EP0891425A2 - Procedes et compositions pour la regulation de sous-ensembles de lymphocytes t par la modulation de l'activite du facteur de transcription - Google Patents

Procedes et compositions pour la regulation de sous-ensembles de lymphocytes t par la modulation de l'activite du facteur de transcription

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Publication number
EP0891425A2
EP0891425A2 EP97926377A EP97926377A EP0891425A2 EP 0891425 A2 EP0891425 A2 EP 0891425A2 EP 97926377 A EP97926377 A EP 97926377A EP 97926377 A EP97926377 A EP 97926377A EP 0891425 A2 EP0891425 A2 EP 0891425A2
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Prior art keywords
protein
nip45
cell
maf
ofthe
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German (de)
English (en)
Inventor
Laurie H. Glimcher
Martin R. Hodge
I-Cheng Ho
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Harvard College
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Harvard College
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Priority claimed from US08/636,602 external-priority patent/US5958671A/en
Priority claimed from US08/755,584 external-priority patent/US5858711A/en
Priority claimed from US08/755,592 external-priority patent/US6537810B1/en
Application filed by Harvard College filed Critical Harvard College
Publication of EP0891425A2 publication Critical patent/EP0891425A2/fr
Withdrawn legal-status Critical Current

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    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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Definitions

  • T helper cells are not a homogeneous population but can be divided on the basis of cytokine secretion into at least two subsets termed T helper type 1 (Thl) and T helper type 2 (Th2) (see e.g., Mosmann, T.R. et al. (1986) J. Immunol. 136:2348-2357; Paul, W.E. and Seder, R.A. (1994) Cell 76:241-251 ; Seder, R.A. and Paul, W.E. (1994) Ann. Rev. Immunol. 12:635-673).
  • Thl T helper type 1
  • Th2 T helper type 2
  • Thl cells secrete interleukin-2 (IL-2) and interferon- ⁇ (IFN- ⁇ ) while Th2 cells produce interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin- 10 (IL-10) and interleukin- 13 (IL-13). Both subsets produce cytokines such as tumor necrosis factor (TNF) and granulocyte/macrophage-colony stimulating factor (GM- CSF).
  • TNF tumor necrosis factor
  • GM- CSF granulocyte/macrophage-colony stimulating factor
  • Thl and Th2 cells are thought to have differing functional activities. For example, Thl cells are involved in inducing delayed type hypersensitivity responses, whereas Th2 cells are involved in providing efficient "help" to B lymphocytes and stimulating production of IgGl and IgE antibodies.
  • Thl Th2 cells
  • Thl to Th2 switch is observed coincident with the release of eggs into the tissues by female parasites and is associated with a worsening of the disease condition (Pearce, E.J., et al. (1991 ) J. Exp. Med. 173:159-166; Grzych, J- M., et al. (1991) J Immunol. 141: 1322-1327; Kullberg, M.C., et al. (1992) J. Immunol. 148:3264-3270).
  • Thl to Th2 switch see e.g., Shearer, G.M. and Clerici, M. (1992) Prog. Chem. Immunol. 54:21-43; Clerici, M and Shearer, G.M. (1993) Immunology Today 14:107-1 1 1 ; Yamamura, M., et al. (1993) J Clin. Invest. 91:1005-1010; Pisa, P., et al. (1992) Proc. Natl. Acad. Sci.
  • autoimmune diseases have been shown to be associated with a predominant Thl response.
  • patients with rheumatoid arthritis have predominantly Thl cells in synovial tissue (Simon, A.K., et al. (1994) Proc. Natl. Acad. Sci. USA £:8562-8566) and experimental autoimmune encephalomyelitis (E ⁇ E) can be induced by autoreactive Thl cells (Kuchroo, V.K., et al. (1993) J. Immunol. 151 :4371-4381).
