Dendritic cell compositions and methods
Methods are provided for the production of dendritic cells from monocytes that have been incubated at a temperature of 1° C.-34° C. for a period of approximately 6 to 96 hours from the time they are isolated from a subject. After the incubation period, the monocytes can then be induced to differentiate into dendritic cells. Mature dendritic cells made by the methods of the invention have increased levels of one or more of CD80, CD83, CD86, MHC class I molecules, or MHC class II molecules as compared to mature dendritic cells prepared from monocytes that have not been held at 1° C.-34° C. for at least 6 hours from the time they were isolated from a subject. Dendritic cells made by the methods of the invention are useful for the preparation of vaccines and for the stimulation of T cells.
1. A composition comprising dendritic cells manufactured by a method comprising:
a) providing monocytes that have been incubated without culturing at a temperature maintained between 16° C.-20° C. for a period of approximately 24 hours from the time they are isolated from a subject;
b) inducing the differentiation of said monocytes into immature dendritic cells; and
c) maturing the immature dendritic cells into mature dendritic cells.
2. The composition of claim 1 , wherein the dendritic cells are antigen-loaded.
3. The composition of claim 2 , wherein the dendritic cells are transfected with mRNA encoding CD40L.
4. The composition of claim 1 , wherein the mature dendritic cells have increased levels of one or more of CD80, CD83, CD86, MHC class I molecules, or MHC class II molecules as compared to mature dendritic cells prepared from fresh monocytes.
5. The composition of claim 1 , wherein said mature dendritic cells have a steady state ratio of ALOX15 RNA to β-actin RNA or GAPDH RNA of less than 1.0.
6. The composition of claim 5 , wherein the ratio is between 0.2 to 0.7.
7. The composition of claim 1 , wherein said mature dendritic cells have a steady state ratio of CD52 RNA to β-actin RNA or GAPDH of greater than 1.0.
8. The composition of claim 7 , wherein the ratio is between 1.2 to 5.0.
9. The composition of claim 1 , wherein said mature dendritic cells have a steady state ratio of TLR1 RNA, TLR2 RNA, IL- 1 βRNA or CD69 RNA to β-actin RNA or GAPDH RNA of less than 1.0.
10. The composition of claim 9 , wherein the ratio is between 0.5 to 0.8.
11. The composition of claim 1 , wherein the monocytes are human monocytes.
12. The composition of claim 1 , wherein the monocytes are present together with peripheral blood mononuclear cells (PBMCs) during all or a portion of the incubation period.
13. The composition of claim 12 , wherein the monocytes are enriched from the PBMCs during or following the incubation period.
14. The composition of claim 13 , wherein the monocytes are enriched from the PBMCs by elutriation, dilute Ficoll gradient centrifugation, dilute Percoll density gradient centrifugation or magnetic bead sorting.
15. The composition of claim 13 , wherein said monocytes are frozen after enrichment.
16. The composition of claim 1 , wherein step (c) comprises contacting said immature dendritic cells with PGE 2 , TNFα, IFN-γto produce mature dendritic cells and transfecting said mature dendritic cells with RNA encoding CD40L and RNA encoding one or more antigen(s) or epitope(s) of interest.