WO2005069791A2

WO2005069791A2 - Compositions and methods for enhancing the th1 response in connection with dendritic cell vaccines

Info

Publication number: WO2005069791A2
Application number: PCT/US2004/043116
Authority: WO
Inventors: John S. Yu; Gentao Liu
Original assignee: Cedars-Sinai Medical Center
Priority date: 2004-01-08
Filing date: 2004-12-21
Publication date: 2005-08-04
Also published as: WO2005069791A3

Abstract

The present invention relates to dendritic cells and their use in a vaccine for treatment of disease conditions. Dendritic cells are transfected with specific IL-10 small-interference RNA (siRNA), which results in suppression of IL-10 gene expression without inducing DC apoptosis or blocking DC maturation. Inhibition of IL-10 by siRNA accompanies increased CD40 expression and IL-12 production after maturation, which has the result of siRNA transfected dendritic cells enhancing allogenic T cell proliferation and Th1 response.

Description

COMPOSITIONS AND METHODS FOR ENHANCING THE TH1 RESPONSE IN CONNECTION WITH DENDRITIC CELL VACCINES

FIELD OF THE INVENTION The invention relates to the use of siRNA in the production of dendritic cell vaccines useful in treating and preventing disease conditions, such as cancer.

BACKGROUND OF THE INVENTION Cancer is the second leading cause of death in the United States, and over one million people are diagnosed with cancer each year. Approximately one out of every two American men and one out of every three American women will have some type of cancer during their lifetime. However, while substantial progress has been made in identifying some of the likely environmental and hereditary causes of cancer, the morbidity rates associated with this disease indicate a need for substantial improvement in the therapeutic interventions for cancer and related diseases and disorders. A promising means of eliciting tumor specific antigen presentation to the immune system is the use of dendritic cell (DC)-based vaccines. DCs abundantly express stimulatory molecules that are essential for activation of naϊve T cells, and they also possess the ability to process and present antigenic peptides in conjunction with cell-surface major histocompatability complex (MHC) to initiate cytolytic T cell function in vitro and in vivo (Banchereau, J. and Steinman, R. M., "Dendritic cells and the control of immunity," Nature, Vol. 398, pp. 245-252 (1998)). The DC-based immunotherapy strategy appears promising as an approach for inducing antitumor immune responses and increasing survival in patients with cancer. Furthermore, DCs are thought to be the most potent of all antigen-presenting cells (APCs) (Steinman, R. M., "The dendritic cell system and its role in immunogenicity," Annu. Rev. Immunol, Vol. 9, pp. 271-296 (1991); Banchereau, J. and Steinman, R. M., 1998). Several variables, however, including cytokine and co-stimulatory molecule levels, the origin of the DCs, and the antigen dose, strongly affect the initiation and intensity of the immune response (Constant, S. et al, "Extent of T cell receptor ligation can determine the functional differentiation of naive CD4+ T cells," J. Exp. Med., Vol. 182, pp. 1591-1596 (1995); Hosken, N. A. et al, "The effect of antigen dose on CD4+ T helper cell phenotype development in a T cell receptor-alpha beta-transgenic model," J. Exp. Med., Vol. 182, pp. 1579-1584 (1995); Freeman, G. J. et al, "B7-1 and B7-2 do not deliver identical costimulatory signals, since B7-2 but not B7-1 preferentially costimulates the initial production of IL-4," Immunity, Vol. 2, pp. 523-532 (1995); Jonuleit, H. et al, "Induction of interleukin 10-producing, nonproliferating CD4(+) T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells," J. Exp. Med., Vol. 192, pp. 1213-1222 (2000)). Interleukin (IL)-10 was initially identified as a critical cytokine that suppresses multiple immune response activities, including the synthesis of Thl -derived cytokines. Further, studies have demonstrated that the immunosuppressive properties of IL-10 are mediated by its suppressive effects on the maturation of DCs (Corinti S. et al, "Regulatory activity of autocrine IL-10 on dendritic cell functions," J. Immunol, Vol. 166, pp. 4312-4318 (2001)). These suppressive effects include the inhibition of MHC class II, CD86 and CD54 expression, and suppression of IL-1 and tumor necrosis factor (TNF)-α transcription (Steinbrink, K. et al, "Induction of tolerance by LL-10-treated dendritic cells," J. Immunol, Vol. 159, pp. 4772-4780 (1997); Waal, M.R. de et al, "Interleukin 10(IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes," J. Exp. Med., Vol. 174, pp. 1209-1220 (1991)). Evidence suggests that a T-helper 1 (Thl) cell response is desirable for optimal tumor rejection and a body of literature suggests that DCs can control the differentiation of regulatory T cells in vivo (Zirvogel, L. et al, "IL-12-engineered dendritic cells serve as effective tumor vaccine adjuvants in vivo," Ann. NYAcad. Sci, Vol. 795, p. 284-293 (1996); Kawakami, Y. et al, "Identification of a human melanoma antigen recognized by umor- infiltrating lymphocytes associated with in vivo tumor rejection," Proc. Nail. Acad. Sci. U S A, Vol. 91, pp. 6458-6462 (1994)). For example, in DCs generated from peripheral blood mononuclear cells (PBMCs) using granulocyte macrophage colony stimulating factor (GM-CSF) and IL-4, IL-10 inhibits IL-12 production and induces a state of antigen-specific anergy in T cells. This anergy is characterized by inhibited T cell proliferation and reduced interferon (LFN)-γ production (Steinbrink, K. et al, "Induction of tolerance by LL-10-treated dendritic cells," J. Immunol, Vol. 159, pp. 4772-4780 (1997)). The inducible cytokine IL-12 is crucial to promoting the development of Thl cells and cell mediated immunity (Trinchieri, G., "Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity," Annu. Rev. Immunol, Vol. 13, pp. 251-276 (1995); Magram, J. et al, "IL-12-deficient mice are defective in LFN gamma production and type 1 cytokine responses," Immunity, Vol. 4, pp. 471-481 (1996); Murphy, K. M. et al, "Signaling and transcription in T helper development," Annu. Rev. Immunol, Vol. 18, pp. 451-494 (2000)). Furthermore, the negative immunoregulatory effects of IL-10 on the Thl response are exerted at the level of APCs (Igietseme, J. U. et al, 2000). IL-10 exerts its immunosuppressive properties on DCs by reducing the expression of antigens and markers on MHC class II molecules and several co-stimulatory and adhesion molecules. Analysis of supematants from IL-10 treated human DC cultures demonstrated an inhibited production of inflammatory cytokines and a lack of IL-12 synthesis. Moreover, tumors escape immunosurveillance by secreting IL-10 to inhibit cytokine production and the antigen- presenting functions of DCs. Another result of tumor generated "escape" is that secreted IL- 10 induces a state of antigen-specific anergy in CD4^" and CD8⁺ T cells, which is characterized by inhibited T-cell proliferation and reduced production (Kim, J. et al, 1995; Fiorentino, D.F. et al, 1991; Steinbrink, K. et al 1997; Enk, A.H. et al. 1993). The DCs of cancer patients exhibit dysfunction as a result of tumor cells secreting immunosuppressive cytokines, such as transforming growth factor (TGF)-β (Constam, D. B. et al, "Differential expression of transforming growth factor-beta 1, -beta 2, and -beta 3 by glioblastoma cells, astrocytes, and microglia," J. Immunol, Vol. 148, pp. 1404-1410 (1992)), vascular endothelial growth factor (NEGF) (Gabrilovich, D. I. et al, "Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function," Clin. Cancer Res., Vol. 5, pp. 2963-2970 (1999)) and IL-10 (Chen, Q. et al, "Production of IL-10 by melanoma cells: examination of its role in immunosuppression mediated by melanoma," Int. J. Cancer, No. 56, pp. 755-760 (1994)). This discovery has created a need in the art for a means to influence T cell response using DC vaccines. RΝA interference (RΝAi) is the process of inducing the degradation of mRΝA to silence gene expression mediated by double-stranded RΝAs (dsRΝAs) (Fire, A. et al., "RΝA as a target of double-stranded RΝA-mediated genetic interference in Caenorhabditis elegans," Proc. Natl Acad. Sci. U S A, Vol. 95, Issue 26, pp. 15502-7 (1998)). RΝAi has received considerable attention because of its capability to silence the target gene expression. Furthermore, RΝAi has been studied in a variety of systems including C. Elegans, mouse embryos and Drosophila (Fire, A. et al, 1998; Wianny, F. and M. Zernicka-Goetz, "Specific interference with gene function by double-stranded RΝA in early mouse development," Nat. Cell Bio., Vol. 2, Issue 2, pp. 70-5 (2000); Hammond, S.M. et al, "An RΝA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells," Nature, Vol. 404, Issue 6775, pp. 293-6 (2000)). In each of these systems, silencing of target gene expression was confirmed by introducing exogenous dsRNA in these organisms. It is well known that immature DCs are highly phagocytic and can internalize a variety of molecules, including nucleic acids. However, RNAi has been attempted in mammalian cells by similar methods, and it was found that long dsRNA (>38bp) could induce a nonspecific type-I IFN response, leading to arrest in transcription and cell death. Studies show that this result means DCs may be particularly sensitive to dsRNA via the expression of the toll-like receptor 3 (Kadowaki, N. et al, "Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens," J Exp. Med., Vol. 194, pp. 863-869 (2001); Honda K, S. et al, "Selective contribution of LFN-alpha/beta signaling to the maturation of dendritic cells induced by double-stranded RNA or viral infection," Proc. Natl. Acad. Sci. USA, Vol. 100, pp. 10872-10877 (2003)). Although much of the success of RNAi is described in the aforementioned systems, RNAi has recently been applied to mammalian cells by using small interfering RNA (siRNA) (Elbashir, S. M. et al, "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells," Nature, Vol. 441, pp. 494-498 (2001); Harborth, j. et al, "Identification of essential genes in cultured mammalian cells using small interfering RNAs," J Cell Sci., Vol. 114, pp. 4557-4565 (2001); Caplen, N. J. et al, "Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems," Proc. Natl. Acad. Sci. U S A, Vol. 98, pp. 9742-9747 (2001)). Recent studies have shown that siRNA inhibits a variety of reporter, endogenous, or infectious gene products following transfection of chemically synthesized siRNAs in cultured mammalian cells (McManus, M.T. et al, 2002). Moreover, there are several successful reports on siRNA being used to silence IL-12 on murine DCs, and silence NF-κB/Rel proteins and Tripeptidyl peptidase II on human DCs (Hill, J.A. et al, 2003; Laderach, D. et al, 2003). In mammals, the mediators of RNAi are siRNAs consisting of 21-23 nucleotide (nt) RNA duplex effector molecules able to recognize and to guide the degradation of complementary mRNA sequences, which are generated by Rnase III cleavage from longer dsRNA (Tuschl, T., "RNA interference and small interfering RNAs," Chembiochem., Vol. 2, pp. 239-245 (2001); McManus, M. T. and Sharp, P. A., "Gene silencing in mammals by small interfering RNAs," Nat. Rev. Genet., Vol. 3, pp. 737-747 (2002); Harmon, G. 1, "RNA interference," Nature, Vol. 418, p. 244 (2002)). Transfection of 21 nt chemically synthesized siRNAs has been shown to knock down the expression of the translated protein in cultured mammalian cells (Elbashir, S. M. et al, 2001; Harborth, J. et al, 2002; McManus, M. T. et al, "Small interfering RNA-mediated gene silencing in T lymphocytes," J. Immunol, Nol. 169, pp. 5754-5760 (2002)). Furthermore, siRΝA technology has been used as a powerful tool to modulate the immune response in DCs (Hill, J. A. et al., "Immune modulation by silencing IL-12 production in dendritic cells using small interfering RΝA," J. Immunol, Nol. 171, pp. 691-696 (2003); Laderach, D. et al, "RΝA interference shows critical requirement for ΝF-kappaB p50 in the production of IL-12 by human dendritic cells," J. Immunol, Nol. 171, pp. 1750-1757 (2003)). It has been shown that RΝA transfection using GenePorter reagents (available from Gene Therapy Systems; San Diego, CA) in bone marrow-derived murine DCs is extremely efficient (Hill, J.A. et al., 2003). Moreover, siRΝA has been successfully transfected into human monocyte-derived DCs by electroporation (Laderach, D. et al., 2003), and siR As' small size (<30 nt) reportedly fails to activate the LFΝ-induced protein kinase R (Williams, B. R. et al, "PKR: a sentinel kinase for cellular stress," Oncogene, Nol. 18, pp. 6112-6120 (2001)) or to elicit a type I LFΝ response in mammalian cells (Caplen, ΝJ. et al., 2001).