  • Thl and Th2 subsets require an understanding ofthe mechanisms by which the differentiation of CD4 T helper precursor cells (Thp), which secrete only IL-2, choose to become Thl or Th2 effector cells. It is clear that the cytokines themselves are potent Th cell inducers and form an autoregulatory loop (see e.g., Paul, W.E. and Seder, R.A. (1994) Cell 76:241-251; Seder, R.A. and Paul, W.E. (1994) Ann. Rev. Immunol. 12:635-673). Thus, IL-4 promotes the differentiation of Th2 cells while preventing the differentiation of precursors into Thl cells, while IL-12 and IFN- ⁇ have the opposite effect.
  • Thp CD4 T helper precursor cells
  • Thl :Th2 ratios One possible means therefore to alter Thl :Th2 ratios is to increase or decrease the level of selected cytokines.
  • Direct administration of cytokines or antibodies to cytokines has been shown to have an effect on certain diseases mediated by either Thl or Th2 cells.
  • administration of recombinant IL-4 or antibodies to IL-12 ameliorate EAE, a Thl -driven autoimmune disease (see Racke; M.K. et al. (1994) J. Exp. Med 180: 1961-1966; and Leonard, J.P. et al. (1995) J. Exp. Med. 181:381-386)
  • anti-IL-4 antibodies cure the Th2-mediated parasitic disease, Leishmania major (Sadick, M.D. et al. (1990) J. Exp. Med. 171 :115- 127).
  • systemic administration of cytokines or antibodies may have unwanted side effects and, accordingly, alternative approaches to manipulating Thl/Th2 subsets are still
  • NF-AT is a multisubunit transcription complex that contains a cyclosporin A sensitive cytoplasmic phosphoprotein and an inducible nuclear component composed of AP-1 family member proteins (Flanagan, W.M. et al. (1991) Nature 352:803-807; Jain, J. et al. (1992) Nature 356:801-804).
  • NF-ATp Purification and cloning of NF-ATp revealed a region of limited sequence identity to the Rel Homology Domain (RHD) ofthe NFKB family of transcription factors (McCaffrey, P.G. et al. (1993) Science 262:750-754). Subsequent cloning and sequencing of three related genes, NF-ATc, NF-AT4/x/c3, and NF-AT3/c4 revealed similar domains. NF-AT family members share approximately 70% sequence similarity within this domain and approximately 18% sequence similarity to the RHD ofthe Rel/NF ⁇ B family of transcription factors.
  • RHD Rel Homology Domain
  • NF-AT proteins are not likely to be directly responsible for mediating Thl- or Th2-specific cytokine transcription. Most, if not all, NF-AT binding sites in cytokine promoter regulatory regions are accompanied by nearby sites that bind auxiliary transcription factors, usually members ofthe AP-1 family. It has been shown that NF-AT and AP-1 proteins bind coordinately and cooperatively and are required for full activity ofthe IL-2 and IL-4 promoters. Different AP-1 proteins, specifically c-Jun, c-Fos, Fra-1, Fra-2, Jun B and Jun D, have been shown to bind to these sites (Rao, A. et al. ( 1994) Immunol.
  • This invention pertains to methods for regulating production of Th2-associated cytokines and for regulating Thl or Th2 subsets by modulating the activity of one or more transcription factors that regulate expression of Th2-specific cytokine genes, and to compositions useful for such modulation
  • tissue-specific expression of IL-4 in Th2 cells is not due to a repressor protein but rather to a Th2-specific transactivator protein.
  • the proto-oncogene c-Maf has now been demonstrated to be responsible for the tissue-specific expression of the Th2-associated cytokine interleukin-4.
  • c-Maf ectopic expression of c-Maf in cells other than Th2 cells (e.g., Thl cells, B cells and non-lymphoid cells) leads to activation of the IL-4 promoter and, under appropriate conditions, production of endogenous IL-4. It further has been discovered that c-Maf and NF-AT synergize to activate Th2-associated cytokine gene expression.
