MX2008007152A - Method for generating dendritic cells employing decreased temperature - Google Patents

Abstract

The invention relates in certain embodiments to a method for generating dendritic cells by employing temperatures below 37ºC during the development of progenitor cells and immature dendritic cells. In some embodiments the invention relates to populations of dendritic cells and its use.

Description

METHOD OF GENERATION OF DENDRITIC CELLS USING A REDUCED TEMPERATURE FIELD OF THE INVENTION This invention relates to methods and means useful for inducing immune responses against malignancies and infectious diseases. More particularly, the invention pertains to improved methods for the generation of antigen presenting cells. BACKGROUND OF THE INVENTION Immune therapies based on dendritic cells that exploit the natural mechanisms of the presentation of antigens represent the most promising non-toxic method of cancer treatment. They can be used as a single treatment, or as an adjuvant for other types of therapies such as for example surgery, irradiation and chemotherapy. The strategy is based on the manipulation and ex vivo reintroduction of cellular products to avoid immune competencies for the purpose of inducing specific immune responses to the tumor. Thus, the ultimate goal of such immune therapies based on dendritic cells is the induction of tumor-specific effector cells in vivo and recent advances have focused on CD8 + cytotoxic T lymphocytes (CTL) capable of recognizing and killing the cells of the tumor. tumor. In addition, the treatment of infectious diseases such Ref.193677 for example, HIV can benefit from vaccination strategies based on dendritic cells. Presentation of antigens The induction of tumor-specific immune responses requires the coupling of antigen-presenting, professional (APC) cells expressing Molecules of the Major Histocompatibility Complex (MHC) as well as the costimulatory membranes secreted and bound to the membrane. In addition, such APC must be capable of absorbing, processing and presenting antigens in association with MHC molecules. Dendritic cells (DCs) are the professional APCs of the immune system with the ability to activate both natural and memory T cells. The stages that lead to the maturation of DC are associated with certain properties of the cell. Immature DCs are particularly good at absorbing extracellular antigens by phagocytosis or pinocytosis and processing antigens to peptides in the endocytotic compartment such as endosomes and phagosomes. Here, the peptides are linked to MHC class II molecules. The immature DCs also have the unique ability to load the peptides from the exogenous proteins to the MHC class II route of presentation, a process called cross-presentation.

The ability to efficiently stimulate an immune response by the activation of CD4 + type 1 helper cells (Thl cells) and CD8 + cytotoxic T cells (CTL) depends crucially on the mature DC. Only a fully mature DC equipped with a panel of membrane-bound accessory and costimulatory molecules such as for example CD40, CD80, CD83, CD86 and MHC class II can efficiently induce the proliferation and differentiation of specific T-lymphocytes for the antigen1. A significant role of the co-stimulatory activity of DC is provided by the secreted cytokines, in particular IL-12p70. Its role in the activation of T cells and their polarization to a Thl type response was clearly demonstrated by Heufler et al (1996) 1. In addition, a good correlation between the presence of mature DC expressing IL-12 in the tumor and patient survival was reported by Inoue et al. (2005). Mature DC for vaccination purposes should produce limited amounts of the inhibitory cytosine IL-10 of Thl cells. CCR7 is the receptor for the CCL19 and CCL21 chemokines that are produced by the stromal cells in the lymph nodes. DC that expresses sufficient levels of activated CCR7 migrates to the lymph node in response to CCL19 or CCL212. Here they find the T lymphocytes and can initiate an immune response. Protocols for the generation of mature DC Many protocols for the generation of mature DC have already been described. The "standard" protocol most frequently used today for the induction of DC employs a maturation cocktail consisting of IL-lbeta, IL-6, TNF-alpha and prostaglandin E2. Despite the migratory activity due to CCR7 and the immunostimulatory activity in vivo. The DC matured by this cocktail generates DC with a reduced capacity to produce IL12p703. A second group of DC maturation protocols comprises polyinosinic acid: polycytidyl, poly (I: C). It is usually used in combination with cytokines such as TNF alpha, IL-lbeta, IFN-gamma and IFN-alpha. The DCs generated by this method produce IL-12p70, but they usually express low levels of CCR7. Low levels of CCR7 expression characterized by DC obtained in the presence of poly- (I: C) restrict their migration in vivo to lymph nodes. Recently, a published patent application US2005 / 0003533A1 described a method for the maturation of dendritic cells expressing CCR7 subsequent to stimulation with CD40L that could be induced to produce IL-12p70. Therefore there is still a requirement not satisfied with the development of standardized methods to generate mature dendritic cells that express high levels of activated CCR7 and that also produce a sufficient amount of IL12p70. Furthermore, despite the efforts of many researchers, dendritic cell-based vaccines for use in cancer therapy have generally provided immune responses with modest clinical efficacy. These vaccines have been produced mainly by ex vivo manipulation and the loading of the autologous DC antigen. Increasing demands regarding patient safety require a high level of reproducibility and adaptability with regulatory tissues. Accordingly, there is a strong need for methods that generate appropriately equipped DCs that efficiently induce immune responses and that provide in particular improved clinical