SUMMARY OF THE INVENTION Described herein are methods for creating a DC vaccine that enhances the Thl response. The methods include transfecting DCs with IL-10 siRNA and administering the cultured DCs as a vaccine. The invention further includes compositions that are formulated for delivery as a vaccine, including the transfected DCs of the present invention and additional components such as a carrier. The DCs of the present invention may be derived from human tissue sources such as blood or bone marrow. In various other embodiments of the invention, methods for treating a disease condition are included. The methods of treating such a disease condition include administering a DC vaccine. The DC vaccine of the methods may include DCs transfected with IL-10 siRNA and additional components including carriers and cytokines. Further, in various embodiments, the DCs of the present invention may have the effect of downregulating IL-10 expression and upregulating IL-12 expression. Still further, the DCs of the present invention may present antigenic peptides on the surface of the DCs. The DC vaccines embodied in the methods of the present invention are formulated for administration to a mammal for the treatment of a disease condition. Further, the disease condition may be any disease condition that benefits from treatment with a DC vaccine, including cancer. h another embodiment, a host cell transfected with siRNA is included. Further, the host cell of the present invention may be a dendritic cell. In still other embodiments, a kit is included with a volume of DCs transfected with siRNA and instructions for its use. The kit of the present invention may include the DC vaccines embodied in the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 shows by flow cytometry analysis that siRNA efficiently transfects into human monocyte-derived DCs using FITC-labeled IL-10 siRNA in accordance with an embodiment of the present invention. Figure IA depicts the results on day 6 of culture with GM-CSF and IL-4, when monocyte-derived DCs (1 X 10⁶) were transfected with GeneSilencer reagent only. Figure IB depicts the results when FITC-labeled IL-10 siRNA (200 nM) was added to DCs without transfection reagents. Figure 1C depicts the results with GeneSilencer reagent and FITC-labeled IL-10 siRNA at 200 nM. DCs were activated with 100 ng/ml LPS and 10 ng/ml TNF-α on day 7 and the transfection efficacy was assessed by flow cytometry on day 8. FITC-siRNA was successfully transfected into 91.1% of the cells after 48 hours and only 5% of DCs incorporated siRNA in the absence of the transfection agent. This data is representative of three independently performed experiments. FIGURE 2 shows that DC viability is not affected by siRNA transfection or transfection reagent in accordance with an embodiment of the present invention. On day 6 of culture with GM-CSF and IL-4, monocyte-derived DCs (lxlO⁶) were unmanipulated (received no treatment), transfected with GeneSilencer alone (mock); 200 nM non-silencing siRNA or transfected 200 nM LL-IO siRNA for 24 hours. Then, the transfected DCs were activated with 100 ng/ml lipopolysaccaride (LPS) and 10 ng/ml TNF-α for 24 h. Cell apoptosis and necrosis were assessed by flow cytometry using annexin V and propidium iodine staining, respectively. This data is representative of three independently performed experiments. FIGURE 3 shows that IL-10 siRNA transfection of DCs specifically blocks IL-10 expression and increases LL-12 production in accordance with an embodiment of the present invention. This is demonstrated through an analysis of LL-10 and IL-12 production in mature DCs after siRNA transfection. On day 6 of culture with GM-CSF and IL-4, monocyte- derived DCs s (lxlO⁶) were unmanipulated (received no treatment), transfected with GeneSilencer alone (mock); 200 nM non-silencing siRNA or transfected 10, 50, 100 and 200 nM LL-10 siRNA for 24 hours. Then, the transfected DCs were activated with 100 ng/ml LPS and 10 ng/ml TNF-α for 24 h. RNA from the transfected DCs was extracted using a RNAeasy kit. Figure 3 A depicts the results of a RT-PCR analysis performed to assess IL-10 and β-actin expression. The expression levels were assessed using SEQ LD NO. 4, IL-10 forward primer, SEQ LD NO.5, IL-10 reverse primer, SEQ ID NO.6, β-actin forward primer, and SEQ ID NO.7, β-actin reverse primer. IL-10 siRNA had the effect of decreasing IL-10 mRNA expression with increasing concentrations. No effect was seen with β-actin mRNA expression. Figure 3B depicts the graphical results of an ELISA test of supematants harvested from culture medium and analyzed for IL-10 production. IL-10 siRNA decreased IL-10 expression by DCs at all doses. Figure 3C depicts the graphical results of an ELISA test of supematants harvested from culture medium and analyzed for IL-12p70 production. A positive correlation was found between the downregulation of IL-10 by IL-10 siRNA and IL- 12 expression demonstrating that LL-10 actively inhibits LL-12 expression. In Figure 3B and Figure 3C, the "a" indicated p<0.01 compared with no treatment, non-silencing siRNA and mock groups; "b" indicated pO.Ol compared with 10 nM IL-10 siRNA; "c" indicated p<0.01 compared with 50 nM IL-10 siRNA; and "d" indicated pO.Ol compared with 100 nM IL-10 siRNA. This data is representative of three independently performed experiments. FIGURE 4 depicts the effects of LL-10 siRNA transfection on DC phenotype after maturation in accordance with an embodiment of the present invention. DC phenotype was assessed using flow cytometry for the expression of maturation markers, CD83, HLA-DR, CD 86, CD40 and CD54 levels on the surface of the DCs. On day 6 of culture with GM-CSF and IL-4, monocyte-derived DCs (lxlO⁶) were unmanipulated (received no treatment), transfected with GeneSilencer alone (mock); 200 nM non-silencing siRNA or transfected with 200 nM LL-10 siRNA for 24 hours. Then, the transfected DCs were activated with 100 ng/ml LPS and 10 ng/ml TNF-α for 24 h. Transfection with IL-10 siRNA had no effect on the expression of these markers except to increase the expression of CD40. This data is representative of three independently performed experiments. FIGURE 5 shows that LL-10 siRNA transfection increases DC allogenic T cell proliferation in accordance with an embodiment of the present invention. DCs were unmanipulated (received no treatment), transfected with GeneSilencer alone (mock), transfected with 200 nM non-silencing siRNA or transfected with 200 nM IL-10 siRNA for 24 hours. Allogenetic peripheral blood mononuclear cells (PBMCs) (2xl0⁵/well) were incubated with siRNA-treated DCs at the indicated ratio of DCs:T cells for six days. Proliferation was determined using a WST-1 assay. IL-10 siRNA was found to increase DC allo stimulatory effect on T cells which was consistent with the decrease in IL-10 expression and upregulation of IL-12. This data is representative of three independently performed experiments. FIGURE 6 shows the effect of IL-10 siRNA transfection of DCs on antigen presentation and specific Thl response activation in accordance with an embodiment of the present invention. On day 6 of culture with GM-CSF and IL-4, monocyte-derived DCs (lxlO⁶) were unmanipulated (received no treatment), transfected with GeneSilencer alone (mock), transfected with 200 nM non-silencing control siRNA or transfected with 200 nM IL-10 siRNA for 24 hours. The transfected DCs were incubated with keyhole limpet hemocyanine (KLH) (50 ug/ml) for three hours, then activated with 100 ng/ml LPS and 10 ng/ml TNF-α for 24 h. After washing, siRNA-treated DCs (10⁶) were seeded in cultures with autologous CD45RO^"CD4⁺ T cells (lOxlO⁶) for six days, and supematants were collected from the medium. Figure 6A depicts the results of an ELISA test for LFN-γ production. The (*) indicates pO.Ol, by one-way ANOVA compared with the other three groups. Figure 6B depicts the results of an ELISA test for IL-4 production. LFN-γ production was increased and IL-4 production was undetectable in cultures of IL-10 siRNA treated DCs and naϊve CD4⁺ T cells, supporting the Thl response. This data is representative of three independently perfonned experiments.