  • NIP45 for NF-AT Interacting Protein 45
  • RHD Rel Homology Domain
  • NIP45 potentiates gene expression mediated by c-Maf and NF-AT such that when all three factors (c-Maf, NF-AT and NIP45) are active in a cell, high levels of endogenous IL-4 production is stimulated.
  • a small maf protein lacking a transactivation domain, such as pl 8 can repress Th2-associated cytokine gene expression, e.g., expression mediated by c-Maf.
  • this invention pertains to methods for modulating Th2-associated cytokines expression by modulating the expression or activity of one or more transcription factors that cooperate with an NF-AT family protein to regulate the expression of Th2-associated cytokine genes.
  • the transcription factor that cooperates with an NF-AT family protein to regulate the expression of a Th2- associated cytokine gene, and thus whose expression or activity is modulated is a Th2- specific transcription factor (e.g. , a Th2-specific maf family protein).
  • the transcription factor that cooperates with an NF-AT family protein to regulate the expression of a Th2-associated cytokine gene, and thus whose expression or activity is modulated is a maf family protein, such as c-Maf.
  • the transcription factor that cooperates with an NF-AT family protein to regulate the expression of a Th2-associated cytokine gene, and thus whose expression or activity is modulated is a protein that interacts with an NF-AT family protein, such as NIP45.
  • the expression or activity of a small maf protein, such as pl 8 is modulated.
  • the methods ofthe invention may involve modulating the expression or activity of one transcription factor (e.g., c-Maf or NIP45 or pl 8) or a combination of transcription factors (e.g., c-Maf + NF-AT, or NF-AT + NIP45, or c-Maf + NF-AT + NIP45).
  • the modulatory methods ofthe invention generally involve contacting a cell with an agent that modulates the expression or activity of a transcription factor(s) such that production ofthe Th2-associated cytokine by a cell is modulated.
  • preferred agents ofthe invention act intracellularly to modulate the activity ofthe transcription factor.
  • the modulatory method of the invention stimulates production of a Th2-associated cytokine.
  • Th2-associated cytokine production can be stimulated in Thl cells, B cells or non-lymphoid cells.
  • the modulatory method ofthe invention inhibits production of a Th2-associated cytokine.
  • a Th2-associated cytokine modulated in the method preferably is interleukin-4.
  • a variety of agents can be used to stimulate the expression or activity of a transcription factor that regulates expression of a Th2-associated cytokine gene.
  • a stimulatory agent ofthe invention can be a nucleic acid molecule encoding the transcription factor that is introduced into and expressed in the cell.
  • chemical agents that enhance the expression or activity ofthe transcription facto can be used as stimulatory agents.
  • agents can be used to inhibit the expression or activity of a transcription factor that regulates expression of a Th2-associated cytokine gene.
  • inhibitory agents include antisense nucleic acid molecules that are complementary to a gene encoding the transcription factor, intracellular antibodies that bind the transcription factor (e.g., in the cell nucleus), inhibitory forms ofthe transcription factor (e.g., dominant negative forms) and chemical agents that inhibit the expression or activity ofthe transcription factor.
  • a cell is contacted with at least one additional agent that modulates the activity of at least one additional transcription factor that contributes to the regulation of the Th2 -associated cytokine gene.
  • the at least one additional transcription factor whose expression or activity is modulated is selected from the group consisting of NF-AT family proteins, NF-AT-interacting proteins, maf family proteins and AP-1 family proteins. Cytokine production by a cell can be modulated in vitro or in vivo in accordance with the methods ofthe invention.
  • a cell is contacted with a modulating agent(s) in vitro and then the cell is administered to a subject to thereby regulate the development of Thl and/or Th2 subsets in the subject.
  • the invention provides methods for regulating the development of Thl or Th2 subsets in a subject.
  • these methods involve direct administration to the subject of an agent that modulates the activity of one or more transcription factors that regulate expression of a Th2-associated cytokine gene such that development of Thl or Th2 cells in the subject is modulated.
  • the modulatory methods ofthe invention can be used to manipulate Thl :Th2 ratios in a variety of clinical situations.
