HK1079989B - Compositions and methods for priming monocytic dendritic cells and t cells for th-1 response - Google Patents

Description

Compositions and methods for eliciting TH-1 responses in monocytic dendritic cells and T cells

Background

Antigen Presenting Cells (APCs) are important in eliciting an effective immune response. They not only present antigens to T cells with antigen-specific T cell receptors, but also provide the signals necessary for T cell activation. These signals are still not fully interpreted, but include many cell surface molecules as well as cytokines or growth factors. The factors necessary for naive or unpolarized T cell activation may differ from those necessary for memory T cell reactivation. The ability of APCs to present antigen and deliver T cell activation signals is often described as accessory cell function. Although monocytes and B cells have been shown to be competent APCs, their antigen presenting capacity in vitro appears to be limited to the reactivation of previously primed T cells. Thus, they are not capable of functionally directly activating naive or unprimed T cell populations.

Dendritic Cells (DCs) are professional antigen presenting cells in the immune system thought to be able to activate naive and memory T cells. Dendritic cells are increasingly prepared ex vivo for immunotherapy, particularly of cancer. The preparation of dendritic cells with optimal immunostimulatory properties requires an understanding and exploitation of the biology of ex vivo culture of these cells. Various protocols for culturing these cells have been described, with the various advantages of each protocol. Recent protocols include the use of serum-free media, and the use of maturation conditions that confer predetermined immunostimulatory properties to the cultured cells.

Maturation of dendritic cells is the process of converting immature DCs, phenotypically similar to skin langerhans cells (langerhansscells), into mature antigen presenting cells that can migrate to lymph nodes. This process results in the loss of the strong antigen uptake capacity characteristic of immature dendritic cells, as well as the upregulation of co-stimulatory cell surface molecules and the expression of various cytokines. Known maturation protocols are based on the in vivo environment believed to be encountered by the DC during or after exposure to the antigen. The best example of this method is the use of Monocyte Conditioned Medium (MCM) as the cell culture medium. MCM is produced in vitro by culturing monocytes and is used as a source of maturation factors. The major components of MCM involved in maturation have been reported to be (pro) inflammatory cytokines interleukin 1 β (IL-1 β), interleukin 6(IL-6) and tumor necrosis factor α (TNF α). Other maturation factors include prostaglandin E2(PGE2), poly-dldc, Vasoactive Intestinal Peptide (VIP), bacterial Lipopolysaccharides (LPS), and mycobacterial or mycobacterial components, such as specific cell wall components.

Fully mature dendritic cells differ from immature DCs in both quality and quantity. Fully mature DCs express higher levels of MHC class I and class II antigens, as well as T cell costimulatory molecules, namely CD80 and CD 86. These changes enhance the ability of dendritic cells to activate T cells because they increase the antigen density on the cell surface and the magnitude of the T cell activation signal by T cell costimulatory molecular counterparts, such as, for example, CD 28. In addition, mature DCs produce large amounts of cytokines, which stimulate and direct T cell responses. Two of these cytokines are interleukin 10(IL-10) and interleukin 12 (IL-12). These cytokines have an opposite effect on the direction of the induced T cell response. IL-10 products lead to the induction of Th-2 type responses, whereas IL-12 products lead to Th-1 type responses. The latter response is particularly desirable in situations where a cellular immune response is desired, such as, for example, in cancer immunotherapy. Th-1 type responses result in the induction and differentiation of Cytotoxic T Lymphocytes (CTL), which are effector arms of the cellular immune system (effector arm). This effector arm is most effective in resisting tumor growth. IL-12 also induces the growth of Natural Killer (NK) cells and has anti-angiogenic activity, both of which are potent anti-tumor weapons. Therefore, the use of IL-12 producing dendritic cells is theoretically most useful for immune stimulation.

Certain dendritic cell maturation factors, such as, for example, bacterial lipopolysaccharides, bacterial cpgdnas, double-stranded RNAs, and CD40 ligands have been reported to induce IL-12 production by immature DCs and sensitize immature DCs to produce Th-1 type responses. In contrast, anti-inflammatory molecules such as IL-10, TGF- β, PGE-2, and corticosteroids inhibit IL-12 production and sensitize cells to produce Th-2 type responses.

It has recently been reported that IL-12 production by dendritic cells is enhanced by interferon gamma in combination with certain dendritic cell maturation factors, such as bacterial Lipopolysaccharide (LPS) and CD 40. However, both LPS and CD40 have the known ability to induce small amounts of IL-12 during maturation. Thus, it is possible that the addition of IFN γ merely enhances production. Interferon gamma signaling utilizes the Jak2-Stat1 pathway, including tyrosine phosphorylation of the tyrosine residue at Stat1701 before it migrates to the nucleus and subsequent transcriptional enhancement of interferon gamma responsive genes. However, the signal transduction pathway of dendritic cells derived from human monocytes is poorly understood. The mechanism of action of interferon gamma in these cells has not been determined.

Attenuated bovine strains of Mycobacterium tuberculosis (Mycobacterium bovis), currently known as bacillus calmette-guerin (BCG), have been used for cancer immunotherapy. In one example, intravesical administration of live BCG has proven effective in treating bladder cancer, although the mechanism of such treatment is not known. The effects of BCG administration are hypothesized to be mediated by inducing an immune response that attacks, e.g., cancer cells. The specific role of BCG in this response is thought to be that of a generalized inducer of immune responses and has an adjuvant function in presenting tumor antigens to the immune system.

BCG was also found to be a potent maturation factor for dendritic cells, with the ability to up-regulate the maturation marker CD 83. BCG is also able to up-regulate MHC molecules as well as co-stimulatory molecules CD80 and CD86, with a concomitant decrease in endocytic capacity. In addition, BCG or BCG-derived lipoarabinomannan (lipoarabidomannan) has been reported to enhance cytokine production, although IL-12 production was found to be specifically inhibited, in contrast to the results found for other DC maturation factors. This latter property, i.e., inhibition of IL-12 production, reduces the appeal of using BCG to mature dendritic cells for immunotherapy expecting a strong cell-mediated cytotoxic response (Th-1 response).

Therefore, the application of BCG in active immunotherapy has the potential to induce dendritic cell maturation. However, there is (still) a need for compositions and methods of using such compositions that induce maturation of such dendritic cells while providing broad immune stimulation and priming of those dendritic cells to generate a type 1 (Th-1) immune response with a strong cytotoxic T cell response.

Summary of The Invention

The present invention provides methods and compositions for inducing maturation of immature Dendritic Cells (DCs) and priming those cells for an antigen-specific cytotoxic T cell response, using factors that simultaneously provide broad immune stimulation (i.e., BCG). In one aspect, a method of producing a population of mature dendritic cells is provided, comprising providing immature dendritic cells; and contacting the immature dendritic cells with an effective concentration of BCG and interferon gamma (IFN γ) under culture conditions suitable for maturation of the immature dendritic cells to form a mature dendritic cell population. The mature dendritic cell population produces an increased ratio of interleukin 12 to interleukin 10 than an immature dendritic cell population that has not been contacted with BCG and IFN γ during maturation. The immature dendritic cells can be contacted with a predetermined antigen prior to or during contact with BCG and IFN γ. The predetermined antigen can be, for example, a tumor-specific antigen, a tumor-associated antigen, a viral antigen, a bacterial antigen, a tumor cell, a bacterial cell, a recombinant cell expressing an antigen, a cell lysate, a membrane preparation, a recombinantly produced antigen, a peptide antigen (e.g., a synthetic peptide antigen), or an isolated antigen.

