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 method for producing dendritic cells from monocytes, comprising:
b. providing 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; and
c. inducing the differentiation of said monocytes into dendritic cells.
2 . The method of claim 1 , wherein the monocytes are human monocytes.
3 . The method of claim 1 , wherein the incubation temperature is 6° C.-28° C.
4 . The method of claim 4 , wherein the incubation temperature is 8° C.-26° C.
5 . The method of claim 1 , wherein the period of incubation is 8 to 48 hours.
6 . The method of claim 5 , wherein the period of incubation is 10 to 30 hours.
7 . The method of claim 1 , wherein the period of incubation is 26 to 72 hours.
8 . The method of claim 1 , wherein the period of incubation is 48 to 80 hours
9 . The method of claim 1 , wherein the differentiation is induced by contacting the monocytes with a culture medium comprising an effective amount of a composition that induces the differentiation of monocytes into immature dendritic cells.
10 . The method of claim 9 , wherein the composition is GM-CSF and IL-4; GM-CSF and IL-13; GM-CSF and IL-15; or IFNα.
11 . The method of claim 1 , wherein the monocytes are present together with peripheral blood mononuclear cells (PBMCs) during all or a portion of the incubation period.
12 . The method of claim 11 , wherein the PBMCs are isolated from the subject by leukapheresis.
13 . The method of claim 11 , wherein the monocytes are enriched from the PBMCs during or following the incubation period.
14 . The method of claim 13 , wherein the monocytes are enriched from said PBMCs by elutriation, dilute Ficoll gradient centrifugation, dilute Percoll density gradient centrifugation or magnetic bead sorting.
15 . The method of claim 13 , wherein the monocytes are enriched from the PBMCs following the incubation period by allowing the monocytes to adhere to plastic.
16 . The method of claim 9 , further comprising a step of maturing the immature dendritic cells into mature dendritic cells.
17 . The method of claim 16 , further comprising loading one or more antigens into said mature dendritic cells.
18 . The method of claim 16 , wherein the step comprises contacting the immature dendritic cells with PGE 2 , TNFα, IL-6 and IL-1β.
19 . The method of claim 16 , wherein said step comprises signaling said immature dendritic cells with IFN-γ, followed by signaling the dendritic cells with CD40L.
20 . The method of claim 19 , wherein said signaling with CD40L is effected upon translation of a recombinant CD40L mRNA within the dendritic cells.