COMPOSITIONS FOR THE PREPARATION OF MATURE DENDRITIC CELLS

Abstract

The invention relates to a method for in vitro maturation of at least one immate dendritic cell, comprising stimulating said immature dendritic cell with TNF, IL-1, IFN, a TLR7/8 agonist and prostaglandin E2(PG). Furthermore, the invention elates to a composition comprising these factors as well as to mature dendritic cells produced by a method of the invention.

Claims

1-22. (canceled)

23. A mature dendritic cell or a population of mature dendritic cells, obtainable by a method for in vitro maturation of at least one immature dendritic cell, comprising stimulating said immature dendritic cell with TNF, IL-1, IFN, a TLR7/8 agonist and prostaglandin E2 (PG2).

24. The mature dendritic cell or a population of mature dendritic cells of claim 23, wherein the method further comprises stimulating said immature dendritic cell with a TLR3 agonist.

25. The mature dendritic cell or a population of mature dendritic cells of claim 24, wherein the TLR3 agonist is polyI:C.

26. The mature dendritic cell or a population of mature dendritic cells of claim 23, wherein said substances are part of a composition added to the culture medium of said immature dendritic cell.

27. The mature dendritic cell or a population of mature dendritic cells of claim 23, wherein said TLR7/8 agonist is an imidazoquinilone type immune response modifying compound.

28. The mature dendritic cell or a population of mature dendritic cells of claim 27, wherein said imidazoquinilone type immune response modifying compound is 4-amino-2-ethoxymethyl-,-dimethyl-1H-imidazol[4,5-c]quinoline-1-ethanol (R848).

29. The mature dendritic cell or a population of mature dendritic cells of claim 23, wherein said immature dendritic cell is a monocyte derived immature dendritic cell, or wherein said immature dendritic cell is obtained directly from peripheral blood.

30. The mature dendritic cell or a population of mature dendritic cells of claim 29, wherein said immature dendritic cell is derived from human peripheral blood mononuclear cells, monocytes, other myeloid progenitor cells, or from CD34 positive progenitor cells by in vitro differentiation to CD14 positive cells.

31. The mature dendritic cell or a population of mature dendritic cells of claim 23, wherein the immature dendritic cell is obtained by incubating human peripheral blood mononuclear cells, monocytes or other myeloid progenitor cells with GM-CSF and IL-4 or IL-13.

32. The mature dendritic cell or a population of mature dendritic cells of claim 23, wherein the immature dendritic cell is of human origin.

33. The mature dendritic cell or a population of mature dendritic cells of claim 23, wherein the method comprises the following steps: a) preparing mononuclear cells from peripheral blood, preferably wherein said mononuclear cells are obtained by leukapheresis from peripheral blood, b) incubating the mononuclear cells of step a) with GM-CSF and IL-4 or IL-13, c) incubating the cells obtained in step b) with a cocktail comprising TNF, IL-1, IFN, a TLR7/8 agonist, prostaglandin E2 (PG), and, optionally, a TLR3 agonist, preferably poly I:C, and d) harvesting the mature dendritic cell or cells.

34. The mature dendritic cell or a population of mature dendritic cells of claim 33, wherein in step a) the mononuclear cells are obtained by leukapheresis from peripheral blood, wherein said incubation in step b) takes 1 to 9 days, 2 to 9 days, or 2 to 6 days, or wherein said incubation in step c) takes 12 h to 72 h, 20 h or 24 h.

35. The mature dendritic cell or a population of mature dendritic cells of claim 23, wherein the mature dendritic cell or cells is/are further loaded in vitro with one or more antigens.

36. The mature dendritic cell or a population of mature dendritic cells of claim 35, wherein said antigen or antigens are supposed to trigger the effector T cell maturation within the lymph nodes.

37. The mature dendritic cell or a population of mature dendritic cells of claim 35, wherein said loading is performed by incubating the mature dendritic cell or cells with at least one peptide of said antigen or by transfecting the dendritic cell or cells with antigen encoding RNA or DNA.

38. A pharmaceutical composition comprising the mature dendritic cell or the population of mature dendritic cells of claim 23.

39. A method of treating a disease selected from the group consisting of tumorigenic and infectious diseases in a patient in need of such treatment, comprising administering to said patient a therapeutically effective dose of the mature dendritic cell or the population of mature dendritic cells of claim 23.

40. A method for the preparation of the mature dendritic cell or the population of mature dendritic cells of claim 23 comprising the combined use of TNF, IL-1, IFN, a TLR7/8 agonist, prostaglandin E2 (PG2) and, optionally, a TLR3 agonist, preferably poly I:C.

41. The method of claim 40, wherein said TLR7/8 agonist is an imidazoquinilone type immune response modifying compound.

42. The method of claim 40, wherein said imidazoquinilone type immune response modifying compound is 4-amino-2-ethoxymethyl-,-dimethyl-1H-imidazol[4,5-c]quinoline-1-ethanol (R848).

Description

SHORT DESCRIPTION OF THE FIGURES

[0054] FIGS. 1A-C:

Generation of Different Matured Dendritic Cells (DCs)

[0055] A. Recovery of harvested DCs after primary cell culture (6 days differentiation+24 h maturation) calculated on seeded total cells (mononuclear cells) or CD14 positive monocytes detected by manual counting within Neubauer chamber and FACS analysis (CD14). Viability detected due to 7AAD incorporation quantitative measured within FL-3 of FACS calibur. Broken line indicated levels of DCs matured with gold standard Jonuleit cocktail.

[0056] B. Surface expression of DC specific molecules for DCs after primary DC culture. Low CD14 expression in comparison to high expression of the DC-specific molecule CD83 indicates a mature status of DCs, as detected by FACS analysis (percentage of all cells without gating, aquisition of 10000 cells total).

[0057] C. Surface expression of co stimulatory molecules, CD80 and CD86, after primary DC culture detected by FACS analyses. Expression of chemokine receptor 7 (CCR7=CD197) as indication for the migratory potential of DCs into lymph nodes. Positive percentage is detected according to overlay with the isotype control antibody.