In certain embodiments, the method optionally further comprises isolating the monocytic dendritic cell precursors; and culturing the precursor in the presence of a differentiation factor to form the immature dendritic cells. Suitable differentiation factors include, for example, GM-CSF, interleukin 4, a combination of GM-CSF and interleukin 4, or interleukin 13. Monocytic dendritic cell precursors can be isolated from a human subject. In particular embodiments, the mature dendritic cells produce IL-12 to IL-10 in a ratio of at least 1: 1.

In another aspect, methods of preparing a population of mature dendritic cells are provided. The methods generally include providing immature dendritic cells; and contacting the immature dendritic cells with an effective amount of BCG and interferon gamma (IFN γ) under culture conditions suitable for maturation of the immature dendritic cells to form a mature dendritic cell population. The resulting population of mature dendritic cells produces a type 1 immune response. The immature dendritic cells can be contacted with a predetermined antigen prior to or during contact with BCG and IFN γ. The predetermined antigen can be, for example, a tumor-specific antigen, a tumor-associated antigen, a viral antigen, a bacterial antigen, a tumor cell, a bacterial cell, a recombinant cell expressing an antigen, a cell lysate, a membrane preparation, a recombinantly produced antigen, a peptide antigen (i.e., a synthetic peptide), or an isolated antigen.

In certain embodiments, the method optionally further comprises isolating the monocytic dendritic cell precursors; and culturing the precursor in the presence of a differentiation factor to form the immature dendritic cells. Suitable differentiation factors include, for example, GM-CSF, interleukin 4, a combination of GM-CSF and interleukin 4, or interleukin 13. Monocytic dendritic cell precursors can be isolated from a human subject. In particular embodiments, the mature dendritic cells produce IL-12 to IL-10 in a ratio of at least about 1: 1.

In yet another aspect, compositions for activating T cells are provided. The composition can include a population of dendritic cells matured with effective concentrations of BCG and IFN γ under suitable maturation conditions; and a predetermined antigen. The dendritic cell population is capable of producing an increased ratio of interleukin 12(IL-12) to interleukin 10(IL-10) compared to a mature dendritic cell population that was contacted with BCG during maturation without IFN γ. In certain embodiments, the dendritic cell population is capable of producing IL-12 to IL-10 in a ratio of at least about 10 to 1. In another embodiment, the dendritic cell population is capable of producing IL-12 to IL-10 in a ratio of at least about 100 to 1 as compared to a similar immature dendritic cell population cultured in the presence of BCG and in the absence of IFN γ during maturation.

In another aspect, an isolated population of immature dendritic cells is provided. The cell population includes immature monocytic dendritic cells, and BCG and IFN γ in effective concentrations to induce maturation of the immature dendritic cells. The resulting mature dendritic cells produce more interleukin 12(IL-12) than interleukin 10(IL-10) as compared to a similar population of immature dendritic cells cultured in the presence of BCG and in the absence of IFN γ during maturation. The cell population may optionally include a predetermined antigen and/or isolated T cells, such as naive T cells. T cells may optionally be present in an isolated lymphocyte preparation.

Methods of making activated T cells are also provided. The methods generally include providing immature dendritic cells; contacting the immature dendritic cells with a predetermined antigen; and contacting the immature dendritic cells with effective concentrations of BCG and IFN γ under culture conditions suitable for maturation of the immature dendritic cells to form mature dendritic cells. Mature dendritic cells can be contacted with naive T cells to form activated T cells that produce IFN γ and/or polarize towards a type 1 (Th-1) response. Suitable antigens include, for example, tumor specific antigens, tumor associated antigens, viral antigens, bacterial antigens, tumor cells, bacterial cells, recombinant cells expressing antigens, cell lysates, membrane preparations, recombinantly produced antigens, peptide antigens (e.g., synthetic peptide antigens), or isolated antigens.

The immature dendritic cells can be contacted with the predetermined antigen, BCG and IFN γ simultaneously, or the cells can be contacted with the predetermined antigen prior to contacting with BCG and IFN γ. In certain embodiments, the method may further comprise isolating the monocytic dendritic cell precursors; and culturing the precursor in the presence of a differentiation factor to induce the formation of immature dendritic cells. Suitable differentiation factors include, for example, GM-CSF, interleukin 4, a combination of GM-CSF and interleukin 4, or interleukin 13. Monocytic dendritic cell precursors can optionally be isolated from a human subject. In particular embodiments, the immature dendritic cells and the T cells are autologous with respect to each other (autologous).

Also provided are isolated mature dendritic cells that produce more interleukin 12(IL-12) than interleukin 10 (IL-10). The mature dendritic cells can be provided by maturing immature dendritic cells with a composition comprising an effective concentration of BCG and IFN γ under conditions suitable for dendritic cell maturation. The isolated mature dendritic cells can optionally include a predetermined antigen. Isolated mature dendritic cells loaded with a predetermined antigen are also provided. The dendritic cells are capable of producing more interleukin 12(IL-12) than interleukin 10(IL-10), such as, for example, at least 10-fold more IL-12 than IL-10 as compared to a similar population of immature dendritic cells cultured in the presence of BCG and in the absence of IFN γ during maturation.

Also provided are methods of generating a type 1 (Th-1) immune response in an animal. The methods generally include providing immature dendritic cells; contacting the immature dendritic cells with effective amounts of BCG and interferon gamma (IFN γ) and a predetermined antigen under culture conditions suitable for maturation of the immature dendritic cells to form mature dendritic cells. The mature dendritic cells can be administered to an animal or contacted with naive T cells to form activated T cells characterized by production of interferon gamma (IFN γ) and/or tumor necrosis factor α (TNF α). The activated T cells can be administered to an animal in need of stimulation of a cytotoxic T cell response against a particular antigen. Suitable antigens include, for example, tumor specific antigens, tumor associated antigens, viral antigens, bacterial antigens, tumor cells, bacterial cells, recombinant cells expressing antigens, cell lysates, membrane preparations, recombinantly produced antigens, peptide antigens (e.g., synthetic peptide antigens), or isolated antigens. The immature dendritic cells can optionally be contacted with the predetermined antigen, BCG, and IFN γ simultaneously, or the immature dendritic cells can be contacted with the predetermined antigen prior to contacting with BCG and IFN γ.

In certain embodiments, the method may further comprise isolating the monocytic dendritic cell precursors from the animal; and culturing the precursor in the presence of a differentiation factor to form the immature dendritic cells. The differentiation factor may be, for example, GM-CSF, interleukin 4, a combination of GM-CSF and interleukin 4, or interleukin 13.

The immature dendritic cells and T cells can be autologous to the animal or allogeneic to the animal (allogenic). Alternatively, immature dendritic cells and T cells can have the same MHC haplotype as the animal, or share MHC markers. In certain embodiments, the animal may be a human, or may be a non-human animal.

Detailed description of the invention

The present invention provides methods for inducing maturation of immature Dendritic Cells (DCs) and priming those cells for an antigen-specific cytotoxic T cell response (Th-1 response). The invention also provides dendritic cell populations useful for activating and producing T cells polarized towards the production of type 1 cytokines (e.g., IFN γ, TNF α, and/or IL-2). Such dendritic cell populations include immature monocytic dendritic cells contacted with BCG, IFN γ and a predetermined antigen under suitable maturation conditions. The immature dendritic cells can be contacted with the antigen during or prior to maturation. Alternatively, immature monocytic dendritic cells that have been exposed to an antigen (e.g., in vivo) can be contacted with BCG and IFN γ under suitable maturation conditions. The resulting mature dendritic cells are primed to activate and polarize the T cells towards a type 1 response. Type 1 responses include the production of type 1 cytokines (e.g., IFN γ and/or IL-2), the production of more IL-12 than IL-10, cytotoxic T cell responses, the production of Th-1 cells, and the production of certain types of antibodies. And also up-regulate tumor necrosis factor alpha (TNF α). In contrast, type 2 responses are characterized by production of IL-4, IL-5 and IL-10, production of more IL-10 than IL-12, production of Th2 cells, and lack of induction of CTL responses.

