WO2016120651A1 - Runx3 dependent enhanced dendritic cell generation - Google Patents

Runx3 dependent enhanced dendritic cell generation Download PDF

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Publication number
WO2016120651A1
WO2016120651A1 PCT/HU2016/050002 HU2016050002W WO2016120651A1 WO 2016120651 A1 WO2016120651 A1 WO 2016120651A1 HU 2016050002 W HU2016050002 W HU 2016050002W WO 2016120651 A1 WO2016120651 A1 WO 2016120651A1
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cells
cell
differentiation
runx3
dcs
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French (fr)
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István SZATMÁRI
Erika TAKÁCS
Pál BOTÓ
Bianka Mónika TÓTH
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Debreceni Egyetem
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the invention relates to a method of producing dendritic cells from cells capable of differentiation in a cell culture.
  • the invention also provides dendritic cells produced by the method of the invention and medical uses of such cells.
  • DC Dendritic cells
  • APCs antigen presenting cells
  • DCs are abundant in the epithelia, connective tissue, peripheral lymphoid nodes, the circulation system and in the T cell rich areas of the spleen.
  • immature DCs are able to take up pathogens, membrane fragments and soluble proteins by macropinocytosis.
  • the activated DCs migrate to the peripheral lymphoid organs and may come into contact with T cells.
  • Stationary DCs sample and process antigens directly in the lymphoid organs.
  • Contact with a T cell results in the loss of the ability of antigen uptake and differentiation.
  • Maturation is associated with upregulation of MHC II molecules and costimulatory molecules such as CD80 and CD86 (Banchereau and Steinman, 1998).
  • Mature DCs have the potential to stimulate TH and TC cells as well as B cells.
  • DCs are very heterogeneous with respect to their cell-surface markers and function but as stated above, maturation can be characterized by the increased expression of MHC molecules and costimulatory molecules on the cell surface (e.g. CD80, CD86, CD40) and their ability to stimulate mixed lymphocyte reaction and capability to process and present protein antigen to T cells.
  • MHC molecules and costimulatory molecules e.g. CD80, CD86, CD40
  • DCs are also involved in the maintenance of self-tolerance in the periphery, inducing regulatory T cells or anergy of autoreactive T cells. Studies point to a primary role of DC in regulating in vivo allergic responses. (Senju 2010)
  • DCs are derived from hematopoietic bone marrow progenitor cells.
  • TF transcription factors
  • Runt-related transcription factor 3 belongs to the runt domain family of transcription factors that are key regulators of lineage-specific gene expression in major developmental pathways.
  • the Runx3 TF can either activate or suppress transcription.
  • Runx3 protein is barely detected in immature DC, and is highly expressed in mature DC. Runx3 is specifically expressed in mature DCs and mediates their response to TGF- ⁇ . In the absence of Runx3, DCs become insensitive to TGF- -induced maturation inhibition, and TGF- -dependent Langerhans cell development is impaired. Maturation of Runx3 knock out DCs is accelerated and accompanied by increased efficacy to stimulate T cells and aberrant expression of 2-integrins. Expression of Runx3 was also demonstrated in B- and myeloid cell lines (Levanon et al., 1994; Shi and Stavnezer, 1998; 1999; Bangsow et al., 2001). The role of the Runx complex in development and cell fate specification is discussed inter alia in Dominic Chih-Cheng Voon et al., 2015.
  • DCs to prime responses to tumor antigens provides a promising approach to cancer immunotherapy as well as the treatment of autoimmune diseases. Since DCs have an important role in the pathogenesis of several viruses, they have significance as a therapeutic target. Antigen-specific negative regulation of the immune response by DCs is a likely approach for treatments of autoimmune diseases, allergies and for regulation of allo-reactive immune response causing graft rejection and graft versus host disease.
  • Embryonic stem cells are characterized by pluripotency and infinite propagation capacity.
  • Non-virus mediated methods for gene transfer including targeted gene integration and procedures for isolation of appropriate transfectant cell clones, have been established for ES cells. (Senju 2003) Genetic modification of ES cells and subsequent in vitro differentiation to DCs is a promising strategy means for analysis of gene functions and differentiation of DCs.
  • Fainaru et al. generated DCs from bone marrow.
  • Their nonadherent fraction of bone marrow derived DC culture consisted at day 7 of immature DCs and granulocytes, and at days 11-14 of immature and spontaneously matured DCs. They also found that Runx3 protein was barely present at day 7, but readily detected when mature DCs arose.
  • Senju et al. describe another method to generate DCs from mouse embryonic stem cells.
  • ES cells were cultured on feeder cell layers of OP9, in the presence of granulocyte-macrophage colony-stimulating factor. The resulting cells were irregularly shaped floating cells with protrusions, expressed major histocompatibility complex class II, CDl lc, CD80, and CD86, with the capacity to process and present protein antigens to T cells.
  • Senju et al. designated them ES-DCs (ES cell-derived DCs). ES-DCs became mature DCs, characterized by a typical morphology (Senju et al., 2003.).
  • Senju et al. reported the generation and characterization of DCs derived from mouse and human iPS cells (Senju et al., 2009; Senju 2010).
  • the human iPS-DCs exhibited characteristics of DCs, as human ES-DC do, in morphology, surface molecules, and T cell-stimulating capacity.
  • the studies using mice have demonstrated that by in vivo transfer of genetically modified mouse ES-DCs immune responses can be effectively modulated both positively and negatively, making iPS-ECs a promising candidate in stem cell therapy.
  • Stem cells are cells capable of differentiating into more than one cell type of the endoderm, mesoderm and/or ectoderm germinal layer and are also capable of self-renewal and/or cell division by mitosis without differentiation. Stem cells are able to give rise to differentiated cells and are capable of both symmetric and asymmetric cell division. Stem cells also have the capacity to replace damaged tissue.
  • embryonic stem cells have a far greater differentiation potential and can develop into almost every type of cell in the body.
  • Adult stem cells can develop into a limited number of cell types only.
  • Embryonic stem cells are pluripotent stem cells which can be isolated from the inner cell mass or can be derived from somatic cells by cell reprogramming induced pluripotent stem (iPS) cells.
  • ES cells express markers that may be used to detect the presence of undifferentiated cells in a culture and are easily recognized by the skilled person. Such characteristic markers are Oct3/4, SSEA-1, Nanog, Zfp42, Esrrb. Alkaline phosphatase staining is also commonly used.
  • iPS cells are stem cells derived from somatic cells inducing the reprogramming of these cells with the overexpression of Oct3/4, Sox2, Klf4 and c-Myc.
  • the iPS cell lines exhibit similar morphology and growth properties as ES cells and express ES cell-specific genes.
  • iPS cells can be derived from various somatic cells.
  • Cell differentiation is the process by which a less specialized cell becomes a more specialized cell type.
  • a totipotent or pluripotent cell may become a differentiated cell through the stages of a multipotent stem cell that has a limited differentiation potential, an intermediate progenitor cell that has the capacity of only a limited number of cell divisions and a postmitotic, differentiated cell.
  • a "cell type” is a cell classification unit used to distinguish between classes (types) of functionally differentiated cells having functionally, as well as morphologically or phenotypically identical properties which are distinct from other classes within a multicellular, preferably an animal species. Said identical properties correspond to the expression pattern of said particular class of cells in the organism. Thus, a given cell type show well defined functional and gene expressional properties. While variances within cell types may occur, and various classifications may be applied, cell types are distinctive and discrete categories and clearly distinguishable from each other within a classification method or system.
  • DCs are specialized cells of the immune system with the unique capacity for initiating primary and secondary T and B lymphocyte responses.
  • Dendritic cells are antigen presenting cells having branched projections ("dendrites”) at certain development stages, resulting in a large contact surface to the surroundings.
  • “Immature DCs” are characterized by high endocytotic activity and a lower T cell activating potential. Contact with an antigen results in activation and maturing: DCs present the taken up antigens on their surfaces with MHC molecules and express coactivators.
  • “Mature DCs” lose their ability to take up antigens, have a high T and B cell activating potential and express high levels of MHC molecules and costimulatory molecules, typically CD80, CD86 on their surfaces. Mature DCs have the capacity to stimulate primary mixed leukocyte reaction.
  • Dendritic cell progenitor cells are proliferating cells that may differentiate into dendritic cells in the bone marrow that share phenotypic characteristics with myeloid precursor populations. Macrophage/dendritic cell progenitor cells (MDPs) are found in the bone marrow, give rise to dendritic cells subsets, and typically cannot differentiate into granulocytes. Common dendritic cell precursors (CDPs) are proliferating cells that differentiate into dendritic cells and into precursors for classical DCs (pre-cDCs) in the bone marrow. However, CDPs have lost the potential to give rise to monocytes.
  • MDPs Macrophage/dendritic cell progenitor cells
  • pre-cDCs classical DCs
  • Runx3 and “RUNX3”, indicating the gene product and the gene, respectively, or alternatively, indicating the mouse gene and the human gene, respectively, are used interchangeably.