responses. In addition, DC generated ex vivo could also be implemented as a therapeutic vaccine in the treatment of some chronic infectious diseases such as HIV and hepatitis B and C, where the traditional vaccination method is not working efficiently. The results of the preclinical studies and the first clinical studies4"5 indicate that DC-based immunotherapy could be a promising strategy for the treatment of patients with chronic infections due to HIV-1 and hepatitis B and C. As with immunotherapy against cancer, the efficient clinical response against these intracellular infectious agents is associated with the induction of the helper response of Thl required for the development of effector cells. CD8 + 5 Therefore, it can be expected that the dendritic cells generated ex vivo should have the same characteristics for the treatment of both chronic infectious diseases and cancer. BRIEF DESCRIPTION OF THE INVENTION In a first aspect, the present invention relates to a method of generating dendritic cells by using temperatures below 37 ° C during the development of progenitor cells and immature dendritic cells. In a second aspect, the invention relates to a population of dendritic cells, wherein the cells are generated by the method for the generation of dendritic cells by the use of temperatures below 37 ° C during the development of progenitor cells and cells. immature dendritic cells. In a third aspect, the invention relates to a pharmaceutical composition comprising a population of dendritic cells wherein the cells are generated by the method for the generation of dendritic cells using temperatures below 37 ° C during the development of progenitor cells and immature dendritic cells. In a fourth aspect, the invention relates to the use of the population of cells, wherein the cells are generated by the method for the generation of the dendritic cells by the use of temperatures below 37 ° C during the development of the cells progenitors and immature dendritic cells, for the stimulation and / or expansion of T cells. In a fifth aspect, the invention relates to the use of the population of cells, wherein the cells are generated by the method for the generation of cells dendrites by the use of temperatures below 37 ° C during the development of progenitor cells and immature dendritic cells, by the induction of an immune response in a subject. In a sixth aspect, the invention relates to the use of the population of cells, wherein the cells are generated by the method for the generation of the dendritic cells by the use of temperatures below 37 ° C during the development of the progenitor cells and immature dendritic cells, for the manufacture of a medicament for the treatment or prevention of cancer or infectious diseases.

BRIEF DESCRIPTION OF THE FIGURES The invention is explained in detail later, with reference to the figures, in which: Figure 1 illustrates the effect of temperature on the surface molecules accessory and co-stimulators of DC. Figure 2A, Figure 2B and Figure 2C illustrate the effect of temperature on the amount of IL-10 produced by DC during the initial days of culture (Figure 3A) and produced by immature DC (Figure 2B) and DC mature (figure 3C). Figure 3A, Figure 3B and Figure 3C, illustrate the effect of temperatures of 31 ° C, 34 ° C and 37 ° C on the production of IL-12p70 by immature dendritic cells (Figure 3A) and mature dendritic cells (Figure 3B). Figures 3B and 3C) generated by the new method and a standard method. Figure 4 illustrates the effect of temperature reduction on the expression of CCR7. Figure 5A, and Figure 5B illustrate the immature and mature DC phenotype generated by the method according to the invention (Figure 5A) and compared to the "standard" method (Figure 5B). Figure 6 illustrates the phenotypic stability of mature DC over time. Figure 7 illustrates the phenotype and activity allo- stimulator (MRL) of DC on day 7 and day 10. Figures 8A and 8B illustrate the allo-stimulatory activity of DC generated by the method according to the invention and a "standard" method. Figure 9 illustrates the functional presentation of the CMV antigen as measured by IFN-α induction. (ELISPOT trial). DETAILED DESCRIPTION OF THE INVENTION The present invention is described in detail below. For the purposes of interpretation they will apply the following definitions and where appropriate, the terms used in the singular will also include the plural and vice versa. Definitions "Differentiation step" as used herein, means the stage at which the cells are allowed to differentiate in response to defined differentiation factors. "Maturation stage" as used herein, means the stage where the cells are allowed to mature in response to the presence of maturation factors. "Reduced temperature" or "decreased temperature" as used herein, means that the temperature is below 37 ° C. One method to generate dendritic cells is the Well-known met of J.H. Peters who was the first to describe the ability of monocytes to transform into DC-like cells in vitro, first spontaneously and subsequently in the presence of MG-CSF and IL-46. After the publications by Romani et al., (1994) 7 and Sallusto & Lanzavecchia (1994) 8 monocytes cultured in the presence of these two cytokines become widely used for the preparation of DC. The procedure begins with the isolation of peripheral blood monocytes and their culture in the presence of GM-CSF and IL-4 for 5-7 days. The cells obtained have immature DC properties characterized by low levels of co-stimulatory molecules and high endocytic activity. During the maturation induced by LPS, TNF-alpha or other maturation agents, cells significantly upregulate costimulatory and accessory molecules, such as for example CD40, CD80, CD83 and CD86, and down-regulate endocytic activity. In vitro tissue culture is carried out in general at 37 ° C. It is already known that Langerhans cells are functionally active at room temperature of the skin at 29-31 ° C, and few studies have documented the in vitro biological effect of reduced culture temperatures in cell systems such as example the cells of the ovaries of the Chinese hamster (CH) and the Porcine alveolar macrophages. In contrast to the work with Bassu et al (2003) Int. Immunol. 