In a related aspect, compositions are provided that comprise immature dendritic cell maturation factors (e.g., monocytic dendritic cell precursors) that can also prime those dendritic cells to generate a type 1 response. Such mature primed monocytic dendritic cells are capable of enhancing the presentation of a predetermined antigen, i.e., a predetermined foreign antigen, by the Major Histocompatibility Complex (MHC) class I. Presentation of antigen by MHC class I molecules is expected to induce differentiation of Cytotoxic T Lymphocytes (CTL) and stimulate antigen-specific CTL-mediated lysis of target cells. Such compositions include BCG and IFN γ, which can be mixed with a cell population comprising immature dendritic cells to mature the immature dendritic cells and reverse or overcome the inhibition of IL-12 induced by contacting the immature dendritic cells with BCG. Immature dendritic cells contacted with such compositions undergo maturation and typically produce greater amounts of IL-12 than IL-10 as compared to immature dendritic cell populations contacted with BCG alone.

In another aspect, monocytic dendritic cell precursors obtained from a subject or donor can be contacted with cytokines (e.g., GM-CSF and IL-4) to obtain immature dendritic cells. The immature dendritic cells can then be contacted with a predetermined antigen, either alone or in combination with BCG and IFN γ, or with a cytokine, to mature the dendritic cells and sensitize the cells to induce a T cell type 1 immune response. In certain embodiments, MHC class I antigen processing is stimulated, which can be used to elicit a CTL response against cells displaying a predetermined antigen.

Dendritic cells are a diverse population of antigen presenting cells found in many lymphoid and non-lymphoid tissues (see Liu, Cell 106: 259-62 (2001); Steinman, Ann. Rev. Immunol.9: 271-96 (1991)). Dendritic cells include lymphoid dendritic cells in the spleen, Langerhans cells of the epidermis, and cryptic cells (Veiled cells) in the blood circulation. Dendritic cells are generally classified as a group based on their morphology, high levels of expression of surface MHC class II molecules, and the lack of certain other surface markers expressed on T cells, B cells, monocytes, and natural killer cells. In particular, monocyte-derived dendritic cells (also referred to as monocytic dendritic cells) typically express CD11c, CD80, CD86, and are HLA-DR⁺However, CD14^-。

In contrast, monocytic dendritic cell precursors (typically monocytes) are generallyIs CD14⁺. Monocytic dendritic cell precursors can be obtained from any tissue in which they colonize, particularly lymphoid tissues such as spleen, bone marrow, lymph nodes and thymus. Monocytic dendritic cell precursors can also be isolated from the circulatory system. Peripheral blood is a readily available source of monocytic dendritic cell precursors. Umbilical cord blood is another source of monocytic dendritic cell precursors. Monocytic dendritic cell precursors can be isolated from a number of organisms capable of eliciting an immune response. Such organisms include animals, including, by way of example, humans, as well as non-human animals, e.g., primates, mammals (including dogs, cats, mice, and rats), birds (including chickens), and transgenic species thereof.

In certain embodiments, the monocytic dendritic cell precursors and/or immature dendritic cells can be isolated from a healthy subject or a subject in need of immune stimulation, such as, for example, a prostate cancer patient or other subject for which cellular immune stimulation is beneficial or desirable (i.e., a subject having a bacterial or viral infection, etc.). Dendritic cell precursors and/or immature dendritic cells can also be obtained from HLA-matched (HLA-matched) healthy individuals for administration to HLA-matched patients in need of immunostimulation.

Dendritic cellsPrecursor and immatureDendritic cells

Methods for isolating enriched dendritic cell precursors and immature dendritic cells from various sources, including blood and bone marrow, are well known in the art. For example, heparinized blood can be collected, by apheresis or leukopheresis, by preparing buffy coats, rosettes, centrifugation, density gradient centrifugation (e.g., using Ficoll (e.g., using a Rosetting method), by preparing buffy coat(colloidal silica particles (diameter 15-30mm) coated with non-dialyzing polyvinylpyrrolidone (PVP)), sucrose, etc.), differential cell lysis, filtrationAnd the like, to isolate dendritic cell precursors and immature dendritic cells. In certain embodiments, for example, a population of leukocytes can be prepared by collecting subject blood, defibrinating to remove platelets, and lysing red blood cells. Dendritic cell precursors and immature dendritic cells can optionally be generated, for example, byThe monocytic dendritic cell precursors were enriched by gradient centrifugation.

Dendritic cell precursors and immature dendritic cells can optionally be prepared in a closed, sterile system. As used herein, the term "closed, sterile system" or "closed system" refers to a system in which exposure to non-sterile ambient or air streams or other non-sterile conditions is minimized or eliminated. Closed systems for separating dendritic cell precursors and immature dendritic cells typically preclude density gradient centrifugation in open tubes, transfer of cells in open air (open air), culture of cells in tissue culture plates or unsealed flasks, and the like. In typical embodiments, the closed system allows for the sterile transfer of dendritic cell precursors and immature dendritic cells from a primary collection vessel to a sealable tissue culture vessel without exposure to non-sterile air.

In certain embodiments, monocytic dendritic cell precursors are isolated by adhesion to a monocyte-binding substrate as disclosed in U.S. patent application No.60/307,978 (attorney docket No.020093-002600US), filed 7/25/2001, the disclosure of which is incorporated herein by reference. For example, a population of leukocytes (e.g., isolated by leukapheresis) can be contacted with the monocytic dendritic cell precursor adhesion substrate. When the population of leukocytes is contacted with the substrate, the monocytic dendritic cell precursors in the leukocyte population preferentially adhere to the substrate. Other leukocytes (including other potential dendritic cell precursors) exhibit reduced substrate binding affinity, thereby allowing the monocytic dendritic cell precursors to be preferentially enriched at the substrate surface.

Suitable substrates include, for example, those having a large surface area to volume ratio. Such a matrix may be, for example, a particulate or fibrous matrix. Suitable microparticle matrices include, for example, glass particles, plastic particles, glass-coated polystyrene particles, and other beads suitable for protein adsorption. Suitable fibrous substrates include microcapillaries and microvillous membranes. The particulate or fibrous substrate generally allows the adherent monocytic dendritic cell precursors to be eluted without substantially reducing the viability of the adherent cells. The particulate or fibrous substrate may be substantially non-porous to facilitate elution of the monocytic dendritic cell precursors or dendritic cells from the substrate. A "substantially nonporous" matrix is one in which at least a majority of the pores present in the matrix are smaller than the cells so as to minimize capture of the cells by the matrix.

Adhesion of the monocytic dendritic cell precursors to the substrate can optionally be enhanced by the addition of a binding medium. Suitable binding media include monocytic dendritic cell precursor media (e.g., mediaRPMI 1640、DMEM、 Etc.), either alone or in any combination, supplemented with, for example, cytokines (such as granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin 4(IL-4), or interleukin 13(IL-13)), plasma, serum (such as human serum, e.g., autologous or allogeneic serum), purified proteins, e.g., serum albumin, divalent cations (such as calcium and/or magnesium ions), and other molecules that aid in the specific adhesion of monocytic dendritic cell precursors to a substrate, or prevent the adhesion of non-monocytic dendritic cell precursors to a substrate. In certain embodiments, the plasma and serum can be heat inactivated. The heat-inactivated plasma and the leukocytes may be autologous or heterologous.

After the monocytic dendritic cell precursors adhere to the substrate, the nonadherent leukocytes are separated from the monocytic dendritic cell precursor/substrate complexes. The non-adherent cells can be separated from the complex using any suitable method. For example, the non-adherent leukocyte and complex mixture can be pelleted and the non-adherent leukocytes and media decanted or drained. Alternatively, the mixture can be centrifuged and the supernatant containing non-adherent leukocytes decanted or drained from the precipitated complex.