  • NIP45 compositions For example, inhibition of Th2 formation may be useful in allergic diseases, malignancies and infectious diseases whereas enhancement of Th2 formation may be useful in autoimmune diseases and organ transplantation.
  • This invention provides isolated compositions of NIP45 protein and isolated nucleic acid sequences encoding NIP45, other compositions related thereto and methods of use thereof.
  • the amino acid sequence of NIP45 protein has been determined (shown in SEQ ID NO: 6) and a cDNA encoding NIP45 protein has been isolated (the nucleotide sequence of which is shown in SEQ ID NO: 5).
  • the invention provides isolated nucleic acid molecules encoding NIP45, or fragments thereof.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding NIP45 protein.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence that is homologous to the amino acid sequence of SEQ ID NO: 6 and interacts with the Rel Homology Domain of an NF-AT family protein.
  • the invention provides an isolated nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 5.
  • the invention provides an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 5.
  • the invention provides an isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 6.
  • Isolated nucleic acid molecules encoding NIP45 fusion proteins and isolated antisense nucleic acid molecules are also encompassed by the invention.
  • vectors such as recombinant expression vectors, containing a NIP45 nucleic acid molecule ofthe invention and host cells into which such vectors have been introduced.
  • a host cell is used to produce NIP45 protein by culturing the host cell in a suitable medium. If desired, NIP45 protein can be then isolated from the host cell or the medium.
  • Still another aspect ofthe invention pertains to isolated NIP45 proteins, or portions thereof.
  • the invention provides an isolated NIP45 protein, or a portion thereof that interacts with an NF-AT family protein.
  • the invention provides an isolated protein which comprises an amino acid sequence homologous to the amino acid sequence of SEQ ID NO: 6 and that interacts with an NF-AT family protein.
  • NIP45 fusion proteins are also encompassed by the invention.
  • the NIP45 proteins ofthe invention can be used to prepare anti-NIP45 antibodies. Accordingly, the invention further provides an antibody that specifically binds NIP45 protein. In one embodiment, the antibody is monoclonal. In another embodiment, the antibody is labeled with a detectable substance.
  • the NIP45-encoding nucleic acid molecules ofthe invention can be used to prepare nonhuman transgenic animals that contain cells carrying a transgene encoding NIP45 protein or a portion of NIP45 protein. Accordingly, such transgenic animals are also provided by the invention. In one embodiment, the NIP45 transgene carried by the transgenic animal alters an endogenous gene encoding endogenous NIP45 protein (e.g., a homologous recombinant animal).
  • Another aspect ofthe invention pertains to methods for detecting the presence of NIP45 protein or mRNA in a biological sample.
  • the biological sample is contacted with an agent capable of detecting NIP45 protein (such as a labeled anti-NIP45 antibody) or NIP45 mRNA (such as a labeled nucleic acid probe capable of hybridizing to NIP45 mRNA) such that the presence of NIP45 protein or mRNA is detected in the biological sample.
  • an agent capable of detecting NIP45 protein such as a labeled anti-NIP45 antibody
  • NIP45 mRNA such as a labeled nucleic acid probe capable of hybridizing to NIP45 mRNA
  • these compositions include recombinant expression vectors that encode a maf family protein, wherein the maf-encoding sequences are operatively linked to regulatory sequences that direct expression ofthe maf family protein in a specific cell type, such as lymphoid cells (e.g., T cells or B cells) or hematopoietic stem cells.
  • these compositions include host cells, such as host lymphoid cells (e.g., host T cells or host B cells) or host hematopoietic stem cells, into which a recombinant expression vector encoding a maf family protein has been introduced.
  • the invention further provides transgenic animals that express a c-Maf protein.
  • the transgenic animal is a mouse and the mouse overexpresses c-Maf in T cells, preferably using a trangene that comprises a CD4 promoter/enhancer operatively linked to a c-maf cDNA.