15 (9): 1053-61 investigating the effect of temperatures similar to those of fever on the activation and maturation of DC, the reduced temperatures only in a few cases have proven their effect on the growth of mammalian cells. Dexter et al. (1977) suggested the use of 33 ° C for the culture of hematopoietic stem cells9. Athanasas-Platsis et al. (1995) found that Langerhans cell marker expression on monocytes was up-regulated during a 24-hour culture at 34 ° C when compared to 37 ° C10. No person has the knowledge described by the inventors on how to generate mature or immature dendritic cells by the use of reduced temperatures. In one embodiment, the invention relates to a method for the generation of dendritic cells by the use of temperatures below 37 ° C during the development of progenitor cells and immature dendritic cells. IL-10 is a negative regulator of DC development and is produced during the activation of a monocyte cell line in the presence of G -CSF11. Kirkley et al. (2003) reported that the production of IL-10 by a macrophage cell line stimulated with LPS was reduced significantly in response to a reduction in incubation temperature from 37 ° C to 31 ° C12. The reduced temperature comprised in the method of the present invention can thus provide improved conditions for the generation of DC by means of, for example, a low IL-10 concentration. The effect of culturing monocytes in the presence of GM-CSF and IL-4 at different temperatures (31 ° C, 34 ° C and 37 ° C) on the CDla expression level of immature DC, an extremely sensitive molecule, Inhibitory effect of IL-10 has been tested. It was found that the DC generated at low temperatures had higher levels of its expression. All additional experiments were performed at 34 ° C. The observation of the following principles was that the levels of IL-10 detected in the supernatants of the cultures were actually significantly lower during the culture at a lower temperature. In one embodiment, the invention relates to a method, wherein the dendritic cells generated are mature dendritic cells. In one embodiment, the invention relates to a method, wherein the development of the progenitor cells and the immature dendritic cells comprise the differentiation of the cells. In one embodiment, the invention relates to a method, where the temperature is below 37 ° C during differentiation. In one embodiment, the invention relates to a method, wherein the temperature is from 31 ° C to 37 ° C. The temperature can be any of the temperatures of 31 ° C, 32 ° C, 33 ° C, 34 ° C, 35 ° C, or 36 ° C. In one embodiment, the invention relates to a method, wherein the temperature is 34 ° C. In one embodiment, the invention relates to a method, wherein the progenitor cells are autologous progenitor cells. In one embodiment, the invention relates to a method, wherein the progenitor cells are selected from the progenitor cells or myeloid stem cells. In one embodiment, the invention relates to a method, wherein the myeloid progenitor cells are monocytes. In another embodiment, the invention relates to a population of dendritic cells that are generated by the method according to the invention. In one embodiment, the invention relates to a population of dendritic cells, wherein the cells express CCR7 and / or IL-12p70. In one embodiment, the invention relates to a population of dendritic cells, wherein the cells express CDla, CD14low, CD83, CD86 and IL-10low. In one embodiment, the invention relates to a population of dendritic cells, further comprising at least one antigen presented in association with an MHC molecule on the surface of the cell. In one embodiment, the invention relates to a population of dendritic cells, wherein at least one antigen is a neoplastic antigen. In one embodiment, the invention relates to a population of dendritic cells, wherein the neoplastic antigen is selected from the group comprising; testicular antigen / cancer, lineage-specific differentiation antigen, tumor overexpressed antigen, mutant or aberrantly expressed antigen, and viral antigen. In a further embodiment, the invention relates to the use of the dendritic cell population as defined above, for the stimulation and / or expansion of T cells. In one embodiment, the invention relates to the use of the dendritic cell population. for the stimulation or expansion of T cells, where the T cells are autologous T cells. In one embodiment, the invention relates to the use of the population of dendritic cells for the stimulation or expansion of T cells, where the use is an in-use. vitro In yet a further embodiment, the invention relates to the use of the dendritic cell population to induce an immune response in a subject. In still another embodiment, the invention relates to a pharmaceutical composition comprising a population of dendritic cells wherein the population is as defined above. In one embodiment, the invention relates to the use of the pharmaceutical composition as a medicament. In one embodiment, the invention relates to a pharmaceutical composition comprising a population of dendritic cells which also comprises conventional adjuvants and excipients. In an alternative embodiment, the invention relates to the use of dendritic cells for the manufacture of a medicament for the treatment or prevention of cancer or infectious diseases. In one embodiment, the invention relates to the use of the dendritic cell population for the manufacture of a medicament for the treatment or prevention of cancer or infectious diseases, wherein the cancer is selected from the group comprising: melanoma, breast cancer, colon cancer and lung cancer, or it could be any kind of cancer.