Isolated dendritic cell precursors can be cultured ex vivo for differentiation, maturation and/or expansion (as used herein, isolated immature dendritic cells, dendritic cell precursors, T cells, and other cells refer to cells that exist by man out of their natural environment and are therefore not natural products. Briefly, ex vivo differentiation typically involves culturing dendritic cell precursors or cell populations having dendritic cell precursors in the presence of one or more differentiation factors. Suitable differentiation factors may be, for example, cell growth factors (such as cytokines, e.g., (GM-CSF), interleukin 4(IL-4), interleukin 13(IL-13), and/or combinations thereof). In certain embodiments, the monocytic dendritic cell precursors differentiate to form monocyte-derived immature dendritic cells.

Dendritic cell precursors can be cultured and differentiated under suitable culture conditions. Suitable tissue culture media includeRPMI 1640、DMEM、 And the like. The tissue culture medium may be supplemented with serum, amino acids, vitamins, cytokines, such as GM-CSF and/or IL-4, divalent cations, and the like, to promote cell differentiation. In certain embodiments, the dendritic cell precursors can be cultured in serum-free media. Such culture conditions may optionally exclude any animal-derived products. A typical cytokine combination in a typical dendritic cell culture medium is approximately 500 units/ml for each of GM-CSF and IL-4. Dendritic cell precursors in differentiationImmature dendritic cells, when formed, are phenotypically similar to skin langerhans cells. Immature dendritic cells are typically CD14^-And CD11c⁺Express low levels of CD86 and CD83 and are able to capture soluble antigens by specific endocytosis.

The immature dendritic cells are matured to form mature dendritic cells. Mature DCs lose the ability to take up antigen and appear to co-stimulate the up-regulation of cell surface molecules and the expression of various cytokines. In particular, mature DCs express higher levels of MHC class I and class II antigens than immature dendritic cells, and mature dendritic cells are generally identified as CD80⁺、CD83⁺、CD86⁺And CD14^-. Higher MHC expression leads to an increase in DC surface antigen density, while upregulation of the co-stimulatory molecules CD80 and CD86 potentiates the T cell activation signal by the counterparts of the co-stimulatory molecules (costermart), such as CD28 on T cells.

The mature dendritic cells of the present invention can be prepared (i.e., matured) by contacting the immature dendritic cells with an effective amount or concentration of BCG and IFN γ. An effective amount of BCG is typically about 10 per milliliter of tissue culture medium⁵-10⁷cfu. An effective amount of IFN γ is typically about 100-1000U per ml of tissue culture medium. Bacillus calmette-guerin (BCG) is an attenuated strain of mycobacterium bovis (m.bovis). As used herein, BCG refers to whole BCG, as well as mural components, BCG-derived lipoarabinomannans, and other BCG components associated with induction of type 2 immune responses. BCG is optionally inactivated, e.g., heat inactivated BCG, formalin treated BCG, and the like.

BCG increased the expression of surface maturation markers CD83 and CD86 on dendritic cells, with concomitant suppression of IL-12 production and exclusion of antigen by endocytosis. Without intending to be bound by any particular theory, dendritic cells matured with BCG have also been described as being involved in homotypic aggregation and in the release of tumor necrosis factor-alpha (TNF α) (see Thurnher et al, int.j. cancer 70: 128-34(1997), incorporated herein by reference). Maturation of immature dendritic cells with IFN γ and BCG promotes IL-12 production by DCs and reduces and inhibits IL-10 production, thereby priming the mature dendritic cells for a type 1 (Th-1) response.

The immature DCs are typically contacted with effective amounts of BCG and IFN γ for about 1 hour to about 24 hours. Immature dendritic cells can be cultured and matured under suitable maturation culture conditions. Suitable tissue culture media includeRPMI 1640、DMEM、 And the like. The tissue culture medium may be supplemented with amino acids, vitamins, cytokines, such as GM-CSF and/or IL-4, divalent cations, and the like, to promote cell maturation. A typical cytokine combination is about 500 units/ml for each of GM-CSF and IL-4.

Maturation of dendritic cells can be monitored by methods known in the art. Cell surface markers can be detected by assays familiar to the art, such as flow cytometry, immunohistochemistry, and the like. Cells can also be monitored for cytokine production (e.g., by ELISA, FACS, and other immunoassays). In the DC population matured according to the invention, IL-12 levels were higher than IL-10 levels to promote a type 1 (Th-1) response. For example, DC can produce IL-12/IL-10 ratio from greater than 1: 1 to about 10: 1 and about 100: 1. Mature DCs also lose the ability to take up antigen by pinocytosis, which can be assayed by uptake assays familiar to those of ordinary skill in the art. Dendritic cell precursors, immature dendritic cells and mature dendritic cells, whether primed or unprimed, along with antigen can be cryopreserved for later use. Methods of refrigeration are well known in the art. See U.S. Pat. No.5,788,963, which is incorporated herein by reference in its entirety.

Antigens

Mature primed dendritic cells according to the invention are capable of presenting antigen to T cells. Mature primed dendritic cells can be formed by contacting immature dendritic cells with a predetermined antigen before and during maturation. Alternatively, immature dendritic cells that have been contacted with an antigen (e.g., within an isolation precursor) can be contacted with a composition comprising BCG and IFN γ to form mature dendritic cells that are primed for a type 1 (Th-1) response.

Suitable predetermined antigens may include any antigen for which activation of T cells is desired. Such antigens may include, for example, bacterial antigens, tumor-specific or tumor-associated antigens (e.g., whole cells, tumor cell lysates, isolated tumor antigens, fusion proteins, liposomes, etc.), viral antigens, and any other antigens or antigen fragments, such as peptide or polypeptide antigens. In certain embodiments, the antigen may be, for example, but not limited to, Prostate Specific Membrane Antigen (PSMA), Prostate Acid Phosphatase (PAP), or Prostate Specific Antigen (PSA) (see Pepsidero et al, Cancer Res.40: 2428-32 (1980); McCormac et al, Urology 45: 729-44 (1995)). The antigen may also be a bacterial cell, a bacterial lysate, a membrane fragment of a cell lysate, or any other source known in the art. The antigen may be expressed or recombinantly produced, or even chemically synthesized. The recombinant antigen may also be expressed on the surface of a host cell (e.g., bacteria, yeast, insect, vertebrate, or mammalian), may be present in the lysate, or may be purified from the lysate.

The antigen may also be present in a sample from the subject. For example, a tissue sample from a subject with hyperproliferative or other disease can be used as a source of antigen. Such samples may be obtained, for example, by biopsy or surgical resection. Such antigens may be used as lysates or as isolated preparations. Alternatively, membrane preparations of cells from a subject (e.g., a cancer patient) or established cell lines can also be used as antigens or antigen sources.

In exemplary embodiments, prostate tumor cell lysate recovered from a surgical sample can be used as an antigen source. For example, samples of cancer patient tumors obtained in biopsy or surgical resection procedures may be used directly to present antigens to dendritic cells, or to provide cell lysates for antigen presentation. Alternatively, membrane preparations of tumor cells from cancer patients can be used. The tumor cell may be prostate cancer, lung cancer, ovarian cancer, colon cancer, brain cancer, melanoma, or any other type of tumor cell. Lysates and membrane preparations can be prepared from isolated tumor cells by methods well known in the art.