DESCRIPTION OF THE INVENTION A. DEFINITIONS Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al, Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., J. Wiley & Sons (New York, NY 1992); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001), provide one skilled in the art with a general guide to many of the terms used in the present application. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below. "Alleviating" specific cancers and/or their pathology includes degrading a tumor, for example, breaking down the structural integrity or connective tissue of a tumor, such that the tumor size is reduced when compared to the tumor size before treatment. "Alleviating" metastasis of cancer includes reducing the rate at which the cancer spreads to other organs. "Antigen" means all, or parts thereof, of a protein or peptide capable of causing an immune response in a vertebrate, such as a mammal. Such antigens are also reactive with antibodies from animals immunized with said protein. The potent accessory function of dendritic cells provides for an antigen presentation system for virtually any antigenic epitope which T lymphocytes are capable of recognizing through their specific receptors. "Beneficial results" may include, but are in no way limited to, lessening or alleviating the severity of the disease condition or its complications, preventing it from manifesting, preventing it from recurring, merely preventing it from worsening, curing the disease condition, prolonging a patient's life or life expectancy, ameliorating a disease, or a therapeutic effort to affect any of the aforementioned, even if such therapeutic effort is ultimately unsuccessful. "Cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, prostate cancer, melanoma, kidney cancer, colorectal cancer, lung cancer, non-Hodgkin's lymphoma, breast cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, head and neck cancer, and brain cancer. "Conditions" and "disease conditions," as used herein may include, but are in no way limited to bacterial infections, protozoan such as malaria, listeriosis, microbial infections, viral infections such as HIN or influenza, autoimmune diseases such as psoriasis, ankolysing spondylitis and any form of cancer or malignancy such as melanoma. Furthermore, the disease condition may be any condition that may benefit from treatment with a DC vaccine. "Curing" cancer includes degrading a tumor such that a tumor cannot be detected after treatment. The tumor may be reduced in size or become undetectable, for example, by atrophying from lack of blood supply or by being attacked or degraded by one or more components administered according to the invention. "Cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, Ν-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor (NEGF); integrin; thrombopoietin (TPO); nerve growth factors (ΝGFs) such as ΝGF-β; platelet-growth factor; transforming growth factors (TGFs) such as TGF-α and TGF- β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF); interieukins (ILs) such as IL-1, IL-lα, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, LL-9, IL-10, IL-11, IL-12 and IL-13; a tumor necrosis factor such as TΝF-α or TΝF-β; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines. "Dendritic Cells" or "DCs" refer to immunocompetent cells of the lymphoid and haemopoietic systems and skin. DCs function morphologically and phenotypically by presenting or processing antigens, thereby stimulating cellular immunity. Examples of dentritic cell types include Langerhans cells, found in the skin, and follicular dendritic cells, found in lymphoid tissues. DCs are also referred to as interdigitating, reticular, and veiled cells. "Isolated" as used herein encompasses a purified dendritic cell that is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. "Mammal" as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term. "Pathology" of cancer includes all phenomena that compromise the well-being of the patient. This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, neoplasia, premalignancy, malignancy, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc. "Therapeutically effective amount" as used herein refers to that amount which is capable of at least partially preventing or reversing the symptoms of the disease condition. A therapeutically effective amount can be determined on an individual basis and will be based, at least in part, on consideration of the species of mammal, the mammal's size, the DCs used, the type of delivery system used and the time of administration relative to the progression of the disease condition. "Treatment" and "treating," as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder even if the treatment is ultimately unsuccessful. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. In tumor (e.g., cancer) treatment, a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy. "Tumor," as used herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