In one embodiment, the invention relates to the use of the dendritic cell population for the manufacture of a medicament for the treatment or prevention of cancer or infectious diseases, wherein the infectious diseases are selected from the group comprising: HIV or hepatitis or other chronic infectious diseases. EXAMPLES This invention is now illustrated by the following examples which are not intended to be limiting in any way. Example 1: Generation of the dendritic cells using a reduced temperature The dendritic cells were typically generated from the buffy coat obtained from the blood bank. 60 ml of the buffy coat were diluted with 60 ml of Mg-free, Ca free Dulbecco's phosphate buffered saline solution (DPBS, product No. BE17-512F, Cambrex, Belgium), and applied to four 50 ml tubes that each contains 15 ml of Lymphoprep (product No. 1053980, AXIS-SHIELD PoC AS, Norway). After centrifugation (460 mg, 30 minutes, 20 ° C), 10-20 ml of the upper plasma layer were transferred to separate tubes. It is estimated that this is approximately 40% plasma (diluted plasma). The final plasma preparation includes the addition of heparin (25 IU / ml) and centrifugation (1500 g, 15 minutes, 4 ° C). The mononuclear cells were collected from the interface, diluted 2 times with DPBS containing EDTA and washed by 4-5 centrifugations, the first at 250 g, the second at 200 g and the following at 150 g, all centrifugations at 4 ° C, 12 minutes Before the last centrifugation, the cells were counted using a Coulter Counter (Beckman Coulter, model Z2), and the amount of monocytes was estimated as the number of cells with an average size of approximately 9 μ? P). The cells were stored at -80 ° C (in plasma diluted with 10% DMSO, 107 monocytes per vial) or used immediately in the experiments. The cells were resuspended in the adsorption medium (RPMI 1640 (Cambrex) supplemented with 2 mM L-glutamine 2% plasma) at a concentration of 2 x 10 6 monocytes / ml. 5 ml of the cell suspension are placed in Falcon flasks not treated with TC T25. After one hour of adsorption at 37 ° C, the non-adherent cells were removed, the adherent cells were rinsed twice with warm RPMI 1640, and 7 ml of the culture medium (RPMI 1640 supplemented with 2 mM L-glutamine and 1 % of the plasma) were added to each container. The containers were placed at different temperatures: 31 ° C, 34 ° C and 37 ° C in separate C02 incubators. The differentiation factors of GM-CSF and IL-4 at the final concentrations of 100 ng / ml and 50 ng / ml respectively, they were added on days 1, 3 and 5. TNF-alpha at a final concentration of 10 ng / ml was added on day 6 to induce maturation and the temperature was elevated to 37 ° C for at least 24 hours of incubation. On day 7, the cells were collected and their phenotype was determined by FACS analysis. The cells were stained using the direct conjugated antibodies of CDla-phycoerythrin (PE), fluorescein isothiocyanate-CD14 (FITC), DC83-PE, CD86-PE, HLA-DR, -P, -Q, -FITC (all of Pharmingen, Beckton Dickinson, Brondby, Denmark) and CCR7-FITC (R &; D Systems Europe, Abington, UK). Appropriate isotype controls were used. Samples were analyzed using a FACSCalibur flow cytometer (Beckton Dickinson) and the CELLQuest software (Beckton Dickinson). The result of the representative experiments is shown in Figure 1. More cells cultured at reduced temperatures express CDla when compared to cells cultured at 37 ° C, while fewer CD83 and CD86 positive cells were observed for cell populations grown at lower temperatures. The average fluorescence index (NFI) for CDla was twice as high during cultivation at 31 ° C and 34 ° C compared to 37 ° C. The degree of maturation as it was judged by the percentage of CD83 and CD86 expressing DC was lower than 34-34 ° C. This reflects either a lower sensitivity to the maturation factors of the cells cultured at reduced temperature because the ripening process requires a temperature of 37 ° C. Example 2: Effect of reduced temperature on the production of IL-10 The production of IL-10, which is a negative regulator of DC, was investigated during the differentiation of monocytes in dendritic cells. Its concentration in the supernatant of the culture taken on days 1, 3 and 5 was measured. The production of IL-10 was measured by a sandwich ELISA assay that included a capture antibody (Ab), detection of biotinylated Ab, standard or sample, and HRP-streptavidin using the "Ready-Set-Go" kit "of eBioscience essentially in accordance with the recommendations of the manufacturers with some modifications. After overnight agglutination of the capture Ab to the Nunc maxisorp 96-well plates and washing, the blocking step was extended at least 3 hours at ta. A standard curve was generated by seven serial dilutions of the standard, starting with 200 pg / ml of IL-10. Standards and samples were incubated at RT for 2 hours followed by incubation at 4 ° C