DC1=Jonuleit=TNF-alpha (long/ml)+IL-1beta (10 ng/ml)+IL-6 (15 ng/ml)+Prostaglandin E2 (=PGE2, 1000 ng/ml)

DC2=Kalinski=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+IFN alpha (3000 IU/ml)+IFNgamma (1000IU/ml)+polyI:C (20 ng/ml)

DC3=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+PGE2 (100 ng/ml)+IFNgamma (1000 IU/ml)+R848 (1 g/ml)

DC4=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+PGE2 (250 ng/ml)+IFNgamma (5000 IU/ml)+R848 (1 g/ml)

DC5=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+PGE2 (250 ng/ml)+IFNgamma (5000 IU/ml)+R848 (1 g/ml)+poly I:C (20 ng/ml)

[0058] FIGS. 2A-C:

Maturation Stability (Wash Out Test) of Different Matured DCs

[0059] DCs were washed out from cytokines and cultured additional 40h after maturation within DC culture medium with serum and gentamycin only.

[0060] A. Viability of different matured DCs after Wash out detected due to 7AAD incorporation.

[0061] B. Surface expression of low induced CD14 in comparison to high CD83 expression levels after Wash out.

[0062] C. Expression of co stimulatory molecules CD80 and CD86 and CCR7 after Wash out.

DC1=Jonuleit=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+IL-6 (15 ng/ml)+Prostaglandin E2 (=PGE2, 1000 ng/ml)

DC2=Kalinski=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+IFN alpha (3000 IU/ml)+IFNgamma (1000IU/ml)+polyI:C (20 ng/ml)

DC3=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+PGE2 (100 ng/ml)+IFNgamma (1000 IU/ml)+R848 (1 g/ml)

DC4=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+PGE2 (250 ng/ml)+IFNgamma (5000 IU/ml)+R848 (1 g/ml)

DC5=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+PGE2 (250 ng/ml)+IFNgamma (5000 IU/ml)+R848 (1g/ml)+poly I:C (20ng/ml)

[0063] FIGS. 3A-C:

Cryopreservation of Different Matured DCs.

[0064] DCs were frozen and stored under gas phase of liquid nitrogen and analyzed after thawing.

[0065] A. Viability of different matured DCs after Wash out detected due to 7AAD incorporation.

[0066] B. Low surface expression of CD14 in comparison to high CD83 expression levels after freezing.

[0067] C. Expression of co stimulatory molecules CD80 and CD86 and CCR7 after freezing.

DC1=Jonuleit=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+IL-6 (15 ng/ml)+Prostaglandin E2 (=PGE2, 1000 ng/ml)

DC2=Kalinski=TNF-alpha (10 ng/ml)+IL-lbeta(10 ng/ml)+IFN alpha (3000 IU/ml)+IFNgamma (1000 IU/ml)+polyI:C (20 ng/ml)

DC3=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+PGE2 (100 ng/ml)+IFNgamma (1000 IU/ml)+R848 (1 g/ml)

DC4=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+PGE2 (250 ng/ml)+IFNgamma (5000 IU/ml)+R848 (1 g/ml)

DC5=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+PGE2 (250 ng/ml)+IFNgamma (5000 IU/ml)+R848 (1g/ml)+poly I:C (20ng/ml)

[0068] FIGS. 4A-F:

[0069] Analyses of allostimulatory capacity of different matured DCs in a mixed lymphocyte reaction.

[0070] A. Functional control of proliferative ability of autologeous T cells: T cells within medium only, T cells stimulated with third party (=five mixed MNC donors) (4000 rad irradiated 10e5/well, ratio 1:1 stimulatory cells: responder cells), T-cells +IL-2 (5 IU/ml) and PHA 10 g/ml last 68 h, T cells stimulated with 50 IU/ml IL-2. T cell numbers 10e5/well. Co culture over 7 days, proliferation was measured by 3H-thymidin incorporation of last 24 h. All values are calculated out of five repeated wells.

[0071] B. Functional control of proliferative ability of one exemplary allogenic T cell responder.

[0072] C. Negative control of proliferation of irradiated (4000 rad) different matured DCs (10e4/well, according to cell number of assay ratio 1:10, DCs: responder cells)

[0073] D. Proliferation of autologeous T cells stimulated by different matured DCs (DC numbers 10e4/well, T cell numbers 10e5, ratio 1:10, DCs: responder cells)

[0074] E. Proliferation of one exemplary, allogenic T cell responder stimulated by different matured DCs (DC numbers 10e4/well, T cell numbers 10e5, ratio 1:10, DCs: responder cells).

[0075] F. Summary of proliferation data of three independent T cell responders in comparison to autologeous T cells stimulated by different matured DCs

DC1=Jonuleit=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+IL-6 (15 ng/ml)+Prostaglandin E2 (=PGE2, 1000 ng/ml)

DC2=Kalinski=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+IFN alpha (3000 IU/ml)+IFNgamma (1000 IU/ml)+polyI:C (20 ng/ml)

DC3=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+PGE2 (100 ng/ml)+IFNgamma (1000 IU/ml)+R848 (1 g/ml)

DC4=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+PGE2 (250 ng/ml)+IFNgamma (5000 IU/ml)+R848 (1 g/ml)

DC5=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+PGE2 (250 ng/ml)+IFNgamma (5000 IU/ml)+R848 (1 g/ml)+poly I:C (20ng/ml)

[0076] FIGS. 5A-C.

[0077] Production of IL-12p70 and IL-10 by DCs Matured By Using Different Cocktails Immature DCs were cultured with different maturation cocktails and the amounts of IL-12p70 and IL-10 were determined by standard ELISA. Filled bars indicate IL-12p70 and empty bars IL-10 respectively.

[0078] A: Supernatant medium of primary maturation cultures after 7 days;

[0079] B: Supernatant medium of cultures of washed, matured DCs and CD4OL-transfected fibroblasts following a coculture period of 24hrs, representing a signa13-assay as described in Material and Methods.

[0080] C. The quotients of IL-12p70/IL-10 were determined for the DC populations matured in different cocktails, based on the g/ml-values of the signal-3 assay. For calculation it was assumed that IL-12p70 and IL-10 are theoretically equal biological potential. Filled circles indicate a positive quotient between 0 and 3, 5, pointed lines valued sharp differences of DCs matured with Jonuleit or Kalinski cocktail for IL-12p70 secretion.