B. DETAILED DESCRIPTION All publications are incorporated herein by reference in their entirety. The invention is based on the surprising discovery of a method of using IL-10 siRNA to silence or downregulate the expression LL-10 in DC vaccines to enhance Thl tumor response. Methods of the present invention inhibit JL-10 expression and activity by DCs by interfering with the production or biological activity of IL-10 RNA. One can use these methods to treat any disease in which inhibiting IL-10 expression has a beneficial effect on a patient. In this study, the induction of RNAi was assessed using IL-10 siRNA in human monocyte-derived DCs. It was found that inhibition of IL-10 in monocyte-derived DCs after stimulation with LPS and TNF-α is accompanied by an increase in IL-12 production and CD40 expression. Moreover, IL-10 siRNA treated DCs significantly increased T cell proliferation when DCs were cultured with allogenic T cells. Still further, Thl polarization was enhanced when naϊve CD4⁺ T cells were stimulated by IL-10 siRNA treated DCs. Thus, while not wishing to be bound by any theory, the present invention is based on the premise that knocking-down endogenous IL-10 expression at the level of DCs by siRNA is a practical strategy for enhancing the specific Thl response against pathogens. It was found that IL-10 siRNA treated DCs could significantly decrease IL-10 expression at both the mRNA and protein levels. Moreover, the inventors found that inhibition of LL-10 expression could increase CD40 expression, increasing the allogenic T cell response and antigen presentation to induce a more potent Thl immune response. It was also demonstrated that siRNA transfection did not affect cell viability or DCs ability to induce a type I LFN response (Hill, J.A. et al, 2003; Laderach, D. et al, 2003). Furthermore, unlike antisense oligodeoxyribo-nucleotides, siRNA appears to require doses 10- to 100-fold less than such nucleotides, to achieve a similar effect (Laderach, D. et al, 2003). Although antibody neutralization and LL-10 antisense oligo transfection has been successfully used to block the bioactivity of IL-10 and inhibit IL-10 production, the inventors herein show that RNAi can be used to analyze specifically the role of single gene products in DC antigen presentation. As discussed above, IL-10 prevents antigen-specific T cell proliferation indirectly by reducing the antigen presenting capacity of DCs. This effect is associated with the inhibition of MHC class II, CD86 and CD54 expression on these cells (Steinbrink, K. et al, 1997; Waal, M.R. de et al, 1991). The inventors found that there was no effect on MHC class II, CD86 and CD54 expression when LL-10 was silenced by RNAi. This is in contrast to a report that indicates that allogenic T helper and cytotoxic T cell responses were diminished in CD40L deficient mice despite the fact that injected DCs expressed high levels of MHC class II, B7.1 and B7.2 (Mackey, M.F. et al, "The role of CD40/CD154 interactions in the priming, differentiation, and effector function of helper and cytotoxic T cells," J. Leukoc. Biol, Vol. 63, pp. 418-428 (1998)). CD40 and CD40L interactions play important roles in the priming, differentiation and effector function of helper and cytotoxic T cells (Mackey, M.F. et al, 1998). In addition, DCs are a major IL-12 producer (Rissoan, M. C. et al, "Reciprocal control of T helper cell and dendritic cell differentiation," Science, Vol. 283, pp. 1183-1186 (1999); Liu, Y. L, "Dendritic cell subsets and lineages, and their functions in innate and adaptive immunity," Cell, Vol. 106, pp. 259- 262 (2001)), and IL-12 production after cognate interaction with CD4+ T cells is largely dependent on CD40/CD40L interaction (Koch, F. et al, "High level IL-12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and downregulation by LL-4 and IL-10," J Exp. Med., Vol. 184, pp. 741-746 (1996). It has been shown that triggering CD40 on DCs leads to IL-12 production. The increase in CD40 expression and LL-12 production and the enhanced Thl induction by LL-10 siRNA treated DCs suggests that antigen presentation is an active process, not just a default outcome of a low level of IL-10 in the environment. This finding underscores the basis for the ineffectiveness of an immunotherapy based only on anti-IL-10 antibodies to enhance Thl induction via binding and neutralization of secreted IL-10. The present invention is supported by the findings of studies suggesting an inverse relationship between the production of LL-10 and IL-12 in activated DCs (Xia, C.Q. and Kao, K.J., "Hepatin induces differentiation of CDla+ dendritic cells from monocytes: phenotypic and functional characterization," Journal of Immunology, Vol. 168, pp. 131-1138 (2002)). Thus, neutralization of the secreted IL-10 by antibody and IL-10 anti-sense oligonucleotides in conjunction with DC-based immunotherapy could also enhance the production of LL-12 (Xia, C. Q. and Kao, K. J., "Suppression of interleukin- 12 production through endogenously secreted interleukin- 10 in activated dendritic cells: involvement of activation of extracellular signal-regulated protein kinase," Scand. J. Immunol, Vol. 58, pp. 23-32 (2003)). Moreover, studies have shown that the Thl immune response was significantly enhanced by DCs in a IL-10 knock-out mouse model (Igietseme, J. U. et al, "Suppression of endogenous IL-10 gene expression in dendritic cells enhances antigen presentation for specific Thl induction: potential for cellular vaccine development," J. Immunol, Vol. 164, pp. 4212-4219 (2000)) and previous studies have demonstrated that endogenous IL-10 production is a crucial regulatory step in Thl activation. It has been established that a Thl -like T cell response is desirable for optimal tumor rejection (Zitvogel, L. et al, "LL-12-engineered dendritic cells serve as effective tumor vaccine adjuvants in vivo," Ann. N Y Acad. Sci., Vol. 795, pp. 284-293 (1996); Kawakami, Y. et al, "Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection," Proc. Natl. Acad. Sci. USA, Vol. 91, pp. 6458-6462 (1994)). Transfection of DCs with IL-10 siRNA is efficient and offers the possibility of treating immune-based diseases in a specific and effective manner. The successful use of antigen pulsed LL-10 siRNA modified DCs to enhance a Thl immune response highlights the utility of this approach for DC-based cancer immunotherapy. Furthermore, the present invention provides a novel immunotherapeutic strategy of using IL- 10 siRNA transfected antigen presenting cells (APCs) as vaccine delivery agents to boost the Thl response against pathogens and tumors that are controlled by Thl immunity. In various embodiments, the present invention includes isolated DCs useful in treating various disease conditions. The isolated DCs of the present invention may include those DCs that enhance a Thl immune response. Still further, the isolated DCs of the present invention may include those DCs with the ability to downregulate IL-10 and upregulate IL-12. The isolation of DCs may be readily accomplished by one of skill in the art without undue experimentation. For example, the method of isolating the DCs may involve conventional methods which enrich subpopulations of cell mixtures including, but in no way limited to, density gradient separation, fluorescence activated cell sorting, immunological cell separation techniques such as panning, complement lysis, rosetting, magnetic cell separation techniques, and nylon wool separation. Furthermore, other techniques may be used to isolate the DCs of the present invention such as observing different patterns of expression of cell surface antigens to identify different cell types. Mature DCs can also be obtained by culturing proliferating or non-proliferating DC precursors in a culture medium containing factors which promote maturation of immature DCs to mature DCs. Other techniques for isolation of the DCs of the present invention will be recognized by one of skill in the art. For example, U.S. Patent No. 6,589,526 reports methods and compositions for obtaining DCs and creating a DC vaccine. Further, a procedure for ex vivo expansion of hematopoietic stem and progenitor cells for enrichment of DCs is described in U.S. Pat. No. 5,199,942. Both patents are incorporated herein by reference in their entirety. The DC precursors, from which the immature DCs for use in this invention are derived, are present in blood as PMBCs. Although most easily obtainable from blood, the precursor cells may also be obtained from any tissue in which they reside, including bone marrow and spleen tissue. When cultured in the presence of cytokines such as a combination of GM-CSF and IL-4 or LL-13, the non-proliferating precursor cells give rise to immature DCs for use in this invention. The isolated DCs of this invention are useful for vaccine therapy that can be reinjected into a patient. However, the isolated DCs of the present invention have many other clinical applications including, for example, adoptive immunotherapy for the production of pathogen specific CTL production or in antigen specific T helper cell production, or for enhanced graft acceptance. Optionally, the isolated DCs of the present invention may be antigen pulsed DCs that can be reinjected into a patient. The isolated DCs of the present invention may also be useful for evaluating DC maturation and development, investigating primary immune response by antigen pulsed blood DCs, analyzing tumor immunity by tumor-antigen exposed DCs, and comparing antigen uptake processing and presentation of DCs. Treating of the DCs with the cancer-specific antigen can be by any method which results in the DCs presenting the antigen so as to stimulate host immunity when a vaccine including the DCs is administered to a mammal. For example, the DCs may be pulsed or cultured in the presence of the antigen, contacted with antigenic proteins, transfected to express antigenic proteins, fused with cells carrying antigenic proteins, or combinations thereof, prior to administration of the vaccine composition to a mammal. In one embodiment, the present invention includes a DC vaccine composition for treating disease conditions in a mammal, including a therapeutically effective amount of DCs transfected with siRNA to downregulate a gene product. The DCs may be transfected with IL-10 siRNA so as to downregulate IL-10 expression and thereby upregulate IL-12 expression when the vaccine composition is administered to a mammal. Furthermore, the DCs of the present invention may also be treated with cancer-specific antigen so as to stimulate host immunity to the cancer when the vaccine composition is administered to a mammal. Further, in various embodiments, the DCs of the present invention may be combined with one or more additional components including, without limitation, a vehicle, an additive, a pharmaceutical adjunct, a therapeutic compound, adjuvants, diluents, excipients, a carrier and agents useful in the treatment of cancer or other disease conditions, and combinations thereof. In certain embodiments, the DCs may be administered in a pharmaceutically acceptable carrier which is nontoxic to the cells and the individual. Such a carrier may be a growth medium, or any suitable buffering medium such as phosphate buffered saline (PBS). Such carriers will be nontoxic to recipients at the dosages and concentrations employed. Once so combined, the DCs may be suitable for administration