overnight. The following stages were carried out according to the manufacturer's protocol. The Solution of the tetramethylbenzidine substrate of the same kit was used in the enzymatic reaction of HRP, and after completion of the reaction, the optical density was measured with a correction of the wavelength as the difference between the readings at 490 and 620 mm . The results of one such experiment were presented in Figure 2A. The spontaneous production of IL-10 by monocytes was low during the first day, and was upregulated significantly after the addition of GM-CSF and IL-4 on day 1. Cells cultured at 34 ° C until the Day 5 generally produced significantly lower amounts of IL-10 compared to cells cultured at 37 ° C. The test of several DC cultures on day 5 showed a similar configuration (Figure 2B). The reduced production of IL-10 at 34 ° C when compared to 37 ° C, continued even after washing the cells on day 5, placing them at 37 ° C, adding the ripening agent on day 6 and collecting the supernatants on day 8 (figure 2C). These results indicate that cells cultured at temperatures below 37 ° C acquire a stable phenotype of low IL-10 production. Example 3: The effect of reduced temperature on the production of IL-12p70 The effect of temperature on the production of IL-12p70 has also been investigated. The production of IL-12p70 was measured by ELISA of sandwich that included the capture, standard or sample Ab, the biotinylated detection Ab, and the HRP-estraptividin. The "Duocet Elisa Development System" kits for IL-12p70 (R &D Systems) were used essentially in accordance with the manufacturer's recommendations with some modifications. After overnight agglutination of the capture Ab to the Nunc maxisorp 96-well plates, the blocking step was extended at least 3 hours at RT. The standard curve was generated by seven serial dilutions of the standard, starting with 500 pg / ml of IL-12p70. Standards and samples were incubated at ta for 2 hours followed by incubation at 4 ° C overnight. The following stages were carried out according to the manufacturer's protocol. The hydrogen peroxide-tetramethylbenzidine mixture was used as a substrate solution for HRP, and after completion the optical density of the enzymatic reaction was measured with a correction of the wavelength as the difference between the readings at 490 and 620 nm. Table 1. Effect of the temperature during the first 5 days of the culture on the production of IL-12p70 during the maturation induced by an MCM mimicking substance As can be seen (table 1) the cells generated at 34 ° C produce significantly higher levels of IL-12p70. Example 4: Selection of plastic for tissue culture Two types of plastics have been compared for tissue culture. Polystyrene for growing something other than a fabric (PS) (product No. 353813, T25 BD-Bioscience, USA) and a Primary plastic ™ (Product No. 353813, T25 BD-Bioscience, USA). The experiment was established in a manner similar to the procedure described in Example 1, using pretreated plastic surfaces, for 15-45 min, with 2% autologous plasma as a source for the components such as, for example, extracellular fibrinogen-like components and fibronectin, in serum free AIM-V medium at 34 ° C until day 5, after which cultures were placed at 37 ° C. Maturing agents; TNF alpha, IL-1 beta, IL-6 and prostaglandin E2 were added on day 6, and cultures were collected on day 8. Progenitor cells depending on the growth condition have the option of developing into macrophages or DC. After a few days in culture, the cells destined for development in macrophages will form adherent cell cultures while the cells destined for development in DC will form cultures. cell phones fixed more loosely. Initially an equal number of cells were seeded and adhered to the different plastic for tissue culture. Inspection of DC cultures from day 6 by light microscopy revealed a significantly lower number of adherent cells on the Primary ™ plastic compared to cells grown on another type of plastic. In general, the crops that grew on the Primary ™ plastic also appeared to be more "clean", that is, with less residues, which reflect less the degree of cell death during maturation. The use of a different concentration of plasma for the pre-treatment of plastic was approved. No significant difference in the properties of DC was observed during the treatment of the Primary plastic ™ with 2%, 10%, 20% or 40% of the plasma (data not shown). However, it was noted that the amount of lymphocyte contamination was reduced with the increase in plasma concentration up to 10%. Therefore, the treatment step of the Primary plastic ™ with 10% plasma was included in the method described in experiment 1 in the subsequent experiments. In the following experiments the method of the invention has been compared with a "standard method" which is carried out as described below unless otherwise indicated.