  • the term Runx3 may refer to the mouse Runx3 gene, the human Runx3 gene or a Runx3 gene from another species.
  • forced expression implies the overexpression of a gene product by way of introducing a transgene into a cell and allowing the transgene to express the gene product.
  • the expression of the transgene may be transient or permanent. Forced expression may also mean the induction of the expression of an endogenous gene by chemical or genetic manipulation.
  • the invention relates to methods of modulating cells capable of differentiation, wherein the cells capable of differentiation are cells produced by a method not comprising the step of destructing a human embryo or doing harm to a human embryo.
  • the term "cells capable of differentiation” therefore relates to cells capable of differentiation produced by a method not comprising the step of destructing a human embryo or doing harm to a human embryo, throughout this description inclusive the claims.
  • the invention relates to a method of modulating cells capable of differentiation to develop into DCs comprising forced expression of Runx3 in cultured cells.
  • a method of modulating cells capable of differentiation to develop into DCs comprising forced expression of a Runx3 gene product in said cells capable of differentiation and culturing said cells in a medium appropriate for culturing and differentiation of said cells into DCs.
  • the method of the invention is suitable to produce a high percentage of DCs derived from cells capable of differentiation in a cell culture.
  • the method of the invention is suitable to produce differentiated DCs derived from cells capable of differentiation in a cell culture.
  • the method of the invention is suitable to produce activated DCs derived from cells capable of differentiation in a cell culture.
  • the method of the invention is suitable to produce DCs derived from cells capable of differentiation in a cell culture, wherein said DCs are characterized by that they express MHC and CD (e.g.
  • the method of modulating cells capable of differentiation to develop into DCs comprising the forced expression of Runx3 in cultured cells according to the invention is suitable to produce a higher percentage of DCs derived from cells capable of differentiation in a cell culture than a conventional method of modulating stem cells to develop into DCs without the forced expression of Runx3.
  • previous methods were able to produce 10% of mature DCs while it is possible to produce up to 25% or more mature DCs using the method of the invention.
  • the rate of CD86/MHCII double positive cells in the cell culture is at least 15%. More preferably, the rate of CD86/MHCII double positive cells in the cell culture is at least 20% or at least 25%.
  • the DCs are activated DCs.
  • the DCs produced by the method of the invention may have reached an activated stage of differentiation characterized by expressing higher levels of MHC, e.g. MHCII and costimulatory molecules, e.g. CD80, CD86 than immature DCs.
  • Zhang et al. were able to differentiate functional antigen presenting cells from pluripotent stem cells, e.g. ES cells or iPS cells, but the resulting proliferating myeloid cells (pMC) showed impaired proliferation and differentiated into immature DC-like cells (pMC-DC) expressing low levels of major histocompatibility complex (MHC)-I, MHC-II, CD40, CD80, and CD86 upon treatment with IL4 plus GM-CSF (Zhang et al. 2015). There is no suggestion in the study of Zhang et al. to use forced Runx3 expression to provide higher percentage of mature DCs. Dicken et al.
  • MHC major histocompatibility complex
  • Runx3 was strongly expressed in CD l ib positive splenic DCs and cell specific gene targeting models uncovered that the CDl lb/Esam hl positive DC development was particularly compromised in Runx3 deficient animals (Dicken et al., 2013). This study, however, failed to realize that besides MHCII, expression of CD86 is also augmented by Runx3, and thus Runx3 generally improves the maturation capacity of ES-DCs and may be used in a method to provide higher percentage of ES-derived mature DCs in a cell culture.
  • the elevated expression of the MHC and CD80/86 molecules may be detected upon LPS stimulation in the DCs produced by the method of the invention.
  • the cells to be differentiated into DCs may be any cells capable of differentiating into DCs.
  • the cells capable of differentiation suitable for the method of the invention may be embryonic stem cells, e.g. mouse embryonic stem cells or human embryonic stem cells.
  • the stem cells are induced pluripotent stem cells, e.g. mouse iPS cells or human iPS cells.
  • the cells suitable to be differentiated into activated DCs according to the method of the invention are progenitor cells, such as DC progenitor cells.
  • the Runx3 gene product may be a Runx3 mRNS or a Runx3 protein.
  • the method of modulating cells capable of differentiation to develop into DCs comprising forced expression of Runx3 in cultured cells according to the invention comprises
  • the inducible clones may be selected based on their specific antibiotic resistance (e.g. Geneticin/G418 resistance).
  • a genetically modified ES cell line into which a gene of interest can be inserted by ere mediated recombination is used and the at least the coding region of a Runx3 sequence is transferred to a modified p2Lox targeting plasmid using the Gateway cloning system, then these constructs are electroporated into Zx.1 ES cells, targeting the ES cells with the Neon transfection system.
  • the at least the coding region of Runx3 may be introduced into the cell by any method known in the art to be suitable for the introduction of exogenous genetic material into a cell, such as by lentivirus introduction, by adenovirus vectors, by plasmid transfer, etc.
  • the expression of Runx3 may be induced permanently or transiently.
  • Runx3 is induced with doxycycline.
  • the expression of Runx3 may be induced by e.g. tetracycline or other agents or techniques known in the art.
  • endogenous Runx3 is induced, e.g. by the TGF -pathway.
  • a DC is provided that is produced by the method of the invention as described supra.
  • the DC of the invention may be an activated DC derived from a cell capable of differentiation that has been differentiated by the method of the invention.
  • the DC of the invention may be a DC derived from a cell capable of differentiation characterized by that it expresses MHC and CD (e.g. CD 80, CD86) molecules on its surface.
  • the invention also relates to a cultured DC in which the expression of the endogenous Runx3 has been specifically induced so that it overexpresses a Runx3 gene product relative to a DC in which the expression of a gene product of an endogenous Runx3 has not been induced.
  • the expression of Runx3 is induced with doxycycline.
  • the expression of Runx3 may be induced by e.g. tetracycline or other agents or techniques known in the art.
  • the invention also relates to a DC obtainable by the method according to the invention for use in the treatment of a patient in need of DC administration.
  • the patient may be diagnosed with a disease associated with deficient activity of DCs or may be in need of increased DC activity.
  • the disease is cancer, an autoimmune disease, e.g. rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's disease, Sjorgen's disease, Addison's disease, type 1 diabetes, ankylosing spondylitis, IBD, Crohn's disease, psoriasis; an inflammatory disease, viral infection, e.g.
  • the DC used is autologous to the patient.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a DC obtainable by the methods described above and pharmaceutically acceptable excipients suitable for live cells.
  • the DC comprised in the pharmaceutical composition or the DC for use in the treatment of a disease associated with the activity of DCs according to the invention may comprise other genetic or chemical modifications suitable for use in the treatment of a disease associated with the activity of DCs.
  • modification may be e.g. the genetic engineering of the cell to express immunostimulatory molecules or the loading the DCs with a particular (tumor) antigen.
  • the DC according to the invention may be used in cell therapy as standalone therapeutic agents, i.e.
  • the DC therapy may be combined with other cancer therapy (e.g. radiation, chemotherapy).
  • cancer therapy e.g. radiation, chemotherapy.
  • the patient to be treated may be a vertebrate animal, preferably a mammal, e.g. a companion animal such as a dog, cat or rabbit. More preferably the patient is a human.
  • an exogenous Runx3 sequence is introduced into a DC which is to be used in the treatment of a disease in a patient
  • a Runx3 sequence that is autologous to the species of the patient is used.
  • the invention also relates to a kit comprising
  • Fig. 1 FACS analysis indicates that after 19 day of culture higher percent of MHCII/CD80 double positive DC were obtained upon doxycycline (+dox) treatment (Runx3 induction) with/without LPS stimulation in clone2 ES-DC cells (results from experiment no. 54).
  • Fig. 2 FACS analysis indicates that after 19 day of culture higher percent of MHCII/CD80 double positive DC were obtained upon doxycycline treatment (+dox) with/without LPS stimulation in clone2 ES-DC cells (results from experiment no. 55).
  • FIG. 3 FACS analysis indicates that after 19 day of culture higher percent of MHCII/CD80 double positive DC were obtained upon doxycycline treatment (+dox) with/without LPS stimulation in clone4 ES-DC cells (results from experiment no. 54).
  • FIG. 4 FACS analysis indicates that after 19 day of culture higher percent of MHCII/CD80 double positive DC were obtained upon doxycycline treatment (+dox) with/without LPS stimulation in clone4 ES-DC cells (results from experiment no. 55).
  • Runx3 driven enhanced ES-DC maturation Runx3 transgenic ES cells were differentiated for 19 days.
  • the Runx3 inducible cells were treated with 1 ⁇ g/ml doxycycline (+dox). In addition, the indicated cells were treated with LPS (100 ng/ ⁇ ) at day 18.
  • A Cell surface expression of CD80 and MHCII was assessed by flow cytometry at day 19. The average percent of the CD80/MHCII double positive cells and the SD values were calculated from 8 experiments.