to a mammal to treat a disease condition; although formulation with such an additional component is not required to be administered. Still further, in various embodiments, the transfected DCs of the present invention may be part of a treatment regimen including other immunoregulatory molecules and the treatment regimen may be suitable for administration to a mammal to treat a disease condition. In certain embodiments, the present invention provides methods of using therapeutic compositions including the DCs of the present invention or activated, antigen-pulsed DCs in conjunction with cytokines, or other immunoregulatory molecules. For example, administration of other cytokines along with the DCs of the present invention is contemplated to stimulate an immune response. The DCs may be administered with such factors including, but in no way limited to, growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor (NEGF); integrin; thrombopoietin (TPO); nerve growth factors (ΝGFs) such as ΝGF-β; platelet-growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF); interieukins (ILs) such as E -l, IL-lα, IL-2, IL-3, IL-4, IL-5, IL- 6, LL-7, IL-8, IL-9, LL-10, LL-11, IL-12, LL-13 and IL-15; a tumor necrosis factor such as TΝF-α or TΝF-β; and other polypeptide factors including LLF, kit ligand (KL) and flt-3 ligand; or any biologically active derivatives thereof. In still other embodiments, DΝA encoding such cytokines maybe transfected into the DCs to express the cytokines endogenously. Furthermore, administration of these cytokines may be effectuated simultaneously, separately or sequentially with the DCs of the present invention. A typical DC-based vaccine therapy may include any appropriate dosage of DCs to be administered to an individual in the treatment of a disease condition as will be recognized by one of skill in the art. Furthermore, the mature DCs prepared according to this invention may be particularly potent at activating T cells. While not wishing to be bound by any particular range, it is believed that a volume of between about 1 million and about 10 million mature DCs may be a therapeutically effective dosage in connection with various embodiments of the invention. In addition, while the amount of DCs to T cells necessary for strong T cell activation is typically a ratio of about 1 DC to about 20 T cells, the ratio in various embodiments of the present invention may be about 1 DC to about 50 T cells. Thus, the present invention may require administration of fewer DCs to be therapeutically effective. For activating T cells in vitro, the ratio of DCs to T cells may be between about 1:10 and about 1:100. A ratio of between about 1:20 and about 1:50 maybe particularly advantageous in accordance with alternate embodiments of the invention. Furthermore, single or multiple doses of the DCs of the present invention can be administered over a given time period, depending upon the cancer or disease condition, as can be determined by one skilled in the art without undue experimentation. The DCs can be administered by any method which allows the DCs to reach the appropriate cells or tissue or other location in the body of a mammal. DCs may be administered to a mammal using standard methods including, but in no way limited to, injection, infusion, deposition, implantation, oral ingestion or topical administration. Administration by injection can include, but is in no way limited to, intravenous, intramuscular, intradermal, subcutaneous or intraperitoneal injection. In certain embodiments, the injections can be given at multiple locations. Furthermore, administration of the DCs can be alone or in combination with other therapeutic agents. Still other methods of administration may be used to deliver the inventive compositions to stimulate an immune response, including bolus injection, continuous infusion, sustained release from implants, or other any other suitable technique as will be recognized by one of skill in the art. Efficient gene silencing may be achieved by employing siRNA duplexes which include sense and antisense strands each including approximately 21 nucleo tides, and further paired such that they possess about a 19-nucleotide duplex region and about a 2-nucleotide overhang at each 3' terminus (Elbashir et al, "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells," Nature, Vol. 411, pp. 494-498 (2001)). It will be appreciated by one of skill in the art of RNAi that alternately sized sense or antisense strands and/or variations on the size of the duplex and the overhang region that comprise them may be suitable for use with the methods of the present invention, and are contemplated as being within the scope thereof. Such appropriate alternate sizes may be readily ascertained without undue experimentation by one possessing such skill. Furthermore, the inclusion of symmetric 3 '-terminus overhangs may aid in the formation of specific endonuclease complexes ("siRNPs") with roughly equivalent ratios of sense and antisense target RNA cleaving siRNPs. It is believed that the antisense siRNA strand is responsible for target RNA recognition, while the 3 '-overhang in the sense strand is not involved in this function. Therefore, in a prefened embodiment, the UU or dTdT 3'- overhang of an antisense sequence is complementary to target mRNA, however the symmetrical UU or dTdT 3 '-overhang of the sense siRNA oligo need not correspond to the mRNA. Deoxythymidines may be included in either or both 3 '-overhangs; this may increase nuclease resistance. However, siRNA duplexes that include either UU or dTdT overhangs may be equally resistant to nuclease. The present invention includes double strands of duplex siRNA specific for IL-10 mRNA. The siRNA may span the region adjacent to the initiation site of LL-10 translation, in one embodiment, region 292-310, and in another embodiment, region 441-459. Within this embodiment, the sequence of the siRNA duplex may be designed using siDESIGN software (available from Dharmacon; Lafayette, CO). Additionally, the siRNA may be compared to the human genome database BLAST (available from the National Center for Biotechnology Information; Bethesda, MD) to ensure the sequences will not target other gene transcripts. A particularly appropriate fragment of IL-10 that the IL-10 siRNA may target is set forth herein as SEQ LD NO.l. Inhibition of this RNA, or those substantially similar to it, may correspondingly inhibit the biological activity of IL-10. Furthermore, the IL-10 siRNA may be encoded by the RNA duplex set forth herein as SEQ ID NO.2 and SEQ LD NO.3, corresponding with the sense and antisense strands, respectively. Thus, various embodiments of the present invention are directed to inhibiting the expression of IL-10 RNA. In certain embodiments of the present invention, immature human monocyte-derived DCs are transfected with the siRNA oligonucleotides using a transfection reagent following the manufacturer's protocol. Transfection reagents enable antisense oligonucleotides to be internalized into eukaryotic cells and to bind a target gene therein. Stable complexes are formed between the transfection reagent and oligos, permitting efficient transfection of oligos into cells in a highly specific, non-toxic fashion. Any number of transfection reagents may be used with the DCs of the present invention, including, but in no way limited to Oligofectamine reagent (available from Invitrogen; Carlsbad, CA); GenePorter reagents (available from Gene Therapy Systems; San Diego, CA); GeneJuice, RiboJuice, ProteoJuice Transfection reagents (available from Novagen; Saή Diego, CA); FuGENE Transfection reagent (available from Roche; Indianapolis, IN); PolyFect, Effectene, SuperFect, and TransMessenger Transfection reagents (available from Qiagen; Valencia, CA); and GeneSilencer siRNA Transfection reagent (available from Cambridge Bioscience; Cambridge, UK). The assembly and production of a DC vaccine transfected with siRNA oligonucleotides of the present invention may be completed in two stages. During the first step, siRNA specific for IL-10 is complexed in serum-free media with a transfection reagent. In the second step, the complexes are transfected into immature human monocyte-derived DCs in normal growth medium by any number of means known to those of skill in the art. For example, transfection can be accomplished using microparticle bombardment, lipofection, electroporation or any other method known in the art. Optionally, the DCs of the present invention may be primed with antigens specific for the disease condition for which the vaccine is administered. However, the DCs of present invention may also be administered directly into a tumor or its surrounding tissue, without priming the DCs with tumor antigens ex vivo, as discussed in U.S. patent application No. 10/251,148. In another embodiment of the present invention, a kit is provided, including a vaccine with a volume of DCs transfected with IL-10 siRNA, a carrier, and instructions for its use. The exact nature of the components configured in the inventive kit depends on its intended purpose and on the particular methodology that is employed. For example, some embodiments of the kit are configured for the purpose of treating cancer in a subject. Further, the exact volume of the DCs in the inventive kit depends on its intended purpose and on the condition that it is meant to treat. In one embodiment, the vaccine of the kit is configured particularly for the purpose polarizing T-cell response in favor of Thl used in the treatment of human subjects. Instructions for use may be included with the kits of the present invention. "Instructions for use" typically include a tangible expression describing the steps for creating DC vaccines and/or for using the same in a therapeutic system. Optionally, the kits also contain other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, specimen containers, syringes, stents, catheters, pipetting or measuring tools, paraphernalia for concentrating, sedimenting, or fractionating samples, or antibodies and/or primers and/or probes for controls. The materials or components assembled in the kits can be provided to the practitioner stored in any convenient and suitable way that preserves their operability and utility. For example, the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated, or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase "packaging material" refers to one or more physical structures used to house the contents of the kits. The packaging material is constructed by well known methods, preferably to provide a sterile, contaminant-free environment. The packaging materials employed in the kits are those customarily utilized in the field. As used herein, the term "package" refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. Thus, for example, a package can be a glass vial used to contain suitable quantities of immature human monocyte-derived DCs and/or siRNA oligonucleotides. The packaging material generally has an external label which indicates the contents and/or purpose of the kits and/or its components. EXAMPLES The following examples illustrate a method of transfecting a cell with siRNA oligonucleotides and a transfection reagent. Modifications of these examples will be readily apparent to those skilled in the art who seek to treat patients whose condition differs from those described herein. These examples are included merely for purposes of illustration.