DC1=Jonuleit=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+IL-6 (15 ng/ml)+Prostaglandin E2 (=PGE2, 1000 ng/ml)

DC2=Kalinski=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+IFN alpha (3000 IU/ml)+IFNgamma (1000IU/ml)+polyI:C (20 ng/ml)

DC3=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+PGE2 (100 ng/ml)+IFNgamma (1000IU/ml)+R848 (1 g/ml)

DC4=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+PGE2 (250 ng/ml)+IFNgamma (5000 IU/ml)+R848 (1 g/ml)

DCS=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+PGE2 (250 ng/ml)+IFNgamma (5000 IU/ml)+R848 (1 g/ml)+poly I:C (20 ng/ml)

[0081] FIG. 6: Expression of EGFP in DCs transfected with EGFP-encoding in vitro transcribed RNA

[0082] Flow cytometry histogram overlays show EGFP RNA-transfected into mature DCs (filled curves) 24 h after electroporation and corresponding untransfected DCs (empty curves) as negative controls. DCs were matured in the four cocktails indicated, RNA was introduced by electroporation, the DCs were returned to their corresponding media containing maturation cocktails and harvested for flow cytometry 24 h later. Numbers indicate the percentages of EGFP-positive DCs and their mean fluorescence intensities. These data are representative of two experiments with measurements at 24 and 48 h.

DC1=Jonuleit=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+IL-6 (15 ng/ml)+Prostaglandin E2 (=PGE2, 1000 ng/ml)

DC2=Kalinski=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+IFN alpha (3000 IU/ml)+IFNgamma (1000IU/ml)+polyI:C (20 ng/ml)

DC4=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+PGE2 (250 ng/ml)+IFNgamma (5000 IU/ml)+R848 (1 g/ml)

DC5=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+PGE2 (250 ng/ml)+IFNgamma (5000 IU/ml)+R848 (1 g/ml)+poly I:C (20 ng/ml)

[0083] FIG. 7: Response of autologous lymphocytes from an HLA-A*0201-positive donor responding to virus-peptide pulsed DCs

[0084] T cell responses were assessed in an IFN-ELISPOT experiment using lymphocytes (T cell enriched Elutra fraction 3=54.76% CD3 positive cells) that were first activated for 7 d with mature peptide-pulsed DCs and then restimulated for 24 h with monocytes plus CEF peptides. For the ELISPOT analyses, 410.sup.3 autologous in vitro activated lymphocytes were stimulated with 210.sup.3 monocytes together with the five peptide CEF pool. The mean S.D. was calculated for triplicate wells. Note: Due to insufficient recoveries, lymphocytes activated by DC2 cells were not included in the assay.

DC1=Jonuleit=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+IL-6 (15 ng/ml)+Prostaglandin E2 (=PGE2, 1000 ng/ml)

DC2=Kalinski=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+IFN alpha (3000 IU/ml)+IFNgamma (1000 IU/ml)+polyI:C (20 ng/ml)

DC3=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+PGE2 (100 ng/ml)+IFNgamma (1000 IU/ml)+R848 (1 g/ml)

DC4=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+PGE2 (250 ng/ml)+IFNgamma (5000 IU/ml)+R848 (1 g/ml)

DC5=TNF-alpha (10 ng/ml)+IL-1beta (10 ng/ml)+PGE2 (250 ng/ml)+IFNgamma (5000 IU/ml)+R848 (1 g/ml)+poly I:C (20ng/ml)

[0085] FIG. 8: Interferon-gamma response of autologous lymphocytes stimulated with mature DCs

[0086] Lymphocytes from an HLA-A*0201 donor were stimulated with autologous DCs matured using the Jonuleit cocktail, the Kalinski cocktail and the three new cocktails described herein.

[0087] Peripheral blood lymphocytes (PBLs) were separated by elutriation and contained in fraction 3, in which 54.8% of the cells were CD3-positive T lymphocytes and 17,7% CD56 positive cells, which is characteristic for natural killer (NK) cells. (A) The elutriation fraction 3 PBLs were directly incubated with the different DC populations matured using the different cocktails and their interferon-gamma secretion (IFN-gamma) was measured after 24 h in a standard ELISPOT assay.

DC1=Jonuleit=TNF (10 ng/ml)+IL-1 (10 ng/ml)+IL-6 (15 ng/ml)+Prostaglandin E2 (=PGE2; 1000 ng/ml);

DC2=Kalinski=TNF (10 ng/ml)+IL-1 (10 ng/ml)+IFN (3000 IU/ml)+IFNy (1000 IU/ml)+polyI:C (20 ng/ml);

DC3=TNF (10 ng/ml)+IL-1 (10 ng/ml)+PGE2 (100 ng/ml)+IFN (1000 IU/ml)+R848 (1 g/ml);

DC4=TNF (10 ng/ml)+IL-1 (10 ng/ml)+PGE2 (25Ong/ml)+IFN (5000 IU/ml)+R848 (1 g/ml);

DC5=TNF (10 ng/ml)+IL-1 (10 ng/ml)+PGE2 (250 ng/ml)+IFN (5000 IU/ml)+R848 (1 g/ml)+poly I: C (20 ng/ml).

EXAMPLE

[0088] The following example represents the description of one experiment as a representative example of at least three independent experiments performed using different donor cells.

1. Material and Methods

Leukapheresis and Elutriation

[0089] To obtain monocytes as a progenitor cell population for generation of human dendritic cells, we used a closed system of elutriation by ELUTRA (Gambro BCT, Lakewood, USA). After informed consent, healthy, unmobilized donors underwent 180 minute leukaphereses with the COBE Spectra cell separator (Gambro BCT, Inc. Lakewood, USA.) using a modified MNC program (V6.1): separation factor was set to 700 with a collection rate of 0.8 ml/min and a target hematocrit of only 1-2%. Resulting blood cells were analysed by automatic blood counter ACT Dif (Beckman Coulter, Krefeld, Germany) to set up conditions for ELUTRA system.