  • Figure 1 A is a schematic ofthe cell fusion approach used to demonstrate that cytokine expression is not due to a repressor
  • Figure IB is a reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of 11-2 and IL-4 cytokine, and control ⁇ -actin, mRNA expressed by an unfused Thl clone (D 1.1), an unfused Th2 clone (D10), Thl and Th2 homokaryons and Thl-Th2 heterokaryons.
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • Figure 2 A is a Northern blot analysis depicting expression of an isolated cDNA clone in Thl cells, Th2 cells or B lymphoma cells. A control probe specific for GAPDH was used to show equal loading of RNA.
  • Figure 2B is a Northern blot analysis depicting upregulated expression ofthe isolated cDNA clone during in vitro differentiation of normal naive spleen cells into Th2 cells. Total RNA was isolated from cells harvested at the indicated time points. Culture supernatant at the appropriate dilution was measured for cytokine (IL-10) production by ELISA to determine differentiation into the Thl or Th2 lineage.
  • IL-10 cytokine
  • Figure 3 A is a bar graph depicting transactivation of the IL-4 promoter by c-Maf in a Thl clone (AE7).
  • AE7 cells were cotransfected with a wild-type IL-4 CAT reporter construct and either a control plasmid (pMEX-NeoI), a c-Maf expression plasmid (pMEX-Maf) or a c-Fos expression plasmid (pMEX-c-Fos).
  • pMEX-NeoI control plasmid
  • pMEX-Maf a c-Maf expression plasmid
  • pMEX-c-Fos a c-Fos expression plasmid
  • Figure 3B is a photograph of a thin layer chromotography plate depicting the relative CAT activity in M12 B lymphoma cells cotransfected with a wild-type IL-4 CAT reporter construct and either two control plasmids (pMEX-NeoI and pREP4), a c- Maf expression plasmid and a control plasmid (pMEX-Maf and pREP4), a c-Fos expression plasmid and a control plasmid (pMEX-c-Fos and pREP4), a c-Jun expression plasmid and a control plasmid (pMEX-c-Jun and pREP4), a control plasmid and an NF- ATp expression plasmid (pMEX-NeoI and pREP-NF-ATp), a c-Maf expression plasmid and an NF-ATp expression plasmid (pMEX-M
  • Figure 4 is a bar graph depicting endogenous production of IL-4 in Ml 2 cells by ectopic expression of c-Maf and NF-ATp.
  • Cells stably transfected with the indicated control or expression plasmids were either unstimulated or stimulated with PMA and ionomycin for 24 hours. 200 ⁇ l of supernatant from each sample was subjected to ELISA for cytokine quantitation.
  • Figure 5 A is a photograph of a DNAse I footprint gel of the IL-4 promoter performed using nuclear extracts from Th2 (D10, CDC35) or Thl (AE7, S53) clones harvested at the indicated time points after stimulation with anti-CD3 antibodies, which depicts a Th2-specific footprint immediately downstream ofthe putative MARE site in the IL-4 promoter. Two Th2-specific hypersensitive residues on the non-coding strand of the IL-4 promoter are indicated by *. Five lanes of a DNAse I digestion of the IL-4 promoter probe (without nuclear extract) and a Maxam-Gilbert A/G ladder were run next to the DNAse I treated samples.
  • Figure 5B is a schematic representation ofthe proximal regulatory region ofthe murine IL-4 promoter.
  • the top portion shows the primary sequence ofthe murine IL-4 promoter. The numbers indicated are relative to the start site of transcription at +1. Asterisks denote the Th2-specific hypersensitive residues seen on DNAse I footprint.
  • the bottom portion shows the sequence ofthe wild type (-59 to -28) oligonucleotide and the 4 bp mutants used in EMSA and the functional assays shown in Figures 6 and 7. Altered nucleotides are shown in lowercase bold and correspond to the numbering system shown in the top portion.
  • FIG. 6 is a photograph of an electrophoretic mobility shift assay (EMSA) demonstrating that c-Maf but not c-Jun binds to the proximal IL-4 promoter and forms a complex with NF-ATp.