EXAMPLE 1 DCs are efficiently transfected with siRNA To establish a protocol for IL-10 RNAi in human monocyte-derived DCs using GeneSilencer siRNA transfection reagent (obtained from Gene Therapy Systems; San Diego, CA), the efficacy of siRNA transfection was evaluated using FITC-labeled IL-10 siRNA. The transfection efficiency was quantified by flow cytometry. As seen in Figure 1, FITC- siRNA was successfully transfected into 91.1% of the cells after 48 hours whereas only 5% of the DCs incorporated siRNA in the absence of the transfection agent.

EXAMPLE 2 siRNA transfection does not reduce DC viability or induce IFN expression To evaluate the toxicity of siRNA and the transfection reagent the viability of the DCs and their ability to induce type I IFN expression was measured. On day 6 of culture in GM- CSF and IL-4, DCs were treated with transfection reagent alone, transfected with non- silencing siRNA or transfected with IL-10 siRNA. After 24h of transfection, apoptosis and necrosis were assessed using annexin N and propidium iodine staining, respectively. As shown in Figure 2, compared with untreated DCs, neither the transfection reagent alone nor siRΝA affected cell viability. Furthermore, there was no detectable IFΝ-α by ELISA assay in the culture medium of transfected DCs (Data not shown).

EXAMPLE 3 IL-10 and IL-12 production in mature DCs after siRNA transfection The specificity of siRΝA gene inhibition in DCs after transfection with IL-10 siRΝA was investigated. Immature human DCs were prepared by culture of adherent mobilized peripheral blood monocytes with GM-CSF and IL-4, and were transfected with 10, 50, 100 and 200 nM of anti-IL-10 or 200 nM control siRNAs. DCs were matured by 100 ng/ml LPS and 10 ng/ml TNF-α after siRNA transfection for 24 hours. Twenty-four hours after maturation, DCs were collected to analyze IL-10 mRNA expression by RT-PCR, and LL-10 and IL-12 production in the culture medium were measured by ELISA. As shown in Figure 3 A and Figure 3B, IL-10 siRNA transfection decreased IL-10 mRNA expression and LL-10 protein production. A decrease of IL-10 protein in the medium was observed even with 10 nM siRNA, while IL-10 siFiNA at 200 nM decreased IL-10 protein production by more than 95%. IL-12 production, however, was increased when IL-10 was decreased as seen in Figure 3C, which demonstrated that endogenous IL-10 inhibits LL-12 expression.

EXAMPLE 4 Cell surface phenotype analysis after IL-10 siRNA transfection To address the effects of IL-10 siRNA transfection on the DC phenotype after maturation, a homogenous population of immature DCs obtained 6 days in culture with GM- CSF and IL-4 was used. These DCs expressed medium levels of MHC class II or CD86, and barely detectable expression levels of CD83 on the surface. The vast majority of these cells (-90%) did not display CD 14 before maturation (data not shown). DCs were matured with 100 ng/ml LPS and 10 ng ml TNF-α after siRNA transfection for 24 hours. Twenty-four hours later, DCs were collected to analyze their phenotypes by flow cytometry. Maturation of DCs induced dramatic phenotypic changes, which was shown by the up-regulation of MHC class II, CD86, CD40, CD83 and CD54 levels on the surface. Moreover, siRNA transfection did not inhibit DC maturation. As shown in Figure 4, there was no difference between the four groups in MHC class II, CD86, CD83 or CD54 expression. It was found, however, that CD40 expression was increased in DCs after IL-10 siRNA transfection.

EXAMPLE 5 T cell stimulation ability of DCs after IL-10 RNA interference The function of DCs can be characterized in part by their ability to stimulate alloreactive T cells in the mixed lymphocyte reaction (MLR) (Banchereau, J. and Steinman, R. M., 1998). To determine whether IL-10 siRNA transfection affected the allostimulatory activity of DCs, MLR was performed using DCs transfected with IL-10 siRNA, non-silencing control siRNA, mock transfected or untreated control. Allogenic T cells were cultured with siRNA-transfected DCs for 6 days, at which point allostimulation was determined by proliferation. All three control groups showed similar allostimulatory activity. Interference with IL-10 siRNA significantly increased the induction of T cell proliferation, which was significant and consistent with the decrease of DL-IO production and increase of IL-12 production as seen in Figure 5. Because IL-10 siRNA treatment did not change MHC class II or co-stimulatory molecule (CD86) expression on DCs, the results demonstrate that IL-10 and IL-12 levels directly influence allogenic T cell proliferation.