  • B Cell surface expression of CD86 was assessed by flow cytometry at day 19. The average percent of the CD86 positive cells and the SD values were calculated from 8 experiments.
  • DCs to prime responses to tumor antigens provides a promising approach to e.g. cancer immunotherapy.
  • a limited number of DCs can be obtained from adult precursor cells.
  • cell differentiation protocols were developed to generate DCs from embryonic stem cells (Senju et al., 2010) but it remains a major challenge to steer directional DC differentiation because ES cell-derived DCs are typically immature with impaired immunogenicity.
  • ES cell-derived DCs represented less mature cells than adult stem cell-derived DCs.
  • ES-DCs consistently expressed less CDl lc regardless of the genetic background of the ES cell than bone marrow-derived cells differentiated into DCs.
  • Maturation markers CD80 and MHCII
  • CD80 and MHCII were moderately induced upon LPS administration in ES-DCs and less than 25% of the cells became MHCII/CD80 double positive.
  • more than 50% percent of the BM-DCs were MHCII/CD80 double positive.
  • Runx3 in developing ES-DCs was probed using a gain of function approach.
  • a genetically modified mouse ES cell line into which a gene of interest can be inserted by ere mediated recombination was used (Iacovino et al., 2014; Kyba et al., 2002).
  • Runx3 was turned on upon the directed DC development by doxycycline treatment between day 5 and day 19 and the cell phenotype was assessed by flow cytometry at the end of the differentiation. Importantly, the data revealed that 19-day cultured, Runx3 instructed ES-DCs showed a higher percent of MHCII/CD80 double positive cells. The elevated expression of these maturation/activation markers was also detected upon LPS stimulation.
  • Runx3 is strongly expressed in mature bone marrow-derived cultured DC and mediates their response to TGF-beta (Fainaru et al., 2004). In their study Fainaru et al. have found it conceivable that Runx3 functions in DC to restrain spontaneous maturation. The deficiency of Runx3 in DC progenitors led to a substantial decrease in splenic CD4+/CDl lb+ DC (Dickenet al., 2013). Whether the induction of Runx3 in a stem cell derived DC has a positive effect on differentiation was not examined.
  • Runx3 is important for the ES cell derived DC differentiation and activation.
  • Our gain of function analysis revealed that the forced expression of Runx3 profoundly stimulated the cytokine driven myeloid ES-DC maturation and activation. Therefore, a method to develop activated DCs from ES cells by the overexpression of Runx3 has been established.
  • Senju et al. reported the successful in vitro differentiation of DCs from mouse and human iPS. They found that human iPS-DCs exhibited characteristics of DCs, comparable to ES-DC, in morphology, surface molecules, and T cell-stimulating capacity.
  • iPS-DCs exhibited characteristics of DCs, comparable to ES-DC, in morphology, surface molecules, and T cell-stimulating capacity.
  • the method of modulating cells capable of differentiation to develop into DCs comprising the forced expression of Runx3 in cultured cells can be applied to iPS cells as well as ES cells.
  • iPS cells offer new opportunities in the biomedical sciences in terms of cell therapies for regenerative medicine and stem cell modeling of human disease. The method of the invention provides mature DCs for these applications to ensure a greater rate of success in therapy.
  • An aspect of the present invention relates to the medical use of the DCs produced by the method of the invention said use comprising administering to a subject an effective number of said DCs.
  • the Runx3 transgene was induced in 5 day co-cultured ES cell-derived differentiated cells. At this stage of the differentiation, the heterogeneous cell contains approximately 10-20 % later plate mesoderm cells (Flkl single positive progenitors). These cells represent the progenitors of the blood cells
  • hES human ES
  • the DCs produced according to the method of the invention may be produced in amounts suitable for use in a range of therapeutic and diagnostic procedures.
  • DCs may be used in cancer immunotherapy as adjuvants or as direct therapy, in autoimmune diseases, graft rejection, allergy, etc.
  • autoimmune or autoimmune-related disorders examples include Addison's disease, celiac disease, multiple sclerosis, myasthenia gravis, rheumatoid arthritis, systemic lupus erythematosus and Type I diabetes.
  • DCs may be used as vectors for anti-tumor and infectious disease vaccines. These strategies are based on the well-known antigen presenting capacity of DCs.
  • DCs produced by the method of the invention may also act as adjuvants for enhancing an immune response. Such an adjuvant function may be useful against tumor cells or other antigens.
  • Non auto-antigens may be derived from prokaryotes, eukaryotes, or viruses.
  • DCs in a suitable state may also carry antigens to induce tolerance in transplantation.
  • DCs produced by the method of the invention may be fused with other cell types e.g. a tumor cell.
  • Such a construct may be applied as a therapeutic or prophylactic vaccine.
  • the cells produced by the method of the invention may also be used as immunogens for the production of antibodies.
  • DCs are suitable for in vitro testing of the immunogenicity of vaccines.
  • DCs may be used as a putative research tool in immunological research, e.g. they can be used to isolate genes and proteins expressed specifically by these cell types.
  • DCs can be cultured in culture media and under conditions known to one skilled in the art.
  • the growth medium for the cells at each step of the method of the invention should allow for the survival, proliferation and differentation of the precursor cells.
  • Cells were differentiated using an OP-9 coculture method as described by Senju et al. 2003 and detailed in the Example section.
  • the term "DC differentation medium” is used for a culture medium for cells which is appropriate for culturing and differentation of DC precursor cells, e.g. ES cells, iPS cells and DC progenitor cells into mature DCs.
  • Various techniques may be used to phenotype and select the cells present in the cultures. These may include morphological analysis, detecting cell type specific antigens with monoclonal antibodies, identifying proliferating cells using tritiated thymidine autoradiography, assaying mixed leukocyte reactions, etc.
  • the morphological features of DCs are e.g. long cytoplasmic processes, large cells with multiple fine dendrites or irregularly shaped membrane.
  • the DC precursors in both blood and marrow lack MHC class II antigens as well as B and T cell and monocyte markers. Mature, nonproliferating DCs have high levels of MHC class II molecules.
  • Antibodies that bind to MHC class I antigens and those bind to the MHC class II antigens are suitable for identifying mature DCs.
  • CD80, CD86 and CD40 are typical antigens which can be used to identify the mature DCs produced by the method of the invention.
  • MHC2-FITC, CD14-PE and CD80-APC are a few examples of the antibodies that can be used for the identification of the DCs produced by the method of the invention, but those with skill in the art will recognize that other antibodies as well may be made and characterized for this purpose.
  • Immature DCs can internalize particles such as bacterial, viral, mycobacterial fragments for processing and presentation. Processing of antigens by DCs includes the fragmentation of the antigens. Thereafter the fragments can be presented. Mature DCs are effective in sensitizing T cells to several different antigens, but have little or no phagocytic activity. The skilled person will understand that mature DCs may be tested for their capacity to stimulate primary mixed lymphocyte reaction and their capacity to process and present protein antigen to T cells. These methods are well known in the art, the skilled person may find information e.g. in Senju et. al., 2003, parts Mixed leukocyte reaction and Antigen presentation assay.
  • the RUNX3 gene sequences of several species are well known from the prior art and sequence databases and are described for example in Bangsow et al.2001 and Bae et al. 1995.
  • One skilled in the art will find information on sequences e.g. in the gene database of NCBI: http://www.ncbi.nlm.nih.gov/gene; NCBI Reference Sequence: NC_000070.6 (Mus musculus); NCBI Reference Sequence: NC_000001.11 (Homo sapiens).
  • the sequence of the RUNX3 gene in the mouse is shown in SEQ ID NO l .
  • the RUNX3 gene sequence that is to be introduced into a cell to be differentiated into a mature DC is preferably of the same species (i.e. autologous to the cell).
  • An effective method of introducing the coding region of Runx3 into the cells to be differentiated is by way of an inducible cassette exchanger.
  • the method of Iacovino et al. is a rapid and efficient recombination system in which an inducible, floxed ere allele replaces itself with an incoming transgene.
  • the inducible cassette exchange (ICE) enables high efficiency integration of genes of interest into cells bearing a single copy ICE locus.
  • the conditional doxycycline-inducible nature of the target locus is well suited to gain-of-function experiments. Detailed description of the method is found in Iacovino et al., 2014.
  • a circular targeting plasmid carrying a LoxP site integrates at a LoxP site on the X chromosome.
  • a tetracycline-responsive promoter resides upstream of the chromosomal loxP site and precise integration places a gene of interest (Runx3) downstream of this promoter, such that it can be expressed in the presence of doxycycline.
  • the reverse tetracycline transactivator (rtTA) is expressed from the Rosa26 promoter enabling inducible expression in various cells.
  • transgene delivery includes e.g. the use of vectors, such as viruses and plasmids, lipofection, electroporation, microinjection.
  • vectors such as viruses and plasmids, lipofection, electroporation, microinjection.