The dendritic cells were typically generated from the buffy coat obtained from the blood bank. 60 ml of the buffy coat were diluted with 60 ml of the Mg-free and Ca free Dulbecco's phosphate-buffered saline solution (DPBS, product No. BE17-512F, Cambrex, Belgium), and applied to four tubes of 50 mi, each containing 15 ml of Lymphoprep (Product No. 1053980, AXIS-SHIELD Poc AS, Norway). After centrifugation (460 g, 30 min., 20 ° C), 10-20 ml of the upper plasma layer was transferred to the separate tubes. Mononuclear cells were collected from the interface, diluted twice with PBS EDTA without calcium and magnesium and washed for 3 centrifugations, the first at 250 g, the second at 175 g and the latter at 110 g, all centrifugations at 4 ° C 12 minutes Before the last centrifugation, the cells were counted using a Coulter Counter (Beckman Coulter, model Z2), and the amount of monocytes was estimated as the number of cells with an average size of approximately 9 μ ??). The cells were resuspended in the absorption medium (RPMI 1640 (Cambrex) supplemented with 2mM L-glutamine and 1% heat inactivated autologous plasma at a concentration of 2 x 108 monocytes / ml. Primary bottles ™ not treated with T25 After one hour of adsorption at 37 ° C, they were removed the non-adherent cells, and 5 ml of the culture medium (RPMI 1640 supplemented with 2mM L-glutamine and 1% plasma) was added to each container. On day 1 the medium was changed by the fresh medium. On day 3, 2 ml of the medium is added. On day 5 all the non-adherent cells were collected and placed in the bottles of T25 Primary ™ with the free medium. The flasks were placed at 37 ° C in a CO2 incubator. The differentiation factors GM-CSF and IL-4 at final concentrations of 100 ng / ml and 50 ng / ml respectively were added on days 1, 3 and 5. TNF-a or the cocktail of cytokines (IL-1, IL-6, TNF-a and PGE-2) were added on day 6 to induce maturation. On day 7, the cells were collected and their phenotype was determined by FACS analysis. Example 5: Production of IL-12p70 Figure 3 illustrates the measurement of IL-12p70 production over two days (days 7 and 8) which were able to show that the dendritic cells generated by the new method produce significantly higher amounts of IL-12p70 than dendritic cells generated by a standard method. Example 6: Expression of CCR7 To investigate the effect of temperature on the expression of CCR7, a maturation cocktail consisting of IL-lbeta, IL-6, TNF-alpha and prostaglandin E2 is used instead of using only TNF-alpha. The result of the experiments presented in figure 4 is the comparison of the different temperatures with the new method and a standard method. It can be seen that the expression of CCR7 is higher with the new method when compared with the standard method. The functionality of the expression of the CCR7 receptor by the dendritic cell generated by the new method was also tested in a standard migration assay (Chemotx Disposable Chemotaxis System (model 116-5) from Neuro Probé, Gaithersburg, MD, USA). Here, the migration of the dendritic cells towards the CCL 19 chemokines was observed with the DC generated by the new method (data not shown) that verify the expression of a functional CCR7 receptor. Example 7: Cell performance The new method described here also showed an increased yield of the cells compared to the standard method. In three different runs, a higher yield of the cells was found at all the temperatures tested (31 ° C, 34 ° C and 37 ° C) with the new method compared with the standard method, see table 2.

Table 2 Example 8: Label Variations from Batch to Batch of the DC Generated by the New Method In accordance with GMP requirements for the production of dendritic cells for medical purposes, there must be low variations from batch to batch in the properties of the dendritic cells . For this purpose, the preparation of the dendritic cells was carried out from the blood of 8 different donors during the period of three weeks, using the same lots of all the reagents used and 0.5% of autologous plasma as well as the AIM-V medium. For comparison, the production of DC using the "standard" method (37 ° C) was carried out. The experiments were carried out on the thawed PBMS. Table 3 summarizes the properties of the DC generated in these experiments. In contrast to the high variability of the DC properties generated by the "standard" method, a very low degree of variability in the properties of DC obtained by the new method was observed. Table 3. Different markers expressed in percentages of dendritic cells generated either by a standard method or by a new method.