EXAMPLE 6 IL-10 siRNA-treated DCs polarize naϊve CD4 cells toward a Thl immune response The process of phenotypic maturation and induction of IL-10 and IL-12 production by DCs is thought to play an important role in the induction of an immune response (Lanzavecchia, A. and Sallusto, F., "Regulation of T cell immunity by dendritic cells," Cell, Vol. 106, pp. 263-266 (2001)). It was observed that interference with LL-10 siRNA inhibited IL-10 secretion and concurrently, increased IL-12 production and CD40 expression after maturation by LPS and TNF-α. When co-cultured with T cells, these DCs appeared optimally suited to induce a Thl immune response. To test the function of siRNA treated DCs, purified CD45RO^" CD4⁺ T cells were stimulated by IL-10 siRNA-transfected DCs pulsed with 50 μg/ml KLH. After six days of stimulation, the IFN-γ level in the culture medium was significantly increased after DCs were treated with IL-10 siRNA. Furthermore, there was no detectable IL-4 in the IL-10 siRNA-treated DCs as seen in Figure 6. The results indicated that DCs matured by LPS and TNF-α polarize CD4⁺ cells toward Thl immune response because IL-4 production was much lower than IFN-γ production by the T cells in every group, hi particular, LL-10 siRNA treated-DCs generated the most significant and strongest Thl immune response as indicated by high LFN-γ production by CD4⁺ cells and the lack of any detectable IL-4 production.

EXAMPLE 7 Generation of monocyte-derived dendritic cells Mononuclear cells were isolated using leukopheresis; a blood filtering process used to remove extra lymphocytes. The COBE Spectra Apheresis System (available from Gambro BCT; Lakewood, CA) was used to harvest the mononuclear cell layer. Leukopheresis yielded between 5xl0⁹ and 10¹⁰ PBMCs. These cells were allowed to become adherent for two hours at 37° C in tissue culture flasks at a concentration of 5xl0⁶ cells/ml in RPMI 1640 media (obtained from Invitrogen; Carlsbad, CA) with 10% autologous heat inactivated serum. After 2 hours at 37°C, non-adherent cells were removed by washing with warm complete medium. To generate autologous DCs, adherent PBMC were cultured in complete medium for 6 days in the presence of 800 U/ml of clinical grade recombinant human GM- CSF (obtained from Berlex; Richmond, CA) and 500 U/ml recombinant human IL-4 (obtained from R&D Systems; Minneapolis, MN).

EXAMPLE 8 siRNA design, synthesis and transfection The 21 nt interfering RNA duplexes with two 3 '-end overhang dT nucleotides in antisense strands of the siRNAs were reported (Elbashir, S. M. et al., 2001; Harborth, J. et al, 2001). The siRNA sequence used for targeted silencing of human LL-10 (Genebank Ace. No.: AY029171) was designed by Qiagen software (available from Qiagen; Valencia, CA), and siRNA sequences were selected according to the method of Elbashir (Elbashir, S. M. et al, 2001). The following sequences were used in this study:

Searches of the human genome database (BLAST) were carried out to ensure the sequence would not target other gene transcripts. siRNA were transfected by GeneSilencer (obtained from Gene Therapy Systems; San Diego, CA), which was used according to the manufacturer's protocol. Non-silencing control siRNA is an irrelevant siRNA with random nucleotides and no known specificity. Sequences were synthesized and annealed by the manufacturer (obtained from Qiagen).

EXAMPLE 9 Extraction and RT-PCR analysis Total RNA was extracted from lxlO⁶ DCs using RNAeasy kit (obtained from Qiagen) according to the manufacturer's instruction. The cDNA was synthesized using oligo dT, dNTP mixture, RNAse inhibitor, and Superscript II Rnase H-Reverse Transcriptase (obtained from Invitrogen). PCR amplification was conducted in 50 μl containing 1-5 μl of cDNA, 1.5 mM MgCl₂, dNTP mixture (0.2 mM each dNTP), 0.5 μM each oligonucleotide primer, and 2 U of Tag DNA polymerase. The following primers were used in this study:

Amplification steps consisted of 35 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 1 min using a DNA cycler (obtained from PerkinElmer; Wellesley, MA). The PCR reaction was analyzed on 1.5% agarose gel electrophoresis stained with ethidium bromide.

EXAMPLE 10 Flow cytometric analysis Directly conjugated mouse mAbs, including FITC-conjugated CD86, CD40, PE-anti- HLA-DR, anti-CD83, and Biotin conjugated anti-CD54 mAbs were used (obtained from BD Pharmingen; San Diego, CA). Cellular staining was measured on a FACSCalibur instrument (obtained from BD Biosciences; San Jose, CA) and data were analyzed using CellQuest software (obtained from BD Biosciences), with results expressed as percentage of cell staining above background staining obtained with isotype control mAbs.

EXAMPLE 11 Allogenic T cell proliferation MLR was set up by culturing PBMCs (5xl0⁴ cell/0.3 ml of ALM-V serum free medium per well in triplicate)(obtained from Invitrogen) with various concentrations of allogenic transfected DCs obtained ~24 h after transfection with the various siRNAs. The mitogenic activity of the growth factors was determined by a colorimetric assay based on formazan dye formation known as WST-1 (obtained from Roche; Indianapolis, LN), which directly correlates with the number of metabolically active cells in the culture. After incubation of the cells for six days, 20 μl/well of the reagent WST-1 were added and incubated for 1.5 h at 37 °C. An increase in the number of viable cells resulted in an increase in the overall activity of mitochondrial dehydrogenases in the sample with an ensuing increase in formazan dye formation. The formazan dye was quantified by measuring the optical density of the dye solution at 450 nm with a scanning multi-well spectrophotometer (obtained from Molecular Devices; Sunnyvale, CA) using 890 nm as the internal reference (Lang, I. et al., "Differential mitogenic responses of human macrovascular and microvascular endothelial cells to cytokines underline their phenotypic heterogeneity," Cell Proliferation, Vol. 34, pp. 143-155 (2001)). All results in the study were based on at least five parallel measurements each time and repeated in up to three independent experiments.

EXAMPLE 12 Naϊve CD4 T cell purification and stimulation by DCs Naϊve CD4 T cells were purified by magnetic separation using anti-CD45RO (clone: UCHL-1) (obtained from Miltenyi Biotech Inc.; Auburn, CA) microbeads to negative selection, then anti-CD4 microbeads for positive selection. Matured, IL-10 siRNA treated DCs were loaded with 50 μg/ml KLH (subunits, _r 350,000/400,000) (obtained from Calbiochem-Novabiochem Corp.; San Diego, CA), then cultured with autologous purified CD45RO^" CD4⁺T cells at 2xl0⁵ DC/ml and 2xl0⁶ T cells/ml in a final volume of 2 ml ALM- V medium for six days. LFN-γ and IL-4 production in the medium was measured by ELISA.

EXAMPLE 13 ELISA assay IL-12 p70, IL-10, IFN-α, LFN-γ, and IL-4 were measured in culture medium using the OptEIA ELISA sets according to manufacturer's instructions (obtained from BD Pharmingen). The coefficient of variation (CV) of inter-assay and intra-assay for ELISA in the experiments was less than 10%.

EXAMPLE 14 Statistics Data are presented as a mean of each triplicate assay. Statistical comparisons between groups were performed using a one-way ANOVA followed by a Dunnett's test, as appropriate. Differences among groups were considered significant when P < 0.05.

While the description above refers to particular embodiments of the present invention, it will be understood that many modifications may be made without departing from the spirit thereof. The accompanying claims are intended to cover such modifications as would fall within the true scope and spirit of the present invention. The presently disclosed embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims, rather than the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

WHAT IS CLAIMED IS:

1. A vaccine composition, comprising: a volume of dendritic cells (DCs) in which IL-10 expression is downregulated; and a carrier.

2. The vaccine composition of claim 1, wherein said volume of DCs is between 1 million and 10 million DCs.

3. The vaccine composition of claim 1, wherein said volume of DCs is transfected with small-interference RNA (siRNA) that targets a fragment of IL-10 mRNA to downregulate said IL-10 expression.