In another exemplary embodiment, purified or semi-purified prostate specific membrane antigen (PSMA, also known as PSM antigen) can be used as the antigen that specifically reacts with monoclonal antibody 7E11-C.5 (see Horoszewicz et al, prog.Clin.biol.Res.37: 115-32(1983), U.S. Pat. No.5,162,504; U.S. Pat. No.5,788,963; Feng et al, proc.am.Assoc. cancer Res.32 (abs.1418) (238 1991); the disclosure of which is incorporated herein by reference). In yet another exemplary embodiment, an antigenic peptide having the amino acid residue sequence Leu Leu His Glu Thr Asp Ser Ala Val (SEQ ID NO: 1) (designated PSM-P1) corresponding to amino acid residues 4-12 of PSMA can be used as an antigen. Alternatively, an antigenic peptide having the amino acid residue sequence Ala Leu Phe Asp Ile GluSer Lys Val (SEQ ID NO: 2) (designated PSM-P2) corresponding to amino acid residue 711-719 of PSMA can be used as the antigen.

In particular embodiments, an antigenic peptide having the amino acid residue sequence Xaa Leu (or Met) Xaa Xaa Xaa Xaa Xaa Xaa Val (or Leu) (designated PSM-PX) where Xaa represents any amino acid residue may be utilized as an antigen. This peptide is similar to the HLA-A0201 binding motif, a 9-10 amino acid residue binding motif found in HLA-A2 patients with "anchor residues" leucine and valine (see Grey et al, Cancer Surveys 22: 37-49 (1995)). The peptide can be used as HLA-A2⁺Patient antigens (see Central Data Analysis Committee "apple Frequencies", section 6.3, edited by Tsuji, K. et al, Tokyo University Press, page 1066-. Likewise, peptides like other HLA binding motifs can be utilized.

Typically, as previously described, immature dendritic cells are cultured under suitable maturation conditions in the presence of BCG, IFN γ and the predetermined antigen. Alternatively, immature dendritic cells can be mixed with a predetermined antigen in a typical dendritic cell culture medium without GM-CSF and IL-4 or maturation factors. After at least about 10 minutes to 2 days of incubation with the antigen, the antigen can be removed and media supplemented with BCG and IFN γ can be added. Cytokines (e.g., GM-CSF and IL-4) may also be added to the maturation medium. Methods of dendritic Cell contact are generally known in the art (see Steel and Nutman, J.Immunol.160: 351-60 (1998); Tao et al, J.Immunol.158: 4237-44 (1997); Dozmorov and Miller, Cell Immunol.178: 187-96 (1997); Inaba et al, J Exp Med.166: 182-94 (1987); Macatonia et al, J Exp Med.169: 1255-64 (1989); De Bruijn et al, Eur.J.Immunol.22: 3013-20 (1992); the disclosure of which is incorporated herein by reference).

The resulting mature primed dendritic cells are then co-incubated with T cells, such as naive T cells. T cells or T cell subsets can be obtained from a number of lymphoid tissues for use as responder cells. Such tissues include, but are not limited to, the spleen, lymph nodes, and/or peripheral blood. The cells can be co-incubated with mature primed dendritic cells as a mixed population of T cells or as a purified subpopulation of T cells. Purification of T cells can be achieved by positive or negative selection, including, but not limited to, using antibodies against CD2, CD3, CD4, CD8, and the like.

By contacting the T cells with mature primed dendritic cells, antigen-reactive or activated polarized T cells or T lymphocytes are provided. As used herein, the term "polarized" refers to T cells that produce high levels of IFN γ or are otherwise sensitized to induce a type 1 (Th-1) response. Such methods typically comprise contacting immature dendritic cells with BCG and IFN γ to prepare mature primed dendritic cells. The immature dendritic cells can be contacted with a predetermined antigen during or prior to maturation. Immature dendritic cells can be co-cultured with T cells (e.g., naive T cells) during maturation, or after maturation and priming of the dendritic cells to induce a type 1 response. The immature dendritic cells or mature dendritic cells can be maturePre-enrichment. In addition, T cells can be enriched from a population of lymphocytes prior to contact with dendritic cells. In particular embodiments, enriched or purified CD4⁺The population of T cells is contacted with dendritic cells. Co-culturing mature primed dendritic cells and T cells results in stimulation of specific T cells, which mature into antigen-reactive CD4⁺T cell or antigen reactive CD8⁺T cells.

In another aspect, a method of restimulating T cells in vitro is provided by culturing cells in the presence of mature dendritic cells primed towards an induced type 1 (Th-1) T cell response. Such T cells may optionally be cultured on feeder cells. The mature primed dendritic cells can optionally be irradiated prior to contact with the T cells. Suitable culture conditions may include one or more cytokines (e.g., purified IL-2, concanavalin A stimulated spleen cell supernatant, or interleukin 15 (IL-15)). By adding immature dendritic cells, BCG, IFN gamma and predetermined antigenIn vitroRestimulation of T cells can be used to promote expansion of T cell populations.

Stable antigen-specific polarized T cell cultures or T cell lines can be restimulated periodicallyIn vitroCan be maintained for a long time. The T cell cultures or T cell lines so established can be stored and, if preserved (e.g., formulated with a refrigerant and frozen), can be used indefinitely to re-provide activated polarized T cells within a desired time interval.

In certain embodiments, activated CD8⁺Or CD4⁺T cells can be produced according to the methods of the invention. Typically, the mature primed dendritic cells used to generate the antigen-reactive polarized T cells are syngeneic with (e.g., obtained from) the subject to which they are to be administered. Alternatively, dendritic cells having the same HLA haplotype as the recipient subject of interest can be prepared in vitro using non-cancerous cells (e.g., normal cells) from an HLA-matched donor. In particular embodiments, antigen-reactive T cells, including CTL and Th-1 cells, are expanded in vitro as a source of immunostimulatory cells.

In vivo administration of cell populations

In another aspect of the invention, methods are provided for administering mature primed dendritic cells, or activated polarized T cells, or a cell population containing such cells, to a subject in need of immune stimulation. Such cell populations may include a population of mature primed dendritic cells and/or a population of activated polarized T cells. In certain embodiments, such methods are performed by obtaining dendritic cell precursors or immature dendritic cells, differentiating and maturing those cells in the presence of BCG, IFN γ and a predetermined antigen to form a mature dendritic cell population that is primed towards a Th-1 response. The immature dendritic cells can be contacted with the antigen prior to or during maturation. Such mature primed dendritic cells can be administered directly to a subject in need of immunostimulation.

In related embodiments, the mature primed dendritic cells can be contacted with lymphocytes from the subject to stimulate T cells within the population of lymphocytes. Activated polarized lymphocytes, optionally clonally expanded in cell culture, with antigen-reactive CD4⁺And/or CD8⁺T cells may be administered prior to administration to a subject in need of immune stimulation. In certain embodiments, the activated polarized T cells are autologous to the subject.

In another embodiment, the dendritic cells, T cells, and recipient subject have the same mhc (hla) haplotype. Methods for determining a subject's HLA haplotype are well known in the art. In related embodiments, the dendritic cells and/or T cells are allogeneic to the recipient subject. For example, dendritic cells can be allogeneic with respect to T cells and recipients, having the same mhc (hla) haplotype. Allogeneic cells typically match at least one MHC allele (e.g., share at least one but not all MHC alleles). In a less typical embodiment, the dendritic cells, T cells, and recipient subject are allogeneic with respect to each other, but all have at least one MHC allele in common.

According to a principleIn embodiments, the T cells are obtained from the same subject from which the immature dendritic cells were obtained. In thatIn vitroFollowing maturation and polarization, the autologous T cells are administered to the subject to prime and/or enhance an existing immune response. For example, T cells may be administered by intravenous infusion, e.g., at about 10⁸-10⁹Cells/m²The dosage of body surface area (see Ridell et al, Science 257: 238-41(1992), incorporated herein by reference). The infusion may be repeated at desired intervals, for example monthly. Any indication of adverse effects of the receptor can be monitored during and after T cell infusion.