  • EMSA was performed using the indicated proteins and labeled double-stranded oligonucleotides.
  • Figure 7A is a bar graph (top) and a photograph of a thin layer chromotography plate (bottom) depicting the relative CAT activity in Ml 2 cells co-transfected with a c- Maf expression vector and either the wild-type IL-4 CAT reporter construct or one ofthe 4 bp mutants shown in Figure 5B, demonstrating that transactivation of the IL-4 promoter by c-Maf maps to the MARE and Th2-specific footprint. The average of three independent experiments and one representative experiment are shown in the top and bottom portions, respectively.
  • Figure 7B is a photograph of an EMSA, performed using recombinant c-Maf, the
  • IL-4 promoter (-59 to -27) probe and the indicated unlabeled double-stranded oligonucleotides as competitors, demonstrating that binding of recombinant c-Maf to the IL-4 promoter maps to the MARE and Th2-specific footprint.
  • Figure 8 is photograph of yeast colonies, in triplicate, transformed with the NIP45 plasmid and either NF-ATp-RHD as "bait" or control baits, Max, CDK2 or pEG202, together with the LacZ reporter plasmid pSHl 8, indicating that only those colonies containing the NIP45 plasmid and the NF-ATp-RHD bait expressed the LacZ reporter gene.
  • Figure 9 is a photograph of an immunoprecipitation/Western blot experiment demonstrating that NIP45 and NF-ATp interact in HepG2 cells.
  • Figure 10 is a schematic diagram comparing the structures ofthe original NIP45 cDNA clone isolated from the yeast two-hybrid screen (top) and the longest NIP45 cDNA clone isolated from a D10.G4 lambda zap II library (bottom).
  • Figure 11 depicts the nucleotide and predicted amino acid sequences ofthe original NIP45 cDNA isolate.
  • Figure 12 depicts the hydrophobicity plot ofthe NIP45 cDNA.
  • Figure 13 is a photograph of an RNA blot analysis of NIP45 transcript levels in various tissues.
  • Figure 14A is a photograph of immunofluorescence analysis of BHK cells transfected with an expression construct encoding an HA-epitope tagged NIP45 protein and probed with a monoclonal antibody specific for the HA peptide as the primary antibody and an indocarbocyanine labelled goat anti-mouse secondary reagent.
  • Figure 14B is a photograph of the same cells depicted in Figure 7A counterstained with the DNA staining dye Hoechst 33258.
  • Figure 14C is a photograph of immunofluorescence analysis of unstimulated
  • BHK cells transfected with an expression construct encoding NF-AT4 and probed with an anti-NF-AT4 specific antibody as the primary antibody and an indocarbocyanine labelled goat anti-mouse secondary reagent.
  • Figure 14D is a photograph ofthe same cells depicted in Figure 7C counterstained with the DNA staining dye Hoechst 33258.
  • Figure 14E is a photograph of immunofluorescence analysis of ionomycin- treated BHK cells transfected with an expression construct encoding NF-AT4 and probed with an anti-NF-AT4 specific antibody as the primary antibody and an indocarbocyanine labelled goat anti-mouse secondary reagent.
  • Figure 14F is a photograph ofthe same cells depicted in Figure 7D counterstained with the DNA staining dye Hoechst 33258.
  • Figure 15 is a photograph of CAT assay results (left) and a bar graph quantitating the relative fold induction of CAT activity (right) in HepG2 cells transfected with a 3X NF-AT-CAT reporter gene construct (containing three NF-AT binding sites) and either a control expression plasmid or an NF-AT family expression plasmid (NF-ATp, NF-ATc, NF-AT3 or NF-AT4), alone (-) or in combination with a NIP45 expression plasmid (+).
  • Figure 16 is a photograph of CAT assay results (left) and a bar graph quantitating the relative fold induction of CAT activity (right) in HepG2 cells transfected with an IL- 4-CAT reporter gene construct (extending to -732 bp of the IL-4 promoter) and combinations of NF-ATp, NIP45 and/or c-Maf expression constructs, as indicated.