[0090] Leukapheresis products were processed by ELUTRA (Gambro BCT, Lakewood, USA) according manufacturer's instructions by a method of counter-flow cenrifugal elutriation using a fixed rotor speed (2400 rpm) and computer controlled stepwise adjustment of media flow rate followed by rotor-off harvesting. Therefore 5000 ml of running buffer containing HANKs buffered salt solution (Biochrom, Berlin, Germany) with 1% human serum albumin (Octalbine, Octapharma, Langen, Germany) were prepared. ELUTRA process resulted in five fractions, with enriched monocytes in the rotor-off fraction. Cellular composition of fractions were characterised by automatic blood counter ACT Diff (Beckman Coulter, Krefeld, Germany) and FACS analysis.

FACS-Analysis of ELUTRA Fractions

[0091] Cells of original leukapheresis product and all five ELUTRA fractions were incubated for 30 minutes with the following fluorescein isothiocyanate (FITC)-and phycoerythrin (PE) conjugated monoclonal mouse antibodies: IgG isotype controls (clone X-40), anti-CD14-FITC (clone: MOP9), anti-CD19-FITC (clone: 4G7), anti-HLA-DR-FITC (clone: L243) (BD Biosciences, Heidelberg, Germany) and anti-CD3-PE (clone UCHT1), anti-CD56-PE (C5.9), anti-CD16-PE (clone: DJ130c), and as a additional control CD14-PE (TK4) (Dako Diagnostics, Hamburg, Germany) and anti-CD67-FITC (clone: 80H3) (Immunotech, Marseille, France). Cells were washed and resuspended in PBS+2% fetal calf serum (Biochrom, Berlin, Germany). Flow cytometry analysis was performed on a FACS Calibur device using Cellquest Pro software (BD Biosciences, Heidelberg, Germany).

Generation of Immature Monocyte-Derived Dendritic Cells from Elutriated Monocytes

[0092] Cells from rotor-off fraction or the subsequently named fraction 5 were used directly for DC generation if CD14 positive cells represented over 60% of all cells detected by FACS analysis. Fraction 5 cells were harvested from ELUTRA collecting bag and washed once with PBS+0.5% human serum and seeded at 3510.sup.6/175 cm.sup.2 cell culture flask (NUNC, Wiesbaden, Germany) in 35ml DC medium containing RPMI 1640 with very low endotoxin (Biochrom, Berlin, Germany), 1.5% human serum (pool of AB-positive adult males) (Blood Bank, University of Tuebingen, Germany) and 10 g/ml Gentamycin (Biochrom, Berlin, Germany) and cultivated for six days by 37 C., 5% CO.sub.2 in a humidified atmosphere. At day 1, 3 and 6 cell cultures were supplemented with 100 ng/ml GM-CSF (Leukine by Berlex, Richmond, USA) and 20 ng/ml recombinant human IL-4 (R&D Systems, Wiesbaden, Germany) in 7 ml fresh DC medium per flask.

Maturation of Dendritic Cells

[0093] Maturation processes were induced by adding different combinations of cytokines and other reagents, as indicated, to immature DCs on day 6 along with additional 7 ml fresh DC medium define here per flask:

[0094] Jonuleit cocktail: 10 ng/ml TNF-, 10 ng/ml IL-1-I, 15 ng/ml IL-6 (R&D Systems, Wiesbaden, Germany) and 1 g/ml prostaglandin E2 (Minprosting, Pharmacia/Pfizer, Erlangen, Germany),

[0095] Kalinski cocktail: 10 ng/ml TNF-, 10 ng/ml IL-1-I (R&D Systems, Wiesbaden, Germany), 3000 IU/ml IFN (Roferon A, Roche, Welwyn Garden City, England), 1000 IU/ml IFNy (ImukinR, Boehringer Ingelheim, Ingelheim, Germany) and 20 ng/ml double-stranded RNA (poly I:C, InVivogen, Toulouse, France).

[0096] New cocktail 1: 10 ng/ml TNF-, 10 ng/ml IL-1-I (R&D Systems, Wiesbaden, Germany), 5000 IU/ml IFN (Imuking, Boehringer Ingelheim, Ingelheim, Germany), 1 g/ml R848 (InVivogen, Toulouse, France) and 250 ng/ml prostaglandin E.sub.2.

[0097] As a variation of cocktail 1, we used the same components expect the concentration of IFN was reduced to 1000 IU/ml and prostaglandin E2 to 100 ng/ml.

[0098] New cocktail 2: similar to cocktail 1 plus 20ng/ml double-stranded RNA (poly I:C, InVivogen, Toulouse, France).

[0099] As a control, one flask received only 7 ml fresh medium only and served as immature DCs (data not shown).

Harvesting of Dendritic Cells

[0100] After incubation of DCs with maturation cocktails for 24 h, cells were harvested by washing twice with PBS+0.5% human serum with light shaking, cells were counted by Neubauer chamber and prepared for the analyses.

Flow Cytometric (FACS)-Analysis

DC Phenotyping:

[0101] DCs were labeled with the following fluorescence-conjugated monoclonal mouse antibodies with specificities for isotype controls (clone X-40), CD14 (FITC, MOP9), CD19 (FITC, clone: 4G7), CD86 (FITC, clone: 2331 FUN-1), CD80 (PE, clone: L307.4) (BD Biosciences, Heidelberg, Germany) and CD209 (PE, clone: DCN46) (Pharmingen, San Diego, USA) and CD3 (FITC, clone: UCHT1), CD56 (FITC, clone: C5.9a), CD1a (FITC, clone: NA1/34) (Dako, Hamburg, Germany) and HLA-DR (PE, clone: B8.12.2, CD40 (PE, clone: mAb89, CD83 (PE, clone: HB15a) (Immunotech, Marseille, France).

[0102] CCR7 staining was performed with a rat hybridoma BLR-2 (clone 8E8) (E.Kremmer, GSF) in comparison to isotype control for IgG2a of hybridoma EBNA-A2 (clone R3) by incubation of DCs in culture supernatant for 60 minutes and followed by after washing, and detection with secondary mouse antibody against rat IgG conjugated with cyanin 5 (Jackson Immuno, West Grove, USA).