According to another embodiment, dendritic cells matured with BCG and IFN γ according to the invention can be injected directly into a tumor or other tissue containing the target antigen. Such mature cells are capable of taking up and presenting antigen to T cells in vivo.

Drawings

FIG. 1 depicts IL-12p70 production by immature DCs in response to an increased number of BCG or an increased number of BCG in combination with interferon gamma (IFN γ).

Examples

The following examples are provided merely to illustrate various aspects of the present invention and should not be construed as limiting the invention in any way.

Example 1: IL-10 and IL-12 produced under different maturation conditions:

in this example, cytokine production by a population of immature dendritic cells contacted with the maturation factors BCG and/or IFN γ was determined. Immature DC are obtained by supplementing peripheral blood mononuclear cells with 1% human plasmaPrepared by contacting the plastic in the presence of medium (Gibco-BRL). Unbound monocytes are removed by washing. Bound monocytes inThe culture was incubated in the presence of 500U GM-CSF and 500U IL-4 per ml for 6 days.

In study 1, immature dendritic cells were matured by addition of inactivated BCG. The resulting mature dendritic cells were assayed for cytokine production. Towards inThe immature dendritic cells in the medium were added with inactivated BCG at various concentrations, followed by culture at 37 ℃ for 24 hours. BCG dilutions added per ml are shown in the table, starting at 4.1X 10⁸A stock solution of cfu/ml. Cytokine production was determined by ELISA assay using antibodies to the cytokine to be detected. Briefly, cytokines on the solid phase surface will be captured using cytokine-specific antibodies (e.g., IL-12 or IL-10). The solid phase surface is then treated with a second labeled antibody against the cytokine to detect the presence of the captured cytokine. The secondary antibody is typically labeled with an enzyme to facilitate detection by colorimetric analysis. Results representative of the experiments are shown in table 1 below. The amount of cytokine, or the production of cytokine, is expressed in pg/ml.

TABLE 1

IL-10 and IL-12 production by BCG-matured DCs

Donor

Detected cytokines

Without added factor

BCG 1∶100

BCG 1∶250

BCG 1∶500

BCG 1∶1000

TNFα+ IL-1β

2

IL-12p70

＜5

393

239

335

＜5

＜5

IL-12p40

nd

nd

2852

nd

nd

nd

2

IL-10

76.5

1206

700

338

153

380

[0072]

2	p70/IL-10	＜0.06	0.33	0.34	1	＜0.03	＜0.01
								3	IL-12p70	249	318	260	74	＜5	＜5
	IL-12p40	nd	nd	12257	nd	nd	nd
								3	IL-10	305	426	162	124	＜5	70
	p70/IL-10	0.82	0.75	1.60	0.60	nd	＜0.07

("nd" means not tested)

Referring to Table 1, the results demonstrate that the addition of BCG can increase the production of cytokines IL-12, IL-10, or subunits thereof, although both the relative and absolute levels of cytokine production are donor dependent. The highest level of increase is seen in IL-12p40 and IL-10, suggesting that the observed low level of IL-12p70 (consisting of p35 and p40 subunits) relative to the level of IL-12p40 is due to the lack of IL-12p35 product. Under all conditions, the ratio of IL-12p70 levels to IL-10 was less than or equal to 1, except in the presence of BCG, indicating that maturation of immature dendritic cells in the presence of BCG alone is likely to polarize naive T cells towards a Th-2 response.

The effect of introducing IFN γ under similar conditions was also determined and is listed in table 2 below. Cytokine production was measured as described above. Comparing tables 1 and 2, it is apparent that the addition of IFN γ in the presence of a maturation factor (such as BCG) increases IL-12p70 production. In particular, the addition of IFN γ together with BCG during maturation increased IL-12p35 production and decreased IL-10 production. As a result, the ratio of IL-12p70 to IL-10 was always greater than 1 when IFN γ was added in conjunction with BCG. In some donors, and under certain conditions, the ratio of IL-12p70 to IL-10 production can be increased to greater than about 100: 1 by the addition of IFN γ in conjunction with BCG. Thus, these results surprisingly demonstrate that the addition of IFN γ together with the maturation factor BCG can significantly increase the production of IL-12p 70.

TABLE 2

IL-10 and IL-12 production by BCG and IFN gamma matured DCs

Donor	Cytokine	IFN gamma alone	BCG1∶100 +IFNγ	BCG1∶250 +IFNγ	BCG1∶500 +IFNγ	BCG 1∶1000 +IFNγ	TNFα +IL1β +IFNγ
								2	IL-12p70	＜5	1223	801	848	461	521
	IL-12p40	＜5	nd	23751	19362	8666	Nd
								2	IL-10	＜5	470	510	394	179	95
	p70/IL-10		2.60	1.57	2.15	2.58	5.48
								3	IL-12p70	212	6858	5380	2934	949	231
	IL-12p40	＜5	nd	48351	nd	16164	25645
								3	IL-10	141	254	241	157	＜5	163
	p70/IL-10	1.50	27	22	18	＞189	1.41

("nd" means not tested)

Example 2: the down-regulation of IL-10 by IFN γ is dose-dependent:

in this example, the ability of IFN γ in combination with BCG to down-regulate the production of IL-10 from a dendritic cell population was demonstrated. Immature dendritic cells were prepared as described above. Immature dendritic cells incubated alone, BCG (from 4.1X 10 cells) at one of two concentrations⁸cfu/ml stock in 1: 1000 or 1: 250 dilution) or exposed alone to IFN γ in the range of 0U to 1000U per ml. Use of commercially available antibodies (e.g.R) by ELISA (supra)&D Systems, Minneapolis, MN) the IL-10 production of the resulting dendritic cells was measured and reported in pg/ml. In the control group, immature DCs cultured alone (without added BCG or IFN γ) produced no detectable IL-10. In contrast, DCs cultured in the presence of IFN γ alone produced small amounts of IL-10 (approximately 20-30 pg/ml). The amount of IL-10 produced is not dose-dependent in the range of 10U to 1000U IFN γ per ml.

In contrast, DCs matured in the presence of BCG alone produced large amounts of IL-10, producing approximately 150pg/ml or > 250pg/ml of IL-10 in the presence of a 1: 1000 or 1: 250 fold dilution of BCG stock, respectively. Addition of IFN γ to BCG during DC maturation resulted in down-regulation of IL-10 production in a dose-dependent manner. For DCs cultured in the presence of a 1: 1000 dilution of BCG, IL-10 production decreased from about 150pg/ml IL-10 (without IFN γ) to about 20-30pg/ml IL-10(1000U IFN γ). For DCs cultured in the presence of the BCG 1: 250 dilution, IL-10 production decreased from about 270pg/ml IL-10 (without IFN γ) to about 50pg/ml IL-10(1000U IFN γ). Thus, maturation of immature DCs in the presence of BCG and IFN γ down-regulated IL-10 production and overcome the apparent stimulation of IL-10 production induced by BCG alone.

Example 3: IL-12 upregulation by IFN γ is dose-dependent:

in this example, the ability of IFN γ to upregulate IL-10 production was demonstrated. Immature dendritic cells were derived from monocyte cultures grown in the presence of GM-CSF and IL-4 for 6 days, as described above. Immature DCs were treated with BCG alone at various dilutions, or with BCG in combination with various concentrations of IFN γ for an additional two days. Culture supernatants were tested for the presence of IL-12p70 by ELISA assay, as described above.