  • RUNX3 may be monitored by measuring the levels of the gene products, e.g. Runx3 mRNS or Runx3 protein.
  • DC differentiation medium (MEM-alpha + 20% FBS; for OP9 co-culture):
  • DC differentiation medium plus GM-CSF 50 ml OP9 medium + GM-CSF (50 ng/ml final cone.) + 2-ME (50 uM final cone).
  • 2-ME beta-mercaptoethanol
  • the Runx3 sequence was transferred to a modified p2Lox targeting plasmid (Iacovino et al.., 2014) using the Gateway cloning system (Life Technologies). Five micrograms of these constructs were electroporated into Zx. l (lacovino et al., 2014) targeting ES cells with the Neon transfection system (Life Technologies). ES cell colonies were selected in 300 ug/mL G418, picked on day 8 and expanded. Five independent colonies were selected and characterized.
  • Mouse ES cells (ZX. l or iRunx3) were maintained on mouse embryonic fibroblast (MEF) layer in knockout Dulbecco's modified Eagle's medium (KO DMEM) (Life Technologies) with 15% ES cell qualified fetal bovine serum (Life Technologies).
  • ES-DC were differentiated with a GM-CSF dependent OP9 co-culture method as described by Senju et al. (Senju et al., 2003.) with minor modifications. In brief, ES cells were harvested and MEFs were removed by 30 minutes adherence to gelatinized dishes. OP9 cell density was setting to 100,000 on T25 flask 1 day before the experiment.
  • Co-culture was started by adding 100,000 ES cells into the OP9 stromal cell layers; cells were co-cultured for 5 days in Alpha Minimum Essential Medium (Alpha- MEM) containing 20% fetal bovine serum (FBS). Half of the medium was replaced at day 3. At day 5, cells were harvested and reseeded to fresh OP9 layers. OP9 cell density was setting to 100,000 cells/T25 flask and 500,000 5-day differentiated ES cells were plated on the OP9 layers. Cells were cultured in Alpha-MEM/20% FBS medium containing GM-CSF (50 ng/ml) and 50 uM beta-mercaptoethanol (2-ME) for additional 6 days.
  • Alpha- MEM Alpha Minimum Essential Medium
  • FBS fetal bovine serum
  • CD45 positive myeloid progenitors were sorted with FACS Aria III (BD Biosciences) and the obtained cells were further cultured for 9 days in RPMI medium containing GM-CSF (50 ng/ml) and 2-ME (50 uM) without OP9 layer.
  • the induction of Runx3 was started at day 5 by treating the cells with doxycycline (1 ⁇ g/ml).
  • Runx3 inducible ES cells were cultured for 19 days to promote DC differentiation as described Senju et al. 2003. 100 ng/ml LPS was added at day 18; in addition runx3 transgene was induced by doxycycline (+dox) treatment starting at day 5. The CD80 and MHCII expression was assessed by flow cytometric analysis. Figs. 1- 4 show the results of two of the clones from parallel experiments (experiment no. 54 and 55).
  • FSC forward scatter
  • SSC side scatter
  • PE conjugated antibody
  • ES-DCs were treated with 1 ⁇ g/ml doxycycline (+dox) and they were co-cultured with 10 ⁇ splenic T cells isolated from BALB/c mice for 5 days. The proliferative response of T cells was assessed by a BrdU Cell Proliferation Assay.
  • HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors. Cell 109, 29-37.
  • Senju S Haruta M
  • Matsunaga Y et al. Characterization of dendritic cells and macrophages generated by directed differentiation from mouse induced pluripotent stem cells. Stem Cells. 2009

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Abstract

The invention relates to a method of producing mature dendritic cells from cells capable of differentiation in a cell culture. The invention also provides dendritic cells produced by the method of the invention and medical uses of such cells.

Description

RUNX3 DEPENDENT ENHANCED DENDRITIC CELL GENERATION
FIELD OF THE INVENTION
The invention relates to a method of producing dendritic cells from cells capable of differentiation in a cell culture. The invention also provides dendritic cells produced by the method of the invention and medical uses of such cells.
BACKGROUND OF THE INVENTION
Dendritic cells (DC) are the most powerful antigen presenting cells (APCs) of the mammalian immune system and as such important mediators of immunity and tolerance. DCs are very efficient in ingesting, transforming and presenting antigens.
They are abundant in the epithelia, connective tissue, peripheral lymphoid nodes, the circulation system and in the T cell rich areas of the spleen. In an early differentiation stage immature DCs are able to take up pathogens, membrane fragments and soluble proteins by macropinocytosis. Upon uptake of an antigen the activated DCs migrate to the peripheral lymphoid organs and may come into contact with T cells. Stationary DCs sample and process antigens directly in the lymphoid organs. Contact with a T cell results in the loss of the ability of antigen uptake and differentiation. Maturation is associated with upregulation of MHC II molecules and costimulatory molecules such as CD80 and CD86 (Banchereau and Steinman, 1998). Mature DCs have the potential to stimulate TH and TC cells as well as B cells.
DCs are very heterogeneous with respect to their cell-surface markers and function but as stated above, maturation can be characterized by the increased expression of MHC molecules and costimulatory molecules on the cell surface (e.g. CD80, CD86, CD40) and their ability to stimulate mixed lymphocyte reaction and capability to process and present protein antigen to T cells.
DCs are also involved in the maintenance of self-tolerance in the periphery, inducing regulatory T cells or anergy of autoreactive T cells. Studies point to a primary role of DC in regulating in vivo allergic responses. (Senju 2010)
DCs are derived from hematopoietic bone marrow progenitor cells. Several transcription factors (TF) affect DC lineage development and function, among them Runx3.
Extensive research have been made to reveal the mechanisms of the production of DC-progenitors and the heterogeneity and functions of their downstream lineage cell subsets (Geissmann F et al., 2010.). However, information about the role of specific TFs in DC development, subset specification and homeostasis is still incomplete.
The mammalian Runt-related transcription factor 3 (Runx3) belongs to the runt domain family of transcription factors that are key regulators of lineage-specific gene expression in major developmental pathways. The Runx3 TF can either activate or suppress transcription.
According to Fainaru et al. Runx3 protein is barely detected in immature DC, and is highly expressed in mature DC. Runx3 is specifically expressed in mature DCs and mediates their response to TGF-β. In the absence of Runx3, DCs become insensitive to TGF- -induced maturation inhibition, and TGF- -dependent Langerhans cell development is impaired. Maturation of Runx3 knock out DCs is accelerated and accompanied by increased efficacy to stimulate T cells and aberrant expression of 2-integrins. Expression of Runx3 was also demonstrated in B- and myeloid cell lines (Levanon et al., 1994; Shi and Stavnezer, 1998; 1999; Bangsow et al., 2001). The role of the Runx complex in development and cell fate specification is discussed inter alia in Dominic Chih-Cheng Voon et al., 2015.
The application of DCs to prime responses to tumor antigens provides a promising approach to cancer immunotherapy as well as the treatment of autoimmune diseases. Since DCs have an important role in the pathogenesis of several viruses, they have significance as a therapeutic target. Antigen-specific negative regulation of the immune response by DCs is a likely approach for treatments of autoimmune diseases, allergies and for regulation of allo-reactive immune response causing graft rejection and graft versus host disease.
In vivo transfer of antigen-presenting DC has proven efficient in initiating T cell responses specific to the antigen. DC-based cellular vaccination is therefore regarded as a potential means for immunotherapy (Senju 2010).
However, only limited number of DCs can be obtained from adult precursor cells and the DC- differentiation potential of monocytes varies depending on the blood donor. To resolve the issue of the cell source for DC therapy, cell differentiation protocols were developed to generate DCs from stem cells.
Embryonic stem cells are characterized by pluripotency and infinite propagation capacity. Non-virus mediated methods for gene transfer, including targeted gene integration and procedures for isolation of appropriate transfectant cell clones, have been established for ES cells. (Senju 2003) Genetic modification of ES cells and subsequent in vitro differentiation to DCs is a promising strategy means for analysis of gene functions and differentiation of DCs.
Fainaru et al. generated DCs from bone marrow. Their nonadherent fraction of bone marrow derived DC culture consisted at day 7 of immature DCs and granulocytes, and at days 11-14 of immature and spontaneously matured DCs. They also found that Runx3 protein was barely present at day 7, but readily detected when mature DCs arose.
In 2000, Fairchild and his colleagues reported a study on the generation of functional DCs from mouse ES cells. They used ESF116 mouse ES cell line derived from a CBA/Ca blastocyst. In their method embryonic bodies (EBs) were formed, and subsequently differentiation of DCs was induced by addition of specific cytokines. (Fairchild et al., 2000.)
Senju et al. describe another method to generate DCs from mouse embryonic stem cells. ES cells were cultured on feeder cell layers of OP9, in the presence of granulocyte-macrophage colony-stimulating factor. The resulting cells were irregularly shaped floating cells with protrusions, expressed major histocompatibility complex class II, CDl lc, CD80, and CD86, with the capacity to process and present protein antigens to T cells. Senju et al. designated them ES-DCs (ES cell-derived DCs). ES-DCs became mature DCs, characterized by a typical morphology (Senju et al., 2003.).