[0103] To test vitality, DCs were pelleted and resuspended for 20 minutes in 7-Aminoactinomycin D (Sigma-Aldrich, Deisenhofen, Germany) at final concentrations of 10 g/ml in PBS+2% fetal calf serum. After washing, cells were analyzed in the third channel of the FACS Calibur machine.

Check of Maturation Stability (WASH OUT Test)

[0104] Matured, harvested and washed DCs were reseeded to 2.5310.sup.6/.sub.9ml fresh DC medium without any cytokines in 25 cm2 cell culture flasks (NUNC, Wiesbaden, Germany).

[0105] After approximately 44 h, DCs were harvested and phenotyped by FACS analyses.

Signal 3-Assay

[0106] DCs were co-cultured with T cell-mimicking cells as described previously (Kalinski, 2004). Briefly, matured, harvested and washed DCs were reseeded in 96 well plates at concentrations of 210.sup.4/well and incubated together with mouse fibroblasts stably transfected with human CD40L Garrone P, Neidhardt E M, Garcia E, Galibert L, van Kooten C, Banchereau J. Fas ligation induces apoptosis of CD40-activated human B lymphocytes. JExp Med. 1995 Nov. 1;182(5):1265-73) at concentrations of 510.sup.4/well. To control proliferation of each cell population alone, DCs with out any additions and CD40L-fibroblasts in standard medium were tested. After 24 h, plates were centrifuged and supernatants of 8 replicate wells were pooled for analyses of IL-10 and IL-12p70 by ELISA.

ELISA (IL-12p70/IL-10)

[0107] Secretion of IL-12p70 and IL-10 by DCs during maturation process (primary DCs) and DCs within Signal 3-assay were detected by standard quantitative ELISA. ELISA was performed utilizing pre-tested antibody duo sets for detection of IL-12p70 and IL-10 (R&D Systems, Wiesbaden, Germany) according to manufacturer's instructions. Colorimetric substrate reaction with tetramethylbenzidine and H.sub.2O.sub.2 was measured after stopping with H.sub.3PO.sub.4 at 450 nm and wavelength correction by 620 nm and analyzed by software easy fit (SLT, Crailsheim, Germany).

Cryopreservation of Dendritic Cells

[0108] After harvesting and washing, 20-2510.sup.6 DCs were collected in 0.5 ml cold 20% human serum albumin (Octalbine, Octapharma, Langen, Germany) and gently mixed with 0.5 ml (equal amounts) freshly prepared freezing solution containing 10% glucose (Braun, Melsungen, Germany), 20% DMSO (CryoSURE, WAK-Chemie, Dessau-Thornau, Germany) in 20% human serum albumin. Cryotubes (NUNC, Wiesbaden, Germany) were stored over night at 80 C. and transferred into the gas phase of a liquid nitrogen container.

Mixed-Lymphocyte Reaction

[0109] DCs were matured in vitro as indicated, washed 2 times in PBS+0.5% human serum, irradiated with 40Gy and plated into 96-well round bottom microplates at 110.sup.4/well (Nunc, Wiesbaden, Germany) in RPMI 1640+1.5% human serum. Cryopreserved cells of fraction 3 after ELUTRA procedure from different donors were used as a source of responder cells and seeded at 110.sup.5/well to the DCs of allogenic donors.

[0110] As control for T cell activation via MHC differences third party cells were used as follows: a mixture of MNCs of 5 independent donors obtained from buffy coats after irradication by 40Gy were used as stimulating cells. General unspecific potential of the T cells to proliferate was controlled by incubation of responder cells with IL-2 (Proleukin by Chiron, Emeryville, USA) at 50 IU/ml and Phycohaemagglutinin at 10 g/ml (Sigma-Aldrich, Deisenhofen, Germany).

[0111] After 6 days, cells were pulsed with 0.5 Ci/well .sup.3H-thymidine (Amersham-Pharmacia, Freiburg, Germany) and uptake of .sup.3H-thymidine was determined after 24 h using a -counter device (Wallac, Freiburg, Germany).

EGFP-RNA Transfection into DCs

[0112] EGFP-RNA was produced in vitro and electroporated into mature DCs at 24 h as described previously (Nair, S. K., Boczkowski, D., Morse, M., Cumming, R. I., Lyerly, H. K. and Gilboa, E. (1998). Induction of primary carcinoembryonic antigen (CEA)-specific cytotoxic T lymphocytes in vitro using human dendritic cells transfected with RNA. Nat Biotechnol. 16:364-369, Javorovic, M., Pohla, H., Frankenberger, B., Wolfel, T. and Schendel, D. J. (2005). RNA transfer by electroporation into mature dendritic cells leading to reactivation of effector-memory cytotoxic T lymphocytes: a quantitative analysis. Mol Ther. 12: 734-743), with the exception that each 0.4 cm electroporation cuvette contained a total volume of 300 , including 8 g of EGFP-RNA and 310.sup.6 DCs. After electroporation, DCs were returned to their original maturation media and incubated in a 24-well plate at 37 C. and 5% CO.sub.2 for 24 or 48 h before flow cytometric analysis.

ELISPOT Assay of Virus-Specific T Cell Activation

[0113] For activation, lymphocytes from ELUTRA fraction 3 were plated at 110.sup.6 cells/well with 110.sup.5 viral peptide-loaded DCs in 24-well plates, in RPMI 1640 medium with 10% human serum; 30 IU/ml IL-2 was added at d3 and lymphocytes harvested at d7. HLA-A*0201-binding peptides included: CMVpp65.sub.495-503 (NLVPMVATV; SEQ ID NO: 1), EBV-BMLF1.sub.280-288 (GLCTLVAML; SEQ ID NO: 2), influenza M1 protein58-66 (GILGFVFTL; SEQ ID NO: 3) or the CEF pool (PANATecs GmbH, Tuebingen, Germany) containing two additional peptides, EBV-LMP-2.sub.426-434 (CLGGLLTMV; SEQ ID NO: 4) and influenza RNA polymerase PA.sub.46-54 (FMYSDFHFI; SEQ ID NO: 5). In vitro activated T cells and autologous monocytes plus CEF peptides were incubated in RPMI 1640 medium containing 2 mM L-glutamine, 1 mM sodium pyruvate, penicillin/streptomycin (100 U/ml), 10% human AB serum (BioWhittaker, Verviers, Belgium) and 20 IU/ml IL-2 at 37 C. with 5% CO.sub.2 for 24 h. IFN-ELISPOT analysis was performed as described (Becker, C.,et. at. (2001). Adoptive tumor therapy with T lymphocytes enriched through an IFN capture assay. Nat Med. 7: 1159-1162, Pohla, H., et at. (2000). Allogeneic vaccination for renal cell carcinoma: Development and monitoring. Bone Marrow Transplant. 25: 83-87), with the exception that antibody precoated PVDF plates (Mabtech AB, Nacka, Sweden) and streptavidin-alkaline phosphatase and a ready-to-use BCIP/NBT-plus substrate solution (Mabtech) were used for detection. Spots were counted using the AID reader system ELRO3 with 3.2.3 software (AID Autoimmun Diagnostika GmbH, Strassberg, Germany).

