canceled) 
     
     
         10 . A vector comprising a nucleic acid encoding a recombinant CD40L polypeptide consisting of amino acid residues 21-261 of SEQ ID NO:2, wherein said vector is pARGCD40L. 
     
     
         11 - 24 . (canceled) 
     
     
         25 . A dendritic cell which preferentially induces a population of CD28 +  CD45RA −  memory/effector T cells from a population of antigen-specific T cells. 
     
     
         26 . A vaccine comprising the dendritic cell of  claim 25 . 
     
     
         27 . 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-γR) agonist and a TNF-αR agonist to produce IFN-γR agonist signaled dendritic cells; and   (b) signaling said IFN-γR agonist signaled dendritic cells with a second transient signal comprising an effective amount of a CD40L polypeptide to produce CCR7 +  mature dendritic cells; wherein the CD40L polypeptide consists essentially of amino acid residues 21-261 of SEQ ID NO:2 or is a polypeptide having at least 80% sequence identity to amino acid residues 21-261 of SEQ ID NO:2.   
     
     
         28 . The method of  claim 27 , wherein the CD40L protein is encoded by an RNA corresponding to the cDNA of SEQ ID NO:30 or SEQ ID NO:33, or a variant which differs due to codon degeneracy, wherein said RNA is optionally capped and/or polyadenylated. 
     
     
         29 . The method of  claim 27 , wherein the dendritic cell is transfected with an mRNA encoding the CD40L polypeptide. 
     
     
         30 . The method of  claim 27 , wherein the dendritic cell is mature at the time of transfection. 
     
     
         31 . The method of  claim 27 , further comprising contacting said iDCs and/or said signaled DCs with PGE 2 . 
     
     
         32 . The method of  claim 27 , wherein steps (a) and (b) further comprise contacting said cells with GM-CSF and at least one of IL-4 or IL-13. 
     
     
         33 . The method of  claim 27 , wherein the cells in step (a) are contacted with TNF-α. 
     
     
         34 . The method of  claim 33 , further comprising contacting the cells in step (a) with PGE 2 . 
     
     
         35 . The method of  claim 27 , wherein the IFN-γR agonist is IFN-γ. 
     
     
         36 . The method of  claim 27 , wherein the first signal comprises IFN-γ, TNF-α and PGE 2 . 
     
     
         37 . The method of  claim 27 , wherein the first signal comprises IFN-γ, TNF-α and PGE 2  and step (b) comprises transfecting said dendritic cells with an RNA encoding said CD40L polypeptide. 
     
     
         38 . The method of  claim 27 , further comprising contacting the immature dendritic cells and/or IFN-γR agonist signaled dendritic cells and/or CCR7 +  mature dendritic cells with a compound selected from the group consisting of: galactosylceramides, glycosylceramides, galactofuranosylceramides, arabinopyranosylceramides, α-C-galactosylceramides and α-S-galactosylceramides. 
     
     
         39 . The method of  claim 38 , wherein the compound is a galactosylceramide. 
     
     
         40 . The method of  claim 39 , wherein the galactosylceramide is (2S,3S,4R)-1-O-(alpha-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetriol (KRN7000). 
     
     
         41 . The method of  claim 27 , wherein the CD40L polypeptide consists of amino acid residues 21-261 of SEQ ID NO:2.