S: Standard method; N: method according to the invention, ND: not determined, X: average value. Finally, figure 5A represents the phenotypes of the immature (day 5) and mature (day 8) dendritic cells generated by the new method. Here, the expression of CD80, the mannose receptor (MR) and the two markers of the Langerhans cells - CD207 (Langerin) and E-cadherin were also measured. As it could be observed, the cells generated according to the method of the invention are not Langerhans cells. Figure 5B depicts the phenotype of immature dendritic cells (day 5) and mature dendritic cells (day 8) generated by the new method and a standard method. Here, cells have been stained for the expression of standard DC markers. Immature cells (day 5) show a cleaner CDLA population. The mature population (day 8) is showing an expression of HLA D, CD83 and CD86 elevated and uniform in the cells generated by the new method when compared with the standard method. Also the expression of CCR7 is expressed more uniformly with the new method. Example 9. Stability of the dendritic cells generated by the new method After the injection in the organism the dendritic cells must migrate and reach the lymphatic node to stimulate the T cells. Therefore, it is very important that the DC maintain its phenotype for several years. days. A common way to perform the stability test is to collect the cells on day 8, wash out the cytokines and continue to grow the cells in the absence of stimulatory cytokines. This kind of experiments have been carried out by culturing the cells without the cytokines for two days. Figure 6 represents the results of the analysis of FACS of the DC collected on day 8 and after two additional days of culture. It seems clear that the expression of the measured parameters: CDla, CD14, CD83, HLA-D and CCR7 remain largely unchanged and thus the phenotype remains stable. In a similar experiment, without removing the cytokines by washing on day 8, the activity of both the phenotype and the allo-stimulatory activity of the dendritic cells is tested on day 7 or on day 10. Figures 7A and 7B show the phenotype activity and allo-stimulatory activity respectively. The results show that the allo-stimulatory effect is still higher after 10 days of the culture and the phenotypic profile on day 10 resembles the profile measured on day 7, verifying the high stability of the generated dendritic cells. Example 10. Allo-stimulation by the dendritic cells generated by the new method The allo-stimulatory capacities of the DC obtained by the "standard" method and the method according to the invention have been compared. Cells were cultured in RPMI 1640 medium with 5% human serum AB. Responding cells were mononuclear cells obtained from healthy donors by density separation of the peripheral blood buffy coat. Stimulating cells were irradiated mature dendritic cells obtained after a 2-day exposure to the maturation cytokine cocktail as described in example 4. The stimulator cells, 0.1 x 108 cells in 100 μ ?, were mixed with concentrated numbers of stimulator cells (in 100 μ! > ) as shown in Figures 8A and 8B and were cultured for 5 days in 96-well microtitre plates with U-bottom. 3H-thymidine was added (0.1 during the last 18 hours. Subsequently, the cells were collected by scintillation counting. The data is provided as the average CPM values of four duplicate crops. The ilcoxon test was used to estimate the differences between the two methods used for the generation of DC. As can be seen, the dendritic cells obtained by the method according to the invention have a 3-10 times higher allo-stimulatory activity. Example 11. Presentation of the antigen by the dendritic cells generated by the new method. In order to discern the DC potential to present the antigen to the T cells, an INFV ELISPOT assay was carried out with the T cells stimulated by DC naked or propelled with a CMV peptide. The INFY ELISPOT assay was chosen as the test that provides a clear result at a single cell level and that the T cells during the encounter with the antigen presented by APC, release the INFy. The CMV peptide used is restricted with respect to HLA-2 and the donor material is known to be positive for HLA-A2, and since 80% of the population has a CMV response this model of virus was chosen. Figure 9 shows the results of an ELISPOT assay showing that there is a strong response of the T cells stimulated with DC loaded with the CMV peptide indicating that these DCs are capable of presenting the antigen to the T cells. References 1. Heufler, C, Koch,, Stanzl, U., Topar, G., Wysocka, M., Trinchieri, G., Enk, A., Steinman, R., Romani, N., and Schuler, G. Interieukin- 12 is produced by dendritic cells and mediates T helper 1 development as well as interferon-gamma production by T helper 1 cells. Eur.J.lmmunol., 26: 659-668, 1996. 2. Scandella, E., Men, Y., Gillessen, S., Forster, R., and Groettrup, M. Prostaglandin E2 is a key factor for CCR7 surface expression and migration of monocyte-derived dendritic cells. Blood, 100: 1354-1361, 2002. 