2. Results and Discussion

[0114] Primary DC culture

[0115] The ELUTRA fraction 5 of the described example (DC034) contained: 80,6% CD14 positive cells and the following contaminants, 2.89% CD3, 2.2% CD56, 1.47% CD19 and 7.72% CD67-positive cells and was therefore appropriate to generate dendritic cells.

[0116] The highest recovery of dendritic cells, based on total seeded cells as well as monocytes (CD14 positive cells), was found using the Jonuleit cocktail, while the lowest was found using the Kalinski cocktail (FIG. 1A). Low cell numbers represent poor ability of to harvest DCs without using cell scrapers or enzymatic digestion. This finding represents a big disadvantage of the Kalinski cocktail in our system which might be due to a higher degree of adherence and fine elongation of dendritic veils of DCs matured with this cocktail. Viability of harvested dendritic cells determined by 7AAD staining showed over 97% live cells using Jonuleit cocktail whereas cells matured with Kalinski cocktail reached only 79,5%, our new cocktails ranged between these two values (FIG. 1A).

[0117] The expression of co stimulatory molecules, like CD80 and CD86, reflected the presence of antigen presenting cells, particularly dendritic cells. FIG. 1B shows a high expression level of these molecules in all DCs matured with different cocktails. CD14 is a monocytic molecule, but under the influence of GM-CSF/IL-4 and maturation cocktails it disappears rapidly from the surfaces of dendritic cells. Here we show that DCs generated with all cocktails loose CD14 expression as a clear evidence for a differentiation in the direction of DCs, instead of the also possible differentiation in the direction of macrophages.

[0118] CD83 on the other hand serves as the most important marker to indicate the maturation status of DCs. Expression of CD83, in combination with nearly undetectable expression of CD14, demonstrated that in all five matured DC populations, cells were of DC identity and highly mature (FIG. 1B).

[0119] The chemokine receptor 7 (CD197) indicates a migratory potential of DCs towards lymph nodes along chemokine gradients of CCL19 and CCL21 within high endothelial venules. All different matured DC populations expressed CCR7 at high levels (FIG. 1C).

DCs after Washing Out Maturation Cytokines (Wash Out-Test)

[0120] Stability of maturation status is an important characteristic of clinically applicable DCs, because patients with malignant diseases often show high serum titers of inhibitory cytokines (e.g. IL-10, TGF-beta, IL-6). These cytokines may influence injected DCs by reversing them to an immature status and tolerize a patient's immune system towards vaccinated tumour antigens.

[0121] To test if our new cocktails induced stable maturation, we tested important DC marker molecules after washing out all cytokines and incubating the DCs at least 40 h following of re seeding in medium only. Remarkably lower viability was found in DCs matured with the Kalinski cocktail (FIG. 2A). FIG. 2A shows, as we expected, that viability is lost over time, because the DCs are exhausted and cells in such a condition after more than 60 h following incubation with maturation cocktail are not suitable for therapy any longer. For test reasons only we checked surface expression of certain molecules, like re-induction of CD14, which would indicate a reverse to immature DCs. As FIG. 2B shows, the five different matured DCs expressing very low levels (under 1%) of CD14 while retaining high levels of the maturation DC marker CD83. Again DCs matured with Kalinski cocktail, showed slightly lower CD83 values than DCs matured with the other cocktails.

[0122] FIG. 2C showed stability of high expression levels of co stimulatory molecules, CD80 and CD86, and of maintenance the migratory potential by CCR7 expression.

DCs after Freezing And Thawing

[0123] In this step, we searched for a method for the generation of high number of DCs that are then cryoconserved.

[0124] It may be conceivable to freeze monocytes or alternatively, to freeze complete mature DCs, even after antigen loading. FIG. 3 shows viability and maturation markers of the different matured DCs after freezing, storage within the gas phase of liquid nitrogen and the thawing procedure. Viability of over 80% is acceptable, which was obtained by DCs matured with Jonuleit and our new cocktails (FIG. 3A), but not by DCs matured with Kalinski and cocktail 5 (both containing poly I:C). DC markers CD83 and CCR7 were expressed at high levels, and only very low numbers of CD14 cells were detected in all thawed DC populations (FIG. 3B and C).

Allostimulatory capacity of DCs

[0125] To test functional capacities of DCs, we used a mixed lymphocyte reaction with DCs as stimulatory cells against allogenic T cells. To control vital abilities of T cells, we tested induction of proliferation against a maximal number of different MHC molecules (third party=5 mixed MNC donors), a mitogenic stimuli (PHA) and the T cell stimulatory cytokine IL-2. FIG. 4 shows these controls for the autologeous T cells (4A) and a exemplary allogenic T cells responder (4B). FIG. 4C shows irradiated DCs without any responder cells and verified that the assay only determined proliferation by responder cells. FIG. 4D indicates low autologeous T cell induction in comparison to one allogenic responder (FIG. 4E).

[0126] In FIG. 4F, we summarized proliferation of 3 independent allogenic responder T cells after co-incubation with different matured DCs in comparison to autologeous T cells of the DC donor. As expected from the MHC differences between the 3 respective responder T cells and DCs we see a high stimulatory capacity. Again, DCs matured with Kalinski cocktail failed to show stimulation levels comparable to the other matured DCs, which may be due to lower viability and a higher percentage of dying cells during the assay procedure.

