Mature Dendritic Cell Compositions and Methods of Culturing Same
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-γR) agonist and/or a tumor necrosis factor alpha receptor (TNF-αR) 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 - 52 . (canceled)
53 . 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 optionally, 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 CD40 agonist to produce CCR7 + mature dendritic cells;
wherein said immature dendritic cells, said signaled dendritic cells, or said CCR7 + dendritic cells are further contacted with a NKT cell ligand.
54 . The method of claim 53 , wherein said NKT cell ligand is a compound selected from the group consisting of: α-galactosylceramides, α-glucosylceramides, α-6-deoxygalactosylceramides, α-6-deoxygalactofuranosylceramides, β-6-deoxygalactofuranosylceramides, β-arabinosylceramides, α-C-galactosylceramides and α-S-galactosylceramides.
55 . The method of claim 54 , wherein said compound is (2S,3S,4R)-1-O-(alpha-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetriol (KRN7000).
56 . The method of claim 53 , wherein said CCR7 + dendritic cells are contacted with said compound.
57 . The method of claim 53 , wherein said immature DCs are contacted with said compound.
58 . The method of claim 53 , wherein said signaled DCs are contacted with said compound.
59 . A method for preparing mature dendritic cells (DCs), comprising sequentially signaling isolated immature dendritic cells with a first signal comprising a tumor necrosis factor alpha receptor (TNF-αR) agonist followed by a second signal comprising a CD40 agonist, wherein said signaling is in the absence of an effective amount of IL-1β and/or IL-6.
60 . The method of claim 59 , wherein said TNF-αR agonist is TNF-α.
61 . The method of claim 59 , further comprising contacting the immature dendritic cells with PGE 2 .
62 . A CD83 + CCR7 − mature dendritic cell.
63 . (canceled)
64 . The method of claim 60 , wherein the TNF-α is a mammalian TNF-α.
65 . The method of claim 64 , wherein the mammalian TNF-α is human TNF-α.
66 . The method of claim 59 , wherein the TNF-αR agonist is an active fragment of human TNF-α.
67 . The method of claim 59 , wherein the CD40 agonist is CD40L.
68 . The method of claim 67 , wherein the CD40L is a human CD40L.
69 . The method of claim 59 , wherein said second signal is effected upon translation of an mRNA encoding a CD40 agonist.
70 . The method of claim 69 , wherein said mRNA comprises a 3′ UTR of simian rotavirus gene 6.
71 . The method of claim 69 , wherein said mRNA comprises a minimal functional element of the 3′ UTR of simian rotavirus gene 6.
72 . The CD83 + CCR7 − mature dendritic cell of claim 62 , wherein said cell is human.
73 . The CD83 + CCR7 − mature dendritic cell of claim 69 .