3. Jonuleit, H., Kuhn, U., Muller, G., Steinbrink, K., Paragnik, L, Schmitt, E., Knop, J "and Enk, AH Pro -inflammatory cytokines and prostaglandins induces maturation of potent immunostimulatory dendritic cells under fetal calf serum-free coriditions. Eur.J.lmmunol., 27: 3135-3142, 1997. 4. Chen,., Li, YG, Zhang, DZ, Wang, ZY, Zeng, WQ, Shi, XF, Guo, Y., Guo, SH, and Ren, H. Therapeutic effect of autologous dendritic cell vacclne on patients with chronic hepatitis B: a clinical study. World J.Gastroenterol., 11: 1806-1808, 2005. 5. Lu, W., Arraes, L. C, Ferreira, W. T., and Andrieu, J.. Therapeutic dendritic-cell vaccine for chronic HIV-1 nfectlon. NatMed., 10: 1359-1365, 2004. 6. Peters, JH, Xu, H., Ruppert, J., Ostermeier, D., Friedrichs, D., and Gieseler, RK Signals required for differentiating dendritic cells from human monocytes in vitro Adv.Exp.Med.Biol, 329: 275-280, 1993. 7. Romani, N "Gruner, S., Brang, D., Kampgen, E., Lenz, A., Trockenbacher, B., Konwalinka, G., Fritsch, P. O., Steinman, R.M., and Schuler, G. Proliferating dendritic cell progenitors in human blood. J.Exp.Med., 180: 83-93, 1994. 8. Sallusto, F. and Lanzavecchia, A. Efficient presentation of soluble antigen by culturad human dendritic cells is maintained by granulocyte / macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor a. J.Exp.Med., 179: 1109-1118, 1994. 9. Dexter, T.M., Alien, T.D., and Lajtha, L. G. Conditions controlling the proliferation of haemopoietic stem cells in vitro. J. Cell Physiol, 91: 335-344, 977. 10. Athanasas-Platsis, S., Savage, N.W., Winning, T.A., and Walsh, L. J. Induction of the CD1a Langerhans cell marker on human monocytes. Arch.Oral Biol., 40: 157-160, 1995. 11. Lehmann,. H. Recombinant human granulocyte-macrophage colony-stimulatlng factor triggers interleukin-10 expression in the monocytic cell line U937. ol.lmmunol., 35: 479-485, 1998. 12. Kirkley, J.E., Thompson, B.J., and Coon, J.S. Temperature alters lipopolysaccharide-induced cytokine secretion by RAW 264.7 cells. Scand.J.lmmunol., 58: 51-58, 2003.

It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (20)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A method of generating mature dendritic cells characterized by using temperatures below 37 ° C during the development of progenitor cells and immature dendritic cells.
2. 2. The method according to claim 1, characterized in that the development of the progenitor cells and the immature dendritic cells comprises the differentiation of the cells.
3. 3. The method according to claim 2, characterized in that the temperature is below 37 ° C during the differentiation.
4. 4. The method according to claims 1-3, characterized in that the temperature is from 31 ° C to 37 ° C.
5. 5. The method according to any of claims 1-3, characterized in that the temperature is 34 ° C.
6. 6. The method according to any of claims 1-5, characterized in that the progenitor cells are autologous progenitor cells.
7. 7. The method of compliance with any of the claims 1-5, characterized in that the progenitor cells are selected from the myeloid progenitor cells or stem cells.
8. 8. The method according to claim 7, characterized in that the myeloid progenitor cells are monocytes.
9. 9. A population of dendritic cells generated by the method according to any of claims 1-8, characterized in that the cells express CDla, CD14baj0, CD83, CD86, IL-10ba] O, CCR7 and IL-12p70 and wherein the cells acquire a stable phenotype of low IL-10 production.
10. 10. The population of cells according to claim 9, characterized in that it also comprises at least one antigen presented in association with an MHC molecule on the cell surface.
11. 11. The population of cells according to claim 10, characterized in that at least one antigen is a neoplastic antigen.
12. The cell population according to claim 11, characterized in that the neoplastic antigen is selected from the group containing: antigen of the testes / cancer, lineage-specific differentiation antigen, neoplastic overexpressed antigen, aberrantly expressed or mutated antigen, and viral antigen
13. 13. The use of the cell population according to any of claims 9-12 for the stimulation and / or expansion of T cells.
14. The use according to claim 13, wherein the T cells are autologous T cells.
15. 15. The use according to claims 13-14, wherein the use is an in vitro use.
16. 16. The use of the cell population according to any of claims 9-12 for the induction of the immune response in a subject.
17. 17. A pharmaceutical composition, characterized in that it comprises a population of dendritic cells according to any of claims 9-12.
18. 18. The use of the cell population according to any of claims 9-12 for the manufacture of a medicament for the treatment or prevention of cancer or infectious diseases.
19. 19. The use according to claim 18, wherein the cancer is selected from the group consisting of: melanoma, breast cancer, colon cancer, and lung cancer.
20. 20. The use according to claim 18, wherein the infectious diseases are selected from the group comprising: HIV and hepatitis.