The results representative of the experiment are shown in figure 1. Results were determined in triplicate for each culture. Mature DCs produced relatively low (< 1000pg/ml) average concentrations of IL-12p70 in response to increasing amounts of BCG alone. The amount of IL-12 produced decreased with increasing amounts of BCG in a dose-dependent manner. In contrast, the amount of IL-12 increased significantly to approximately 5000pg/ml upon addition of IFN γ (10U/ml) together with BCG (stock solution at 1: 1000 dilution). Upon addition of 100U/ml IFN γ together with BCG (stock 1: 1000 dilution), the amount of IL-12 rose to almost 20,000 pg/ml. Addition of 500U/ml or 1000U/ml IFN γ together with BCG (1: 1000 dilution of stock) produced IL-12 at levels of approximately 21,000pg/ml and 22,000pg/ml, respectively.

Taken together, although BCG alone appears to antagonize IL-12 production, maturation of immature DCs in the presence of BCG and IFN γ significantly increases IL-12 production.

Example 4: stimulation of antigen-specific T cells:

in this example, immature DCs matured in the presence of BCG and IFN γ were shown to stimulate antigen-specific T cells to produce IFN γ. Influenza A virus specific T cell lines were obtained by supplementing with 5% human serum2X 10 medium temperature culture⁶Cells/ml Peripheral Blood Mononuclear Cells (PBMC) and 5. mu.g/ml influenza M1 peptide (GILGFVFTL; SEQ ID NO: 3). These culture conditions lead to the selective expansion of those T cells specific for the influenza M1 peptide. After 2 days of culture, 20U/ml IL-2 and 5ng/ml IL-15 were added to the culture. After approximately 7 to 14 days of culture, the T cell lines were placed in cytokine-free medium overnight.

Antigen-specific T cells were then co-incubated with immature DCs, with DCs matured with BCG alone, or with DCs matured with BCG and IFN γ. The ratio of antigen-specific T cells to DCs was 1: 1. T cells and DCs were incubated at 37 ℃ for 24 hours. DC were either directly loaded or osmotically loaded with influenza M1 peptide. Briefly, immature DCs were collected from culture flasks and concentrated by centrifugation. For osmotic loading, cells were suspended in a small volume of hypertonic medium followed by the addition of an equal volume of influenza M1 peptide dissolved in PBS. After incubation on ice for 10 minutes, the cells were washed thoroughly. For direct loading, cells are suspended in equal volumesThe medium and influenza M1 peptide in PBS were incubated for 1 hour at 37 ℃. Cells were incubated at 37 ℃ for 2 hours to allow antigen processing.

After co-incubation of T cells and DCs, T cell responses were measured by ELISA to quantify IFN γ in 100 μ l samples of culture supernatants. The results show that, independent of the DC loading method, DCs stimulated with BCG and IFN γ are super stimulators of antigen specific T cells. T cells co-cultured with immature DCs produced very little IFN γ (< 2,000pg/ml), whereas T cells co-cultured with DCs matured with BCG alone were intermediate producers of IFN γ (> 5,000 pg/ml). T cells co-incubated with DCs matured with BCG and IFN γ produced high levels of IFN γ (> 20,000pg/ml for osmotically loaded DCs, or > 25,000pg/ml for directly loaded DCs).

Thus, DCs matured with BCG and IFN γ are better stimulators of antigen-specific T cells, independent of the method of DC loading with antigen.

Example 5: regeneration of antigen-specific T cell responses in vitro:

keyhole Limpet Hemocyanin (KLH) -specific T cell lines were generated by stimulating PBMCs with a 10: 1 ratio of T cells to DCs matured either from BCG or from BCG and IFN γ and loaded with KLH or a control protein. The T cells and mature DCs were supplied with fresh medium (supplemented with 5% human AB serum, 20U/ml IL-2 and 5ng/ml IL-15) every 3 to 4 daysMedia). Cells can be expanded into larger flasks as needed. Because of the low overall precursor frequency to an antigen and because the naive cells require a powerful stimulus to respond, the stimulus is repeated 3-4 times in a 10-21 day interval. Cells were allowed to recover overnight in cytokine-free medium prior to restimulation.

Stimulated T cell lines were tested for KLH-specific cell proliferation using a standard 3 day thymidine incorporation assay. Stimulated T cells were incubated with KLH-pulsed immature dendritic cells at different DC to T cell ratios. T cell proliferation was measured as Counts Per Minute (CPM). Cell proliferation, or cytokine production in response to a stimulus is considered evidence of an antigen-specific response. To control antigen specificity, a negative control antigen (influenza a) was also included in the experiment.

When T cells were stimulated with BCG matured DCs alone, T cell proliferation was always low (< 5,000 CPM). This low level of proliferation is low whether the DCs are exposed to KLH or influenza a virus. Low levels of proliferation were also observed in incubation with immature DCs. No significant differences were observed between 3 groups of DCs using effector to stimulus ratios of 50: 1, 25: 1, or 12.5: 1(T cell to DC). As a control, dendritic cells matured with BCG and IFN γ were used to stimulate T cells, and KLH-pulsed DCs always induced higher T cell proliferation (approximately 10,000-33,000CPM) compared to immature DCs or mature DCs pulsed with influenza A virus. For mature DCs pulsed with KLH antigen, T cell proliferation increased in proportion to the increase in effector to stimulator ratio.

T cell effector function is also monitored by cytokine secretion. KLH-specific T cell lines (generated as described above) were stimulated with either BCG alone or with BCG and IFN γ matured DCs. Stimulated T cell lines were tested for cytokine production by intracellular cytokine staining after non-specific stimulation of cells with anti-CD 3 antibody (50ng/ml) and PMA (5 ng/ml). Cytokine production is measured as the percentage of cells that produce a particular cytokine. Very low to undetectable percentages (< 5%) of cell samples produce intracellular IL-2, IL-4, IL-5 or IL-10. IL-5 and IL-10 were not detected in T cells stimulated by DCs matured with BCG and IFN γ. T cells stimulated by BCG-matured DCs alone produced low levels of IFN γ (< 10%) and TNF- α (< 15%). In contrast, more T cells stimulated with IFN γ and BCG mature DCs produced IFN γ (approximately 35%) and TNF α (> 45%). IFN gamma is known IL-12 produced by stimulus. Thus, by stimulating T cells with DCs matured with BCG and IFN γ, T cells were polarized towards a type 1 (Th-1) response.

Example 6: induction of the Th-1 cytokine tumor necrosis factor alpha (TNF α):

in this example, the ability of the combination of BCG and IFN γ to upregulate the type 1 cytokine tumor necrosis factor α (TNF α) was demonstrated. Briefly, immature dendritic cells are as described aboveThe cells are obtained and grown in the presence of GM-CSF and IL-4. Immature DCs were incubated with BCG alone or in combination with IFN γ for 24 hours. Subsequently, protein transport inhibitors (GolgiPlug) were added^TMPharMingen) to block transport of the produced cytokines from the golgi complex, and the cells were incubated overnight. Cells were then harvested, permeabilized, and stained internally with a TNF α -specific fluorescently labeled antibody or isotype control antibody using methods well known in the art. Frequency of TNF α positive DCs and fluorescence intensity of cells were determined using FACS analysis (table 3). It was found that DCs matured with BCG in the presence of IFN γ enhanced the ability of DCs to produce the Th 1 cytokine TNF α.

TABLE 3

TNF alpha produced by DC matured in the presence of BCG with or without IFN gamma

Example 7: induction of response to cell-associated antigens:

this example demonstrates that DCs matured in the presence of BCG and IFN γ can elicit significantly higher tumor-specific T cell IFN γ release and similar levels of antigen-specific cytotoxicity than DCs matured with BCG alone. Immature dendritic cells were isolated as indicated above and cultured in the presence of GM-CSF and IL-4. The DCs were then loaded with intact tumor cells previously infected with recombinant adenoviruses expressing either Green Fluorescent Protein (GFP) or influenza A M1 protein (a 549). After 24 hours, the DCs were matured with BCG or BCG in combination with IFN γ. Tumor-bearing DC or GFP or M1-expressing tumor cells were used to stimulate autologous M1-specific T cell lines. After 24 hours, cell culture supernatants were collected and subjected to a standard IFN γ ELISA. Only M1-loaded tumor cell-expressing DCs were able to stimulate IFN γ release, and matured DCs in BCG plus IFN γ induced this response significantly more potently than immature or BCG matured DCs.