Zhan and colleagues showed that generation of DCs from human ES cells is also possible. Similar to the method of Fairchild and colleagues, they induced formation of embryonic bodies by suspension culture of human ES cells, and then cultured the EBs in the presence of hematopoietic cytokines to generate leukocytes with antigen presenting function (Zhan et al., 2004.).
Senju et al. reported the generation and characterization of DCs derived from mouse and human iPS cells (Senju et al., 2009; Senju 2010). The human iPS-DCs exhibited characteristics of DCs, as human ES-DC do, in morphology, surface molecules, and T cell-stimulating capacity. The studies using mice have demonstrated that by in vivo transfer of genetically modified mouse ES-DCs immune responses can be effectively modulated both positively and negatively, making iPS-ECs a promising candidate in stem cell therapy.
However, recent studies have also uncovered that PSC-derived antigen presenting cells often exhibited impaired immunogenicity, e.g. poor T cell activation capacity (Tseng et al., 2009, Senju et al. 2003, Zhang et al., 2015). Yet unpublished data of the present inventors also reveal that ES-DCs have a compromised maturation/activation potential compared to BM-DCs (Takacs et al. Enhanced dendritic cell differentiation from pluripotent stem cells by ectopic expression of Runx3, to be published).
In view of this, steering directional DC differentiation has remained a major challenge because a high percentage of ES cell-derived DCs are typically immature with impaired immunogenicity.
DEFINITIONS
"Stem cells" are cells capable of differentiating into more than one cell type of the endoderm, mesoderm and/or ectoderm germinal layer and are also capable of self-renewal and/or cell division by mitosis without differentiation. Stem cells are able to give rise to differentiated cells and are capable of both symmetric and asymmetric cell division. Stem cells also have the capacity to replace damaged tissue.
Two major groups of stem cells are existing (1) embryonic stem cells (2) adult stem cells.
In general, embryonic stem cells have a far greater differentiation potential and can develop into almost every type of cell in the body. Adult stem cells can develop into a limited number of cell types only.
By definition we distinguish totipotent, pluripotent and multipotent stem cells.
"Embryonic stem cells" are pluripotent stem cells which can be isolated from the inner cell mass or can be derived from somatic cells by cell reprogramming induced pluripotent stem (iPS) cells. ES cells express markers that may be used to detect the presence of undifferentiated cells in a culture and are easily recognized by the skilled person. Such characteristic markers are Oct3/4, SSEA-1, Nanog, Zfp42, Esrrb. Alkaline phosphatase staining is also commonly used.
"Induced pluripotent stem cells" (iPS cells) are stem cells derived from somatic cells inducing the reprogramming of these cells with the overexpression of Oct3/4, Sox2, Klf4 and c-Myc. The iPS cell lines exhibit similar morphology and growth properties as ES cells and express ES cell-specific genes. iPS cells can be derived from various somatic cells.
"Cell differentiation" is the process by which a less specialized cell becomes a more specialized cell type. In the course of cell differentiation, e.g. a totipotent or pluripotent cell may become a differentiated cell through the stages of a multipotent stem cell that has a limited differentiation potential, an intermediate progenitor cell that has the capacity of only a limited number of cell divisions and a postmitotic, differentiated cell.
A "cell type" is a cell classification unit used to distinguish between classes (types) of functionally differentiated cells having functionally, as well as morphologically or phenotypically identical properties which are distinct from other classes within a multicellular, preferably an animal species. Said identical properties correspond to the expression pattern of said particular class of cells in the organism. Thus, a given cell type show well defined functional and gene expressional properties. While variances within cell types may occur, and various classifications may be applied, cell types are distinctive and discrete categories and clearly distinguishable from each other within a classification method or system. "Dendritic cells" (DCs) are specialized cells of the immune system with the unique capacity for initiating primary and secondary T and B lymphocyte responses. Dendritic cells are antigen presenting cells having branched projections ("dendrites") at certain development stages, resulting in a large contact surface to the surroundings. "Immature DCs" are characterized by high endocytotic activity and a lower T cell activating potential. Contact with an antigen results in activation and maturing: DCs present the taken up antigens on their surfaces with MHC molecules and express coactivators. "Mature DCs" lose their ability to take up antigens, have a high T and B cell activating potential and express high levels of MHC molecules and costimulatory molecules, typically CD80, CD86 on their surfaces. Mature DCs have the capacity to stimulate primary mixed leukocyte reaction.
"Dendritic cell progenitor cells" are proliferating cells that may differentiate into dendritic cells in the bone marrow that share phenotypic characteristics with myeloid precursor populations. Macrophage/dendritic cell progenitor cells (MDPs) are found in the bone marrow, give rise to dendritic cells subsets, and typically cannot differentiate into granulocytes. Common dendritic cell precursors (CDPs) are proliferating cells that differentiate into dendritic cells and into precursors for classical DCs (pre-cDCs) in the bone marrow. However, CDPs have lost the potential to give rise to monocytes.
The terms "Runx3" and "RUNX3", indicating the gene product and the gene, respectively, or alternatively, indicating the mouse gene and the human gene, respectively, are used interchangeably. The term inducing "Runx3", therefore, may indicate the induction of the gene, i.e. enhancing gene expression. The term Runx3 may refer to the mouse Runx3 gene, the human Runx3 gene or a Runx3 gene from another species.
The term "forced expression" implies the overexpression of a gene product by way of introducing a transgene into a cell and allowing the transgene to express the gene product. The expression of the transgene may be transient or permanent. Forced expression may also mean the induction of the expression of an endogenous gene by chemical or genetic manipulation.
BRIEF DESCRIPTION OF THE INVENTION
The invention relates to methods of modulating cells capable of differentiation, wherein the cells capable of differentiation are cells produced by a method not comprising the step of destructing a human embryo or doing harm to a human embryo. The term "cells capable of differentiation" therefore relates to cells capable of differentiation produced by a method not comprising the step of destructing a human embryo or doing harm to a human embryo, throughout this description inclusive the claims. The invention relates to a method of modulating cells capable of differentiation to develop into DCs comprising forced expression of Runx3 in cultured cells. A method of modulating cells capable of differentiation to develop into DCs is provided, the method comprising forced expression of a Runx3 gene product in said cells capable of differentiation and culturing said cells in a medium appropriate for culturing and differentiation of said cells into DCs. The method of the invention is suitable to produce a high percentage of DCs derived from cells capable of differentiation in a cell culture. The method of the invention is suitable to produce differentiated DCs derived from cells capable of differentiation in a cell culture. The method of the invention is suitable to produce activated DCs derived from cells capable of differentiation in a cell culture. The method of the invention is suitable to produce DCs derived from cells capable of differentiation in a cell culture, wherein said DCs are characterized by that they express MHC and CD (e.g. CD80) molecules on their surface. The method of modulating cells capable of differentiation to develop into DCs comprising the forced expression of Runx3 in cultured cells according to the invention is suitable to produce a higher percentage of DCs derived from cells capable of differentiation in a cell culture than a conventional method of modulating stem cells to develop into DCs without the forced expression of Runx3. For example, previous methods were able to produce 10% of mature DCs while it is possible to produce up to 25% or more mature DCs using the method of the invention. Preferably, the rate of CD86/MHCII double positive cells in the cell culture is at least 15%. More preferably, the rate of CD86/MHCII double positive cells in the cell culture is at least 20% or at least 25%.
According to a preferred embodiment of the invention the DCs are activated DCs. The DCs produced by the method of the invention may have reached an activated stage of differentiation characterized by expressing higher levels of MHC, e.g. MHCII and costimulatory molecules, e.g. CD80, CD86 than immature DCs.
Zhang et al. were able to differentiate functional antigen presenting cells from pluripotent stem cells, e.g. ES cells or iPS cells, but the resulting proliferating myeloid cells (pMC) showed impaired proliferation and differentiated into immature DC-like cells (pMC-DC) expressing low levels of major histocompatibility complex (MHC)-I, MHC-II, CD40, CD80, and CD86 upon treatment with IL4 plus GM-CSF (Zhang et al. 2015). There is no suggestion in the study of Zhang et al. to use forced Runx3 expression to provide higher percentage of mature DCs. Dicken et al. have showed that Runx3 was strongly expressed in CD l ib positive splenic DCs and cell specific gene targeting models uncovered that the CDl lb/Esamhl positive DC development was particularly compromised in Runx3 deficient animals (Dicken et al., 2013). This study, however, failed to realize that besides MHCII, expression of CD86 is also augmented by Runx3, and thus Runx3 generally improves the maturation capacity of ES-DCs and may be used in a method to provide higher percentage of ES-derived mature DCs in a cell culture.
The elevated expression of the MHC and CD80/86 molecules (i.e. maturation/activation markers) may be detected upon LPS stimulation in the DCs produced by the method of the invention.
