Mature dendritic cell compositions and methods for 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. A method for preparing CD83 + CCR7 + mature dendritic cells mDCs), comprising the sequential steps of:
(a) culturing isolated immature dendritic cells (iDCs) in culture medium comprising an interferon gamma receptor (IFN-γR) agonist and a TNF-αR agonist to produce CD83 + mDCs; and
(b) cotransfecting said CD83 + mDCs with mRNA encoding CD40L polypeptide and mRNA encoding an antigen approximately 12 to 30 hours after initiating step (a) to produce CD83 + CCR7 + mDCs; wherein the CD40L polypeptide comprises amino acid residues:
(i) 51 through 261 of SEQ ID NO: 2, or
(ii) 120 through 261 of SEQ ID NO: 2.
2. The method of claim 1 , wherein said culture medium further comprises PGE 2 .
3. The method of claim 1 , wherein said CD83 + mDCs are cotransfected using electroporation.
4. The method of claim 1 , wherein said iDCs are generated by contacting isolated human monocytes with GM-CSF and at least one of IL-4 or IL-13.
5. The method of claim 1 , wherein said iDCs are prepared from CD34 + hematopoietic stem or progenitor cells.
6. The method of claim 1 , wherein said IFN-γR agonist is IFN-γ.
7. The method of claim 1 , wherein said TNF-αR agonist is TNF-α.
8. The method of claim 1 , wherein said culturing is in the absence of an effective amount of IL-1β and IL-6.
9. The method of claim 6 , wherein said IFN-γ comprises a polypeptide having at least 95% sequence identity with SEQ ID NO:6.
10. The method of claim 6 , wherein said IFN-γ comprises the sequence of SEQ ID NO:6.
11. The method of claim 1 , wherein said mRNA encoding said CD40L polypeptide is in vitro translated mRNA.
12. The method of claim 1 , wherein said mRNA encoding an antigen is in vitro translated mRNA.
13. The method of claim 1 , wherein said mRNA encoding said CD40L polypeptide and the mRNA encoding said antigen are in vitro translated mRNAs.
14. A method for preparing CD83 + CCR7 + mature dendritic cells (mDCs), comprising the sequential steps of:
(a) culturing isolated immature dendritic cells (iDCs) in culture medium comprising an interferon gamma receptor (IFN-γR) agonist and a TNF-αR agonist to produce CD83 + mDCs; and
(b) cotransfecting said CD83 + mDCs with mRNA encoding a CD40L polypeptide and mRNA encoding an antigen approximately 12 to 30 after initiating step (a) to produce CD83 + CCR7 + mDCs, wherein said mRNA encoding said CD40L polypeptide comprises:
(i) a polynucleotide comprising nucleotides 190 to 822 of SEQ ID NO:1, or
(ii) a polynucleotide comprising nucleotides 397 to 822 of SEQ ID NO:1.
15. The method of claim 1 , further comprising cryopreserving said cotransfected CD83 + CCR7 + mDCs.
16. The method of claim 15 , wherein said CD83 + CCR7 + mDCs are cryopreserved 4 hours post cotransfection.
17. The method of claim 1 , wherein said isolated iDCs are cultured for approximately 12 to 30 hours.
18. The method of claim 1 , wherein said mRNA encodes CD40L polypeptide consisting of amino acid residues 51 through 261 of SEQ ID NO: 2.
19. The method of claim 1 , wherein said mRNA encodes CD40L polypeptide consisting of amino acid residues 120 through 261 of SEQ ID NO: 2.