TABLE 4

Induction of a response against a cell-associated antigen

The foregoing examples are provided for illustration and are not intended to limit the scope of the claimed invention. Other variations of the invention will be apparent to those of ordinary skill in the art and are encompassed by the appended claims. All publications, patents, patent applications, and other references cited herein are also incorporated by reference in their entirety.

Claims (39)

1. An in vitro method of preparing a population of mature dendritic cells, comprising:

providing immature dendritic cells; and are

Contacting immature dendritic cells with an effective amount of bcg and interferon gamma under culture conditions suitable for maturation of the immature dendritic cells to form a mature dendritic cell population;

wherein the mature dendritic cell population produces an increased ratio of interleukin 12 to interleukin 10 as compared to a population of immature dendritic cells that have not been contacted with bcg and interferon gamma during maturation.

2. The method of claim 1, further comprising contacting the immature dendritic cells with a predetermined antigen prior to contacting with bcg and interferon gamma.

3. The method of claim 1, further comprising simultaneously contacting the immature dendritic cells with a predetermined antigen, bcg, and interferon gamma.

4. The method of claim 2 or 3, wherein the predetermined antigen is a tumor specific antigen, a tumor associated antigen, a viral antigen, a bacterial antigen, a tumor cell, a bacterial cell, a cell lysate, a membrane preparation, a recombinantly produced antigen, a peptide antigen, or an isolated antigen.

5. The method of claim 1, further comprising:

isolating monocytic dendritic cell precursors; and

culturing the precursor in the presence of a differentiation factor to form immature dendritic cells.

6. The method of claim 5, wherein the differentiation factor is granulocyte/macrophage colony stimulating factor, interleukin 4, a combination of granulocyte/macrophage colony stimulating factor and interleukin 4, or interleukin 13.

7. The method of claim 5, wherein the monocytic dendritic cell precursors are isolated from a human subject.

8. The method of claim 1, wherein the mature dendritic cells produce interleukin 12 to interleukin 10 in a ratio of at least about 1: 1.

9. The method of claim 1, wherein the mature dendritic cells produce interleukin 12 to interleukin 10 in a ratio of at least about 10: 1.

10. The method of claim 1, wherein the mature dendritic cells produce interleukin 12 to interleukin 10 in a ratio of at least about 100: 1.

11. An in vitro method of preparing a population of mature dendritic cells, comprising:

providing immature dendritic cells; and

contacting immature dendritic cells with an effective amount of bcg and interferon gamma under culture conditions suitable for maturation of the immature dendritic cells to form a mature dendritic cell population;

wherein the mature dendritic cell population produces a type 1 immune response.

12. The method of claim 11, further comprising contacting the immature dendritic cells with a predetermined antigen prior to contacting with bcg and interferon gamma.

13. The method of claim 11, further comprising simultaneously contacting the immature dendritic cells with a predetermined antigen, bcg, and interferon gamma.

14. The method of claim 12 or 13, wherein the predetermined antigen is a tumor specific antigen, a tumor associated antigen, a viral antigen, a bacterial antigen, a tumor cell, a bacterial cell, a recombinant cell expressing an antigen, a cell lysate, a membrane preparation, a recombinantly produced antigen, a peptide antigen, or an isolated antigen.

15. The method of claim 11, further comprising:

isolating monocytic dendritic cell precursors; and

culturing the precursor in the presence of a differentiation factor to form immature dendritic cells.

16. The method of claim 15, wherein the differentiation factor is granulocyte/macrophage colony stimulating factor, interleukin 4, a combination of granulocyte/macrophage colony stimulating factor and interleukin 4, or interleukin 13.

17. The method of claim 11, wherein the monocytic dendritic cell precursors are isolated from a human subject.

18. The method of claim 11, wherein the mature dendritic cells produce interleukin 12 to interleukin 10 in a ratio of at least about 1: 1.

19. The method of claim 11, wherein the mature dendritic cells produce interleukin 12 to interleukin 10 in a ratio of at least about 10: 1.

20. The method of claim 11, wherein the mature dendritic cells produce interleukin 12 to interleukin 10 in a ratio of at least about 100: 1.

21. A composition for activating T cells comprising:

a dendritic cell population matured with an effective concentration of BCG and interferon gamma under conditions suitable for maturation; and

a predetermined antigen;

wherein the population of dendritic cells produces an increased ratio of interleukin 12 to interleukin 10 compared to a population of mature dendritic cells contacted with bcg during maturation without interferon gamma.

22. The composition of claim 21, wherein the dendritic cell population produces interleukin 12 to interleukin 10 in a ratio of at least about 10: 1.

23. The composition of claim 21, wherein the dendritic cell population produces interleukin 12 to interleukin 10 in a ratio of at least about 100: 1.

24. An isolated population of cells comprising:

isolated immature monocytic dendritic cells, and an effective concentration of bcg and interferon gamma to induce maturation of the immature dendritic cells;

wherein the resulting population of mature dendritic cells produces more interleukin 12 than interleukin 10.

25. The cell population of claim 24, further comprising a predetermined antigen.

26. The cell population of claim 24, further comprising isolated T cells.

27. The cell population of claim 26, wherein the T cells are naive T cells.

28. The cell population of claim 24, further comprising isolated lymphocytes.

29. An in vitro method for preparing T cells sensitized to induce an anti-naive Th-1 response comprising:

providing immature dendritic cells;

contacting the immature dendritic cells with a predetermined antigen;

contacting immature dendritic cells with an effective concentration of bcg and interferon gamma under culture conditions suitable for maturation of the immature dendritic cells to form mature dendritic cells; and

contacting the mature dendritic cells with naive T cells to form interferon gamma-producing activated T cells.

30. The method of claim 29, wherein the predetermined antigen is a tumor specific antigen, a tumor associated antigen, a viral antigen, a bacterial antigen, a tumor cell, a bacterial cell, a recombinant cell expressing an antigen, a cell lysate, a membrane preparation, a recombinantly produced antigen, a peptide antigen, or an isolated antigen.

31. The method of claim 29, wherein the immature dendritic cells are contacted simultaneously with the predetermined antigen, bcg, and interferon gamma.

32. The method of claim 29, further comprising:

isolating monocytic dendritic cell precursors; and

culturing the precursor in the presence of a differentiation factor to form immature dendritic cells.

33. The method of claim 32, wherein the differentiation factor is granulocyte/macrophage colony stimulating factor, interleukin 4, a combination of granulocyte/macrophage colony stimulating factor and interleukin 4, or interleukin 13.

34. The method of claim 32, wherein the monocytic dendritic cell precursors are isolated from a human subject.

35. The method of claim 29, wherein the immature dendritic cells and the T cells are autologous with respect to each other.

36. An isolated mature dendritic cell producing more interleukin 12 than interleukin 10 prepared by maturing immature dendritic cells with a composition comprising an effective concentration of bcg and interferon gamma under conditions suitable for dendritic cell maturation.

37. The isolated mature dendritic cell of claim 36, further comprising a predetermined antigen.

38. Isolated mature dendritic cells loaded with a predetermined antigen that produce more interleukin 12 than interleukin 10.

39. The isolated mature dendritic cell of claim 38, wherein the cell produces at least 10-fold more interleukin 12 than interleukin 10.