According to an embodiment of the invention the cells to be differentiated into DCs may be any cells capable of differentiating into DCs.
According to a preferred embodiment the cells capable of differentiation suitable for the method of the invention may be embryonic stem cells, e.g. mouse embryonic stem cells or human embryonic stem cells.
According to another preferred embodiment the stem cells are induced pluripotent stem cells, e.g. mouse iPS cells or human iPS cells.
In another embodiment the cells suitable to be differentiated into activated DCs according to the method of the invention are progenitor cells, such as DC progenitor cells.
The Runx3 gene product may be a Runx3 mRNS or a Runx3 protein.
In an embodiment the method of modulating cells capable of differentiation to develop into DCs comprising forced expression of Runx3 in cultured cells according to the invention comprises
• introducing at least the coding region of Runx3 into a cell capable of differentiation to develop into a DC,
• culturing and differentiating said cell under conditions suitable for the differentiation into DCs,
• allowing the cells to express the RUNX3 gene product, and
• selecting DCs, e.g. activated DCs. In a preferred embodiment the method of modulating cells capable of differentiation to develop into DCs comprising forced expression of Runx3 in cultured cells according to the invention comprises
• introducing at least the coding region of Runx3 into a stem cell by means of an inducible cassette exchanger,
• culturing the engineered cell,
• selecting the inducible cell clones,
• differentiating the selected clones into DC by applying a DC differentiation medium to the culture medium of said cell clones,
• inducing the expression of the Runx3 gene product,
• phenotyping the cells and selecting DCs.
The inducible clones may be selected based on their specific antibiotic resistance (e.g. Geneticin/G418 resistance).
In a more preferred embodiment a genetically modified ES cell line into which a gene of interest can be inserted by ere mediated recombination is used and the at least the coding region of a Runx3 sequence is transferred to a modified p2Lox targeting plasmid using the Gateway cloning system, then these constructs are electroporated into Zx.1 ES cells, targeting the ES cells with the Neon transfection system.
The at least the coding region of Runx3 may be introduced into the cell by any method known in the art to be suitable for the introduction of exogenous genetic material into a cell, such as by lentivirus introduction, by adenovirus vectors, by plasmid transfer, etc. The expression of Runx3 may be induced permanently or transiently.
In a preferred embodiment the expression of Runx3 is induced with doxycycline. The expression of Runx3 may be induced by e.g. tetracycline or other agents or techniques known in the art.
In another embodiment the expression of endogenous Runx3 is induced, e.g. by the TGF -pathway.
In another aspect a DC is provided that is produced by the method of the invention as described supra. The DC of the invention may be an activated DC derived from a cell capable of differentiation that has been differentiated by the method of the invention. The DC of the invention may be a DC derived from a cell capable of differentiation characterized by that it expresses MHC and CD (e.g. CD 80, CD86) molecules on its surface.
The invention also relates to a cultured DC in which the expression of the endogenous Runx3 has been specifically induced so that it overexpresses a Runx3 gene product relative to a DC in which the expression of a gene product of an endogenous Runx3 has not been induced. In a preferred embodiment the expression of Runx3 is induced with doxycycline. The expression of Runx3 may be induced by e.g. tetracycline or other agents or techniques known in the art.
The invention also relates to a DC obtainable by the method according to the invention for use in the treatment of a patient in need of DC administration. The patient may be diagnosed with a disease associated with deficient activity of DCs or may be in need of increased DC activity. Preferably, the disease is cancer, an autoimmune disease, e.g. rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's disease, Sjorgen's disease, Addison's disease, type 1 diabetes, ankylosing spondylitis, IBD, Crohn's disease, psoriasis; an inflammatory disease, viral infection, e.g. HIV, hepatitis, a Herpes virus infection; bacterial infection, fungal infection, parasitic infection, graft rejection, graft versus host disease, allergy or other immune reaction related disease. Preferably, the DC used is autologous to the patient.
The invention also relates to a pharmaceutical composition comprising a DC obtainable by the methods described above and pharmaceutically acceptable excipients suitable for live cells.
The DC comprised in the pharmaceutical composition or the DC for use in the treatment of a disease associated with the activity of DCs according to the invention may comprise other genetic or chemical modifications suitable for use in the treatment of a disease associated with the activity of DCs. Such modification may be e.g. the genetic engineering of the cell to express immunostimulatory molecules or the loading the DCs with a particular (tumor) antigen.
The DC according to the invention may be used in cell therapy as standalone therapeutic agents, i.e.
administered in the presence of standard adjuvants or excipients only or together with e.g. cytokine-induced killer cells, adoptively transferred T lymphocytes, immunostimulatory cytokines. When used in cancer therapy, the DC therapy may be combined with other cancer therapy (e.g. radiation, chemotherapy).
The patient to be treated may be a vertebrate animal, preferably a mammal, e.g. a companion animal such as a dog, cat or rabbit. More preferably the patient is a human.
Preferably, if an exogenous Runx3 sequence is introduced into a DC which is to be used in the treatment of a disease in a patient, a Runx3 sequence that is autologous to the species of the patient is used.
The invention also relates to a kit comprising
• cells capable of differentiation to be developed into DCs and a transforming vector comprising at least the coding region of Runx3 or
• cells comprising an exogenous coding region of Runx3, and
• culturing medium suitable for the culturing of stem cells and DC,
• differentiation medium suitable for the differentiation of stem cells into DC cells,
• optionally an agent to induce the expression of a Runx3 gene product.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1. FACS analysis indicates that after 19 day of culture higher percent of MHCII/CD80 double positive DC were obtained upon doxycycline (+dox) treatment (Runx3 induction) with/without LPS stimulation in clone2 ES-DC cells (results from experiment no. 54).
Fig. 2. FACS analysis indicates that after 19 day of culture higher percent of MHCII/CD80 double positive DC were obtained upon doxycycline treatment (+dox) with/without LPS stimulation in clone2 ES-DC cells (results from experiment no. 55).
Fig. 3. FACS analysis indicates that after 19 day of culture higher percent of MHCII/CD80 double positive DC were obtained upon doxycycline treatment (+dox) with/without LPS stimulation in clone4 ES-DC cells (results from experiment no. 54).
Fig. 4. FACS analysis indicates that after 19 day of culture higher percent of MHCII/CD80 double positive DC were obtained upon doxycycline treatment (+dox) with/without LPS stimulation in clone4 ES-DC cells (results from experiment no. 55).
Fig. 5. Runx3 driven enhanced ES-DC maturation. Runx3 transgenic ES cells were differentiated for 19 days. The Runx3 inducible cells were treated with 1 μg/ml doxycycline (+dox). In addition, the indicated cells were treated with LPS (100 ng/μΐ) at day 18. (A) Cell surface expression of CD80 and MHCII was assessed by flow cytometry at day 19. The average percent of the CD80/MHCII double positive cells and the SD values were calculated from 8 experiments. (B) Cell surface expression of CD86 was assessed by flow cytometry at day 19. The average percent of the CD86 positive cells and the SD values were calculated from 8 experiments. (C) The indicated number of 19 day-differentiated, LPS activated, Runx3 -inducible ES-DCs were used as stimulators for MLR (mixed leukocyte reaction). ES-DCs were treated with 1 μg/ml doxycycline (+dox) and they were co- cultured with 105 splenic T cells isolated from BALB/c mice for 5 days. The proliferative response of T cells was assessed by a BrdU Cell Proliferation Assay. The corrected absorbance (450-540 nm) and the SD values were calculated from 4 experiments.
DETAILED DESCRIPTION OF THE INVENTION
The application of DCs to prime responses to tumor antigens provides a promising approach to e.g. cancer immunotherapy. However, only a limited number of DCs can be obtained from adult precursor cells. To resolve the issue of the cell source for DC therapy, cell differentiation protocols were developed to generate DCs from embryonic stem cells (Senju et al., 2010) but it remains a major challenge to steer directional DC differentiation because ES cell-derived DCs are typically immature with impaired immunogenicity.
In consistence with literature data, the present inventors have shown that ES cell-derived DCs represented less mature cells than adult stem cell-derived DCs. ES-DCs consistently expressed less CDl lc regardless of the genetic background of the ES cell than bone marrow-derived cells differentiated into DCs. Maturation markers (CD80 and MHCII) were moderately induced upon LPS administration in ES-DCs and less than 25% of the cells became MHCII/CD80 double positive. In contrast, more than 50% percent of the BM-DCs were MHCII/CD80 double positive.
Cell differentiation programs are governed by lineage determining and stimulus activated transcription factors. Recently we have assessed the gene expression of 17 DC specific transcription factors in ES cell- and bone marrow (BM)-derived DCs (unpublished results). Our data revealed that Runx3 exhibited impaired expression in ES cell-derived DCs suggesting that the forced expression of this transcription factor in ES-DCs might modify the development or function of this ex vivo generated cell type.