4. The vaccine composition of claim 3, wherein said fragment spans the region adjacent to an initiation site of IL-10 translation.

5. The vaccine composition of claim 3, wherein said fragment comprises nucleotides 292-310 of IL-10 mRNA.

6. The vaccine composition of claim 3, wherein said fragment comprises nucleotides 441-459 of IL-10 mRNA.

7. The vaccine composition of claim 3, wherein said siRNA comprises SEQ ID NO.2.

8. The vaccine composition of claim 3, wherein said siRNA comprises SEQ ID NO.3.

9. The vaccine composition of claim 1, wherein said volume of DCs is derived from a human tissue source.

10. The vaccine composition of claim 1, wherein said volume of DCs is derived from human blood, human bone marrow, or both.

11. The vaccine composition of claim 1, wherein said vaccine is formulated for delivery to a mammal.

12. The vaccine composition of claim 1 1, wherein said vaccine is formulated for delivery by a delivery technique selected from the group consisting of injection, infusion, deposition, implantation, oral ingestion, topical administration, and combinations thereof.

13. The vaccine composition of claim 11 , wherein said mammal is a human.

14. The vaccine composition of claim 1 , further comprising an additional component selected from the group consisting of a vehicle, an additive, a pharmaceutical adjunct, a therapeutic compound, adjuvants, diluents, excipients, cytokines, growth hormones, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α and -β, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin, nerve growth factors, platelet-growth factor, transforming growth factors, insulin-like growth factor-I and -II, erythropoietin, osteoinductive factors, interferons, colony stimulating factors, interieukins, tumor necrosis factors, polypeptide factors including LIF, kit ligand and flt-3 ligand, any biologically active derivatives thereof, and combinations thereof.

15. A method for treating a disease condition in a mammal, comprising: providing a vaccine composition, comprising a volume of dendritic cells (DCs) in which IL-10 expression is downregulated, and a carrier; and administering said vaccine composition to said mammal in an amount sufficient to treat said disease condition.

16. The method of claim 15, wherein said volume of DCs is transfected with small- interference RNA (siRNA) that targets a fragment of IL-10 mRNA to downregulate said JL-10 expression.

17. The method of claim 16, wherein said fragment comprises nucleotides 292-310 of IL- 10 mRNA.

18. The method of claim 16, wherein said fragment comprises nucleotides 441-459 of IL- 10 mRNA.

19. The method of claim 16, wherein said siRNA comprises SEQ ID NO.2.

20. The method of claim 16, wherein said siRNA comprises SEQ LD NO.3.

21. The method of claim 15, wherein said volume of DCs is between 1 million and 10 million DCs.

22. The method of claim 15, wherein said volume of DCs is derived from a human tissue source.

23. The method of claim 15, wherein said volume of DCs is derived from human blood, human bone marrow, or both.

24. The method of claim 15, wherein said volume of DCs is prepared for use by a technique selected from the group consisting of pulsing said DCs with antigenic proteins, contacting said DCs with antigenic proteins, transfecting said DCs to express antigenic proteins, fusing said DCs with cells carrying antigenic proteins, and combinations thereof.

25. The method of claim 15, wherein administering said vaccine composition is accomplished by a technique selected from the group consisting of injection, infusion, deposition, implantation, oral ingestion, topical administration, any technique for delivery of a vaccine, and combinations thereof.

26. The method of claim 15, wherein said mammal is a human.

27. The method of claim 15, wherein said disease condition is selected from the group consisting of bacterial infections, protozoan, listeriosis, microbial infections, viral infections, autoimmune diseases, ankolysing spondylitis, cancer, malignancyprostate cancer, melanoma, kidney cancer, colorectal cancer, lung cancer, non-Hodgkin's lymphoma, breast cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, head and neck cancer, and brain cancer.

28. The method of claim 15, wherein said vaccine composition further comprises an additional component selected from the group consisting of a vehicle, an additive, a pharmaceutical adjunct, a therapeutic compound, adjuvants, diluents, excipients, cytokines, growth hormones, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α and -β, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin, nerve growth factors, platelet-growth factor, transforming growth factors, insulin-like growth factor-I and -II, erythropoietin (EPO), osteoinductive factors, interferons, colony stimulating factors, interieukins, tumor necrosis factors, polypeptide factors including LfF, kit ligand (KL) and flt-3 ligand, any biologically active derivatives thereof, and combinations thereof.

29. An isolated dendritic cell (DC) transfected with small-interfence RNA (siRNA) that targets a fragment of IL-10 mRNA.

30. The isolated DC of claim 29, wherein said fragment comprises nucleotides 292-310 of IL-10 mRNA.

31. The isolated DC of claim 29, wherein said fragment comprises nucleotides 441-459 of IL-10 mRNA.

32. The isolated DC of claim 29, wherein said siRNA comprises SEQ ID NO.2.

33. The isolated DC of claim 29, wherein said siRNA comprises SEQ ID NO.3.

34. A kit, comprising: a vaccine composition, comprising a volume of dendritic cells (DCs) in which IL-10 expression is downregulated, and a carrier; and instructions for the use of said vaccine composition in the treatment of a disease condition in a mammal.

35. The kit of claim 34, wherein said volume of DCs is transfected with small- interference RNA (siRNA) that targets a fragment of IL-10 mRNA to downregulate said IL-10 expression.

36. The kit of claim 35, wherein said fragment comprises nucleotides 292-310 of IL-10 mRNA.

37. The kit of claim 35, wherein said fragment comprises nucleotides 441-459 of IL-10 mRNA.

38. The kit of claim 35, wherein said siRNA comprises SEQ JD NO.2.

39. The kit of claim 35, wherein said siRNA comprises SEQ ID NO.3.

40. The kit of claim 34, wherein said volume of DCs is prepared for use by a technique selected from the group consisting of pulsing said DCs with antigenic proteins, contacting said DCs with antigenic proteins, transfecting said DCs to express antigenic proteins, fusing said DCs with cells carrying antigenic proteins, and combinations thereof.

41. The kit of claim 34, wherein said vaccine composition is formulated for delivery to a mammal.

42. The kit of claim 34, wherein said mammal is a human.

43. The kit of claim 34, wherein said vaccine composition further comprises an additional component selected from the group consisting of a vehicle, an additive, a pharmaceutical adjunct, a therapeutic compound, adjuvants, diluents, excipients, cytokines, growth hormones, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α and -β, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin, nerve growth factors, platelet-growth factor, transforming growth factors, insulin-like growth factor-I and -II, erythropoietin (EPO), osteoinductive factors, interferons, colony stimulating factors, interieukins, tumor necrosis factors, polypeptide factors including LLF, kit ligand (KL) and flt-3 ligand, any biologically active derivatives thereof, and combinations thereof.

44. The kit of claim 34, wherein said disease condition is selected from the group consisting of bacterial infections, protozoan, listeriosis, microbial infections, viral infections, autoimmune diseases, ankolysing spondylitis, cancer, malignancyprostate cancer, melanoma, kidney cancer, colorectal cancer, lung cancer, non-Hodgkin's lymphoma, breast cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, head and neck cancer, and brain cancer.

45. A method of creating a vaccine, comprising: providing isolated dendritic cells (DCs); transfecting said DCs with small-interference RNA (siRNA) that targets a fragment of IL-10 mRNA; and formulating said DCs transfected with said siRNA into a composition for delivery as a vaccine.

46. The method of claim 45, wherein said fragment spans the region adjacent to an initiation site of LL-10 translation.

47. The method of claim 45, wherein said fragment comprises nucleotides 292-310 of IL- 10 mRNA.

48. The method of claim 45, wherein said fragment comprises nucleotides 441-459 of IL- 10 mRNA.

49. The method of claim 45, wherein said siRNA comprises SEQ JD NO.2.

50. The method of claim 45, wherein said siRNA comprises SEQ ID NO.3.