IL-12p70 and IL-10 Release of DCs

[0127] During differentiation and maturation processes, DCs secrete cytokines into their culture supernatant. FIG. 5A shows secretion of biologically active IL-12p70 (filled bars) and IL-10 (empty bars) into primary DC cultures. DCs matured with Jonuleit cocktail do not secrete detectable amounts of IL-12p70, whereas Kalinski cocktail as well as our new cocktails induced IL-12p70 in ng-ranges per ml. These results indicate a composition's capability to induce cytokine production in DCs. In FIG. 5B, it is visible that after mimicking encounter of DCs with potential T cells within lymph nodes via CD40 ligand it is still possible to re-induce IL-12p70 from DCs matured in the Kalinski cocktail and to a lesser degree also using our new cocktails.

[0128] IL-10, as a potential Th2 cytokine, counter-regulates Thi polarization. To take this effect into account, we calculated the values of both of these important regulatory cytokines as having theoretically equal biological potential and determined a quotient of IL-12p70/IL-10. FIG. 5C represents as the results showing that Kalinski cocktail is superior to Jonuleit, as in the literature described, but our new cocktails also revealed positive quotients for IL-12 re-induction after 24h of CD40 ligation and thereby secrete more IL-12p70 than DCs matured with Jonuleit cocktail.

[0129] To summarize the results we disclose herein a new combination of substances, including a TLR7/8 ligand and other cytokines and supplements, which is capable to induce fully maturation of DCs and to induce Thi regulatory capacities in these cells. In comparison to the DC maturation with Jonuleit cocktail, our cocktails also showed high cell viabilities after harvesting and freezing, high maturation levels by expression of CD83, co stimulatory molecules and migratory potential by CCR7 expression as well as maturation stability. In contrast to Jonuleit cocktail, our cocktail is able to induce IL-12p70 secretion within primary culture as well as after mimicking T cell interaction by CD40 ligation. Mature DCs generated with our new cocktail combine the best characteristics of Jonuleit cocktail in addition to gaining Th1-inducing capacities via IL-12p70. In contrast to the DCs obtained after maturation with the Kalinski cocktail, our new cocktail results in DCs with IL-12p70 secretion without the negative impact of loss of cell numbers and poor caused by extended cell death processes.

[0130] Our procedures to generate human dendritic cells are compliant with the regulations of good manufactory practice (GMP) and therefore are useful for clinical application to generate vaccines, which could promote Thi polarization of effector T cells against tumour antigenic structures.

Expression of Protein Following RNA-Transfer into DCs by Electroporation

[0131] Several sources of antigens have been considered for use in DC-based tumor vaccines. RNA is an attractive candidate to provide whole proteins to DCs for processing and presentation, thereby bypassing the need to know specific MHC-binding peptides. To test the capacity of DCs to express protein after loading with in vitro transcribed RNA, we analyzed EGFP expression by flow cytometry after transfer of corresponding RNA. DC3 cells were not included because cocktail 3 was identical to cocktail 4, except for lower amounts of IFN and PGE2 (see e.g. legend to FIG. 1). EGFP expression was found previously to peak 12-24 h following RNA transfection into DC1 cells and expression was stable for 48 h (Javorovic, M., Pohla, H., Frankenberger, B., Wolfel, T. and Schendel, D. J. (2005). RNA transfer by electroporation into mature dendritic cells leading to reactivation of effector-memory cytotoxic T lymphocytes: a quantitative analysis. Mol Ther. 12: 734-743), therefore, percentages of EGFP-positive cells and mean fluorescence intensities (MFI) were measured 24 and 48 h after electroporation. DC1 and DC4 cells expressed EGFP whereas DC2 cells did not express EGFP and DC5 cells expressed no (Fig.6) or only very low levels of EGFP (data not shown). This same pattern was seen at 48 h (data not shown). The TLR3 ligand, poly (I:C), was present in Kalinski and cocktail 5 (DC2 and DCS cells, respectively) but was missing in Jonuleit (DC1 cells) and cocktail 4 (DC4 cells). Kalinski cocktail also contained IFN, which was not present in any other cocktail. Interestingly, we found elsewhere that DCs matured only with IFN also failed to express EGFP protein following RNA transfer (Frankenberger, B., et at. (2005). Cell-based vaccines for metastatic renal cell carcinoma: genetically-engineered tumor cells and monocyte-derived dendritic cells. World J Urol. 3:166-174).

[0132] Induction of IFN Secretion by T Cells with Peptide-Pulsed DCs

[0133] Because DCS cells could not be loaded with RNA, their capacity to present peptides was tested as an alternative (FIG. 7). Autologous lymphocytes of ELUTRA fraction 3 of an HLA-A*0201-positive donor were stimulated for 7 days with peptide-pulsed DCs matured in cocktails 3-5 in comparison to Jonuleit cocktail. Cells were then restimulated for 24 h with autologous monocytes plus CEF peptides and analyzed in an IFN-ELISPOT assay. Peptide-specific responses were found with all the DC populations, demonstrating that DC5 cells could present peptides for T cell activation.

[0134] Therefore, we found that the presence of poly (I:C) in maturation cocktails prevented DCs from being able to express protein after loading with exogenous RNA, presumably through TLR3 activation of mechanisms to protect cells from foreign RNA (Kato, H. et at. (2006). Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441: 101-105). Thus, DCs matured in Kalinski medium or cocktail 5 can not be used for RNA-based vaccines, although both are suitable for use with peptides, as shown here for cocktail 5 and published previously for Kalinski cocktail (Mailliard, R. B. et at. (2004). alpha-type-1 polarized dendritic cells: a novel immunization tool with optimized CTL-inducing activity. Cancer Res. 64: 5934-5937). In contrast, cocktails 3 and 4 would be well suited for generating IL-12p70-producing DCs using either peptides or RNA as sources of tumor-associated antigens for cancer vaccine development.

Interferon-Gamma Response of Autologous Lymphocytes Stimulated with Mature DCs.

[0135] Lymphocytes from an HLA-A*0201 donor were stimulated with autologous DCs matured using the Jonuleit cocktail, the Kalinski cocktail and the three new cocktails described in the patent.