The effect of Runx3 in developing ES-DCs was probed using a gain of function approach. A genetically modified mouse ES cell line into which a gene of interest can be inserted by ere mediated recombination was used (Iacovino et al., 2014; Kyba et al., 2002). To introduce the coding region of Runx3, first targeting vectors were constructed then the genetically modified ES cell line was targeted by inducible cassette exchange (Iacovino et al.., 2014). The inducibility of the individual clones was tested, thereafter the well inducible ES cell clones were differentiated to ES-DCs. Runx3 was turned on upon the directed DC development by doxycycline treatment between day 5 and day 19 and the cell phenotype was assessed by flow cytometry at the end of the differentiation. Importantly, the data revealed that 19-day cultured, Runx3 instructed ES-DCs showed a higher percent of MHCII/CD80 double positive cells. The elevated expression of these maturation/activation markers was also detected upon LPS stimulation.
It has been described previously, that Runx3 is strongly expressed in mature bone marrow-derived cultured DC and mediates their response to TGF-beta (Fainaru et al., 2004). In their study Fainaru et al. have found it conceivable that Runx3 functions in DC to restrain spontaneous maturation. The deficiency of Runx3 in DC progenitors led to a substantial decrease in splenic CD4+/CDl lb+ DC (Dickenet al., 2013). Whether the induction of Runx3 in a stem cell derived DC has a positive effect on differentiation was not examined. We compared the mRNA levels of 17 DC/macrophage specific transcription factors (Batf3, Bcl-6, Egrl, Egr2, Id2, Ikzfl, 2, 4, Irf8, Maf, Mafb, Relb, Runx3, Sfpi/Pu. l, Tcf4, Spi-B and Zbtb46) in ES-DCs and BM (bone marrow) derived DCs. Our analysis indicated that three DC affiliated transcription factors (Spi-B, Runx3 and Irf4) showed impaired expression in ES cell-derived DCs comparing to BM-derived DC. Interestingly, our gain of function analysis revealed that one of this factors (Runx3) enhanced the GM-CSF dependent ES-DC maturation.
Our experimental data prove that Runx3 is important for the ES cell derived DC differentiation and activation. Our gain of function analysis revealed that the forced expression of Runx3 profoundly stimulated the cytokine driven myeloid ES-DC maturation and activation. Therefore, a method to develop activated DCs from ES cells by the overexpression of Runx3 has been established.
Senju et al. reported the successful in vitro differentiation of DCs from mouse and human iPS. They found that human iPS-DCs exhibited characteristics of DCs, comparable to ES-DC, in morphology, surface molecules, and T cell-stimulating capacity. One skilled in the art will understand that the method of modulating cells capable of differentiation to develop into DCs comprising the forced expression of Runx3 in cultured cells can be applied to iPS cells as well as ES cells. iPS cells offer new opportunities in the biomedical sciences in terms of cell therapies for regenerative medicine and stem cell modeling of human disease. The method of the invention provides mature DCs for these applications to ensure a greater rate of success in therapy. An aspect of the present invention relates to the medical use of the DCs produced by the method of the invention said use comprising administering to a subject an effective number of said DCs. The Runx3 transgene was induced in 5 day co-cultured ES cell-derived differentiated cells. At this stage of the differentiation, the heterogeneous cell contains approximately 10-20 % later plate mesoderm cells (Flkl single positive progenitors). These cells represent the progenitors of the blood cells
It is possible to generate human ES (hES) cells from single blastomeres (Chung et al.., 2008). Carrying out of the method of the invention does not, therefore, necessitate the destruction of a human embryo or the use of hES cell lines derived from a destructed embryo, when the method of the invention is carried out on hES cells.
The DCs produced according to the method of the invention may be produced in amounts suitable for use in a range of therapeutic and diagnostic procedures. DCs may be used in cancer immunotherapy as adjuvants or as direct therapy, in autoimmune diseases, graft rejection, allergy, etc.
Examples of autoimmune or autoimmune-related disorders include Addison's disease, celiac disease, multiple sclerosis, myasthenia gravis, rheumatoid arthritis, systemic lupus erythematosus and Type I diabetes.
DCs may be used as vectors for anti-tumor and infectious disease vaccines. These strategies are based on the well-known antigen presenting capacity of DCs. DCs produced by the method of the invention may also act as adjuvants for enhancing an immune response. Such an adjuvant function may be useful against tumor cells or other antigens. Non auto-antigens may be derived from prokaryotes, eukaryotes, or viruses. DCs in a suitable state may also carry antigens to induce tolerance in transplantation.
DCs produced by the method of the invention may be fused with other cell types e.g. a tumor cell. Such a construct may be applied as a therapeutic or prophylactic vaccine.
The cells produced by the method of the invention may also be used as immunogens for the production of antibodies. DCs are suitable for in vitro testing of the immunogenicity of vaccines. DCs may be used as a putative research tool in immunological research, e.g. they can be used to isolate genes and proteins expressed specifically by these cell types.
DCs can be cultured in culture media and under conditions known to one skilled in the art. The growth medium for the cells at each step of the method of the invention should allow for the survival, proliferation and differentation of the precursor cells. Cells were differentiated using an OP-9 coculture method as described by Senju et al. 2003 and detailed in the Example section. The term "DC differentation medium" is used for a culture medium for cells which is appropriate for culturing and differentation of DC precursor cells, e.g. ES cells, iPS cells and DC progenitor cells into mature DCs.
Various techniques may be used to phenotype and select the cells present in the cultures. These may include morphological analysis, detecting cell type specific antigens with monoclonal antibodies, identifying proliferating cells using tritiated thymidine autoradiography, assaying mixed leukocyte reactions, etc. The morphological features of DCs are e.g. long cytoplasmic processes, large cells with multiple fine dendrites or irregularly shaped membrane. The DC precursors in both blood and marrow lack MHC class II antigens as well as B and T cell and monocyte markers. Mature, nonproliferating DCs have high levels of MHC class II molecules. Antibodies that bind to MHC class I antigens and those bind to the MHC class II antigens are suitable for identifying mature DCs. CD80, CD86 and CD40 are typical antigens which can be used to identify the mature DCs produced by the method of the invention. MHC2-FITC, CD14-PE and CD80-APC are a few examples of the antibodies that can be used for the identification of the DCs produced by the method of the invention, but those with skill in the art will recognize that other antibodies as well may be made and characterized for this purpose.
Immature DCs can internalize particles such as bacterial, viral, mycobacterial fragments for processing and presentation. Processing of antigens by DCs includes the fragmentation of the antigens. Thereafter the fragments can be presented. Mature DCs are effective in sensitizing T cells to several different antigens, but have little or no phagocytic activity. The skilled person will understand that mature DCs may be tested for their capacity to stimulate primary mixed lymphocyte reaction and their capacity to process and present protein antigen to T cells. These methods are well known in the art, the skilled person may find information e.g. in Senju et. al., 2003, parts Mixed leukocyte reaction and Antigen presentation assay.
The RUNX3 gene sequences of several species are well known from the prior art and sequence databases and are described for example in Bangsow et al.2001 and Bae et al. 1995. One skilled in the art will find information on sequences e.g. in the gene database of NCBI: http://www.ncbi.nlm.nih.gov/gene; NCBI Reference Sequence: NC_000070.6 (Mus musculus); NCBI Reference Sequence: NC_000001.11 (Homo sapiens).
The sequence of the RUNX3 gene in the mouse is shown in SEQ ID NO l .The RUNX3 gene sequence that is to be introduced into a cell to be differentiated into a mature DC is preferably of the same species (i.e. autologous to the cell).
An effective method of introducing the coding region of Runx3 into the cells to be differentiated is by way of an inducible cassette exchanger. The method of Iacovino et al. is a rapid and efficient recombination system in which an inducible, floxed ere allele replaces itself with an incoming transgene. The inducible cassette exchange (ICE), enables high efficiency integration of genes of interest into cells bearing a single copy ICE locus. The conditional doxycycline-inducible nature of the target locus is well suited to gain-of-function experiments. Detailed description of the method is found in Iacovino et al., 2014. Genetically modified mouse ES cell line with a conditional variable locus on the X chromosome was engineered. In this system, a circular targeting plasmid carrying a LoxP site integrates at a LoxP site on the X chromosome. A tetracycline-responsive promoter resides upstream of the chromosomal loxP site and precise integration places a gene of interest (Runx3) downstream of this promoter, such that it can be expressed in the presence of doxycycline. The reverse tetracycline transactivator (rtTA) is expressed from the Rosa26 promoter enabling inducible expression in various cells.
Other methods of delivering the transgene are also known and include e.g. the use of vectors, such as viruses and plasmids, lipofection, electroporation, microinjection.
The expression of RUNX3 may be monitored by measuring the levels of the gene products, e.g. Runx3 mRNS or Runx3 protein.
The invention will be further described by way of the following example.
EXAMPLE
Materials:
Mouse embryonic fibroblast (MEF P2; mitomycin treated)
iRunx3 C2 mouse ES cells
iRunx3 C4 mouse ES cells
OP9 feeder cells
DC differentiation medium (MEM-alpha + 20% FBS; for OP9 co-culture):
5.1 g MEM-alpha, powder (Invitrogen)
1.11 g sodium bicarbonate
500 ml distilled water (Milipore water)
adjust the pH to 7.2 using 0.1 N HC1 (usually 4.5 ml)
500 ml MEM-alpha (redissolved and pH adjusted)
126 ml FBS (Hyclone, final cone. 20%)
6 ml Penicillin, Streptomycin
DC differentiation medium plus GM-CSF: 50 ml OP9 medium + GM-CSF (50 ng/ml final cone.) + 2-ME (50 uM final cone). 2-ME= beta-mercaptoethanol)
RPMI-1640 medium plus GM-CSF:
500 ml RPMI-1640 + Glutamax
50 ml FBS (Hyclone)
6 ml Penicillin, Streptomycin
50 ml RPMI-1640 medium plus GM-CSF (50 ng/ml final cone) + 2ME ( 50 uM final cone)
T25 tissue culture flask and 10 cm plastic Petri dishes
Procedure:
Part 1 Generation ofRunx3 inducible ES cells
The Runx3 sequence was transferred to a modified p2Lox targeting plasmid (Iacovino et al.., 2014) using the Gateway cloning system (Life Technologies). Five micrograms of these constructs were electroporated into Zx. l (lacovino et al., 2014) targeting ES cells with the Neon transfection system (Life Technologies). ES cell colonies were selected in 300 ug/mL G418, picked on day 8 and expanded. Five independent colonies were selected and characterized.
Part2 ES cell differentiation
Mouse ES cells (ZX. l or iRunx3) were maintained on mouse embryonic fibroblast (MEF) layer in knockout Dulbecco's modified Eagle's medium (KO DMEM) (Life Technologies) with 15% ES cell qualified fetal bovine serum (Life Technologies). ES-DC were differentiated with a GM-CSF dependent OP9 co-culture method as described by Senju et al. (Senju et al., 2003.) with minor modifications. In brief, ES cells were harvested and MEFs were removed by 30 minutes adherence to gelatinized dishes. OP9 cell density was setting to 100,000 on T25 flask 1 day before the experiment. Co-culture was started by adding 100,000 ES cells into the OP9 stromal cell layers; cells were co-cultured for 5 days in Alpha Minimum Essential Medium (Alpha- MEM) containing 20% fetal bovine serum (FBS). Half of the medium was replaced at day 3. At day 5, cells were harvested and reseeded to fresh OP9 layers. OP9 cell density was setting to 100,000 cells/T25 flask and 500,000 5-day differentiated ES cells were plated on the OP9 layers. Cells were cultured in Alpha-MEM/20% FBS medium containing GM-CSF (50 ng/ml) and 50 uM beta-mercaptoethanol (2-ME) for additional 6 days. At day 10, CD45 positive myeloid progenitors were sorted with FACS Aria III (BD Biosciences) and the obtained cells were further cultured for 9 days in RPMI medium containing GM-CSF (50 ng/ml) and 2-ME (50 uM) without OP9 layer. The induction of Runx3 was started at day 5 by treating the cells with doxycycline (1 μg/ml).
Part 3 Cell sorting and flow cytometric analysis
Runx3 inducible ES cells were cultured for 19 days to promote DC differentiation as described Senju et al. 2003. 100 ng/ml LPS was added at day 18; in addition runx3 transgene was induced by doxycycline (+dox) treatment starting at day 5. The CD80 and MHCII expression was assessed by flow cytometric analysis. Figs. 1- 4 show the results of two of the clones from parallel experiments (experiment no. 54 and 55).
Cells were analyzed or sorted with a FACS Aria III (BD Biosciences). Live cells were gated based on forward scatter (FSC)/side scatter (SSC) profile to eliminate dead cells or cell debris. For FACS sorting approximately 400,000 CD45+ cells were sorted. For cell sorting the following conjugated antibody was used: CD45-phycoerythrin (PE).
For flow cytometric analysis cells were collected in Eppendorf tube (cell number was 400K) and centrifuged (8000 rpm 2min). Then cells were resuspended in 10 ul/sample SM (staining medium: PBS + 2% FBS) containing Fc-blocker (190 ul SM + 10 ul Fc blocker). Cells were incubated at 4 C on ice for 15 min with SM+Fc blocker. 10 ul cell suspension was transferred to Eppendorf tubes and 10 ul antibody mix was added (antibodies were 20 fold diluted!). Cells were stained for 30 min on ice then 200 ul SM was added. The following conjugated antibodies were used: MHC2-FITC, CD14-PE, CD80-APC (BD Biosciences).
Mixed leukocyte reaction
ES-DCs were treated with 1 μg/ml doxycycline (+dox) and they were co-cultured with 10^ splenic T cells isolated from BALB/c mice for 5 days. The proliferative response of T cells was assessed by a BrdU Cell Proliferation Assay. REFERENCES
Bae et al. Cloning, mapping and expression of PEBP2aC, a third gene encoding the mammalian Runt domain. Gene 159 (1995), 245-248
Bangsow, C, Rubins, N., Glusman, G., Bernstein, Y., Negreanu, V., Goldenberg, D., Ben-Asher, E., Lotem, J., Lancet, D., Levanon, D., Groner, Y., 2001. The RUNX3 gene-sequence, structure and regulated expression, Gene 279:221-232
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Dicken, J., Mildner, A., Leshkowitz, D., Touw, LP., Hantisteanu, S., Jung, S., and Groner, Y. (2013). Transcriptional reprogramming of CD1 lb+Esam(hi) dendritic cell identity and function by loss of Runx3. PloS one 8, e77490.
Fainaru, O., Woolf, E., Lotem, J., Yarmus, M., Brenner, O., Goldenberg, D., Negreanu, V., Bernstein, Y., Levanon, D., Jung, S., et al. (2004). Runx3 regulates mouse TGF-beta-mediated dendritic cell function and its absence results in airway inflammation. The EMBO journal 23, 969-979.
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Senju, S., Hirata, S., Motomura, Y., Fukuma, D., Matsunaga, Y., Fukushima, S., Matsuyoshi, H., and Nishimura, Y. (2010). Pluripotent stem cells as source of dendritic cells for immune therapy. Int J Hematol 91, 392-400.
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Claims

1. A method of modulating cells capable of differentiation to develop into dendritic cells (DC), the method comprising forced expression of a Runx3 gene product in said cells capable of differentiation and culturing said cells in a medium appropriate for culturing and differentiation of said cells into DCs.
2. The method of claim 1 wherein the dendritic cells are activated dendritic cells or mature dendritic cells.
3. The method of claim 1 or 2 wherein the cells capable of differentiation are embryonic stem cells.
4. The method of claim 1 or 2 wherein the cells capable of differentiation are induced pluripotent stem cells.
5. The method of claim 1 or 2 wherein the cells capable of differentiation are dendritic cell progenitor cells.
6. The method of any one of claims 1 -5 further comprising
• introducing at least the coding region of a Runx3 gene into a cell capable of differentiation to develop into a dendritic cell by means of an inducible casette exchanger,
• culturing the engineered cell,
• selecting the inducible cell clones,
• differentiating the selected clones into DCs by applying a DC differentiation medium to the culture medium of said cell clones,
• inducing the expression of the Runx3 gene product and
• selecting the dendritic cells.
7. The method according to claim 6, wherein at least the coding region of the Runx3 gene is transferred to a p2Lox targeting plasmid using the Gateway cloning system, and then the construct is electroporated into Zx. l ES cells, targeting the ES cells with the Neon transfection system.
8. The method according to any one of claims 1 -5, wherein expression of a gene product of an endogenous Runx3 gene is induced.
9. A dendritic cell produced by the method according to any of claims 1 -8.
10. The dendritic cell according to any one of claims 7-8 for use in the treatment of a patient in need of dendritic cell administration.
11. The dendritic cell for use according to claim 10, wherein the patient is diagnosed with a disease associated with the deficient activity of dendritic cells.
12. The dendritic cell for use according to claim 10 or 11, wherein the disease is selected from cancer, autoimmune diseases, inflammatory diseases, infections, graft rejection, graft versus host disease, allergies and other immune reaction related diseases.
13. The dendritic cell for use according to any one of claims 10-12, wherein said dendritic cell is autologous to said patient.
14. A pharmaceutical composition comprising the dendritic cell according to any one of claims 7-8 and a pharmaceutically acceptable carrier suitable for live dendritic cells.
15. A kit comprising
• cells capable of differentiation to be developed into dendritic cells and a transforming vector comprising at least the coding region of Runx3 or
• cells comprising an exogenous coding region of Runx3, and
• culturing medium suitable for the culturing of stem cells and DC,
• differentiation medium suitablefor the differentiation of stem cells into DC cells,
• optionally an agent to induce the expression of a Runx3 gene product.
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