[0136] Peripheral blood lymphocytes (PBLs) were separated by elutriation and contained in fraction 3, in which 54.8% of the cells were CD3-positive T lymphocytes and 17.7% CD56 positive cells, which is characteristic for natural killer (NK) cells. The elutriation fraction 3 PBLs were directly incubated with the different DC populations matured using the different cocktails and their interferon-gamma secretion (IFN-gamma) was measured after 24 h in a standard ELISPOT assay.

[0137] For ELISPOT analysis, PBLs of ELUTRA fraction 3 were plated in 50 l per well in triplicates on antibody precoated PVDF plates (Mabtech AB, Nacka, Sweden), following incubation of the plates for 2 h at 37 C. in RPMI 1640 culture medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, penicillin/streptomycin (100 U/ml) and 10% human AB serum (BioWhittaker, Verviers, Belgium) to block unspecific binding. The capture antibody was the IFN-specific clone 1-D1K (Mabtech). The DC populations were carefully added to the wells. For background evaluation, DCs and lymphocytes were plated alone. The total culture volume was 150 .sub.11.1 and the plates were incubated in a 37 C. humidified incubator with 5% CO.sub.2 for 24 h. After removal of the cells and extensive washing with PB S/0.5% Tween20, incubation with the biotinylated detection antibody, clone 7-B6-1 (Mabtech) and the development of the spots were performed as described previously (1,2), with the exception that streptavidin-alkaline phosphatase and a ready-to-use BCIP/NBT-plus substrate solution were used. Spots were counted using the AID reader system ELRO3 with the software version 3.2.3 (AID Autoimmun Diagnostika GmbH, Strassberg, Germany).

[0138] For activation, lymphocytes from ELUTRA fraction 3 were plated at 110.sup.6 cells/well with 110.sup.5 viral peptide-loaded DCs in 24-well plates, in RPMI 1640 medium with 10% human serum; 30 IU/ml IL-2 was added at d3 and lymphocytes harvested at d7. HLA-A*0201-binding peptides included: CMVpp65.sub.495-503 (NLVPMVATV), EBV-BMLF1.sub.1280-288 (GLCTLVAML), influenza M1 protein.sub.58-66 (GILGFVFTL) or the CEF pool (PANATecs GmbH, Tuebingen, Germany) containing two additional peptides, EBV-LMP-2.sub.426-434 (CLGGLLTMV) and influenza RNA polymerase PA.sub.46-54(FMYSDFHFI). In vitro activated T cells and autologous monocytes with or without CEF peptides were incubated in RPMI 1640 medium containing 2 mM L-glutamine, 1 mM sodium pyruvate, penicillin/streptomycin (100 U/ml), 10% human AB serum (BioWhittaker, Verviers, Belgium) and 20 IU/ml IL-2 at 37 C. with 5% CO.sub.2 for 24 h. IFN-ELISPOT analysis was performed as described (Becker, C., et. at. (2001). Adoptive tumor therapy with T lymphocytes enriched through an IFN capture assay. Nat Med. 7: 1159-1162; Pohla, H., et at. (2000). Allogeneic vaccination for renal cell carcinoma: Development and monitoring. Bone Marrow Transplant. 25: 83-87), with the exception that antibody precoated PVDF plates (Mabtech AB, Nacka, Sweden) and streptavidin-alkaline phosphatase and a ready-to-use BCIP/NBT-plus substrate solution (Mabtech) were used for detection. Spots were counted using the AID reader system ELRO3 with 3.2.3 software (AID Autoimmun Diagnostika GmbH, Strassberg, Germany).

[0139] The dominant interferon-gamma producing cells detected at this early time point (see FIG. 8) are activated NK cells, in contrast to the experiments above, where the responses represent those of peptide-specific T cells stimulated by the DCs during the 7 d activation period (see FIG. 7). This analysis demonstrated that activation of NK cells was about three-fold enhanced using DCs matured in cocktail DC5, compared to cocktails DC1, DC3 and DC4.

[0140] Combined, these studies show that the DCs, and particularly those in cocktail 5 are able to activate natural killer cells (FIG. 8) as well as effectively restimulate peptide-specific effector T cells recognizing epitopes derived from cytomegalovirus, Epstein-Barr virus and influenza virus.

3. Summary of the Example

[0141] Dendritic cell (DC)-based vaccines often utilize monocyte-derived DCs matured with a cytokine cocktail (Jonuleit) of IL-1, TNF, IL-6 and prostaglandin E2 (PG). To obtain DCs that direct T cells to Th1-responses, we sought cocktails yielding DCs that produce biologically active IL-12p70. After elutriation of apheresis products by ELUTRA, we cultured enriched monocytes with GM-CSF and IL-4 for 6 days in GMP-conform medium with human serum. Immature DCs were matured for 24 h with various cocktails, containing TLR7/8 ligands with or without poly I:C and interferon , PG, IL-1 and TNF. Matured DCs expressed >80% CD83, CD86, CD80 and HLA-DR, CD40, >60% CD209 (data not shown), <2% CD14, and >60% lymph node homing chemokine receptor CCR7. DCs retained full maturity and expressed typical surface markers after cryopreservation and after washing out cytokines and reculture for 44 h.

[0142] IL-12p70 and IL-10 were present in supernatants of DCs matured with cocktails containing TLR7/8 ligands. A cocktail of IFN-, IL-1, TNF, PG and the TLR7/8 ligand R848 yielded DCs that secreted IL-12p70 after harvest and 24h coculture with CD40L-transfected fibroblasts, mimicking encounter with T cells in lymph nodes. We calculated the relative amounts of IL-12p70 versus IL-10 and found that our DCs revealed only a slightly lower quotient of IL-12 to IL-10 as reported by Kalinski (IL-1, TNF, IFN, IFN, poly I:C).

[0143] Functionality of DCs matured with our new cocktails was tested by mixed lymphocyte culture and ELISpot assays. Our DCs induced alloresponses and stimulated T cells specific for viral antigens comparable to DCs generated by Jonuleit cocktail (data not shown).

[0144] In summary, this new cocktail for DC maturation combines characteristics of good harvesting, reasonable recoveries, stability of maturation markers and Th1-inducing capacity with GMP-procedures required for high quality DC vaccines.