  • Figure 17 is a bar graph depicting the level of IL-4 (in pg/ml) in the supernatants of Ml 2 B lymphoma cells transiently cotransfected with expression plasmids for NF- ATp, c-Maf and a pCI vector control (top bar) or expression plasmids for NF-ATp, c- Maf and NIP45 (bottom bar).
  • Figure 18 is a Northern blot analysis of transcripts expressed on day 0, 1, 3, 5 or 7 during in vitro differentiation of normal naive spleen cells into Th2 cells, depicting upregulated expression of c-maf over time and downregulated expression of pl 8 over time.
  • Figure 19 is a photograph of a thin layer chromotography plate depicting the relative CAT activity in Ml 2 cells transfected with an IL-4 promoter reporter gene construct and either a c-Maf expression vector alone (5 ⁇ g), a pl 8 expression vector alone (10 ⁇ g) or a constant amount of c-Maf expression vector (5 ⁇ g) together with increasing amounts of a pl8 expression vector alone (2.5, 5 or 10 ⁇ g), depicting repression of IL-4 promoter activity by pl8.
  • Figure 20 is a schematic diagram of a c-maf transgene for overexpression in T cells using a CD4 promoter/enhancer to regulate expression ofthe c-maf cDNA.
  • Figure 21 is a bar graph depicting total cell numbers (x million) in lymph nodes (LN), spleen or thymus from either wild type mice or c-maf transfenic mice, demonstrating that c-maf transgenic mice exhibit decreased cell numbers in lymphoid organs.
  • Figure 22 is a bar graph depicting basal levels of serum IgE (ng/ml) in wild type mice or c-maf transfenic mice, demonstrating that c-maf transgenic mice exhibit increased basal levels of serum IgE.
  • this invention pertains to methods and compositions for regulating cytokine gene expression and T cell subsets by modulating transcription factor activity.
  • the invention is based, at least in part, on the discovery that Th2-specific expression of the interleukin-4 gene does not result from the action of a specific repressor protein (as shown in Example 1 ) but rather from the action of a specific transactivator protein.
  • the transcription factor responsible for Th2-specific expression ofthe interleukin-4 gene has now been identified as the c-Maf proto-oncoprotein, which is selectively expressed in differentiating and mature Th2 cells and absent from Thl cells (see Example 2).
  • Ectopic expression of c-Maf in cells that do not normally express it leads to transactivation of the IL-4 promoter (see Example 3) and, under appropriate conditions, to production of endogenous IL-4 (see Example 4).
  • a protein present in nuclear extracts of Th2 cells, but not Thl cells footprints the IL-4 promoter in the region of a maf response element (MARE) (see Example 5) and recombinant c-Maf binds to the IL-4 promoter in vitro (see Example 6).
  • MARE maf response element
  • the ability of c-Maf to transactivate IL-4 maps to the MARE and Th2-specific footprint in the IL-4 promoter (see Example 7).
  • the invention further is based, at least in part, on the discovery of a protein that interacts with NF-AT and potentiates transcriptional activation by c-Maf and NF-AT.
  • This protein, NIP45 was identified based upon its interaction with the Rel Homology Domain (RHD) of NF-AT (see Example 8). Coimmunoprecipitation experiments demonstrated that NIP45 and NF-AT interact in vivo in mammalian cells (see Example 9).
  • the cDNA encoding NIP45 has been sequenced and characterized (see Example 10). Examination of the tissue expression pattern of NIP45 mRNA revealed that the NIP45 transcript is preferentially expressed in spleen, thymus and testis (see Example 1 1 ).
  • NIP45 protein is evenly distributed throughout the cell nucleus (see Example 12). Functional studies showed that NIP45 synergizes with NF-AT to stimulate transcription from promoters containing NF-AT binding sites and, moreover, synergizes with NF-AT and c-Maf to stimulate transcription from the IL-4 promoter (see Example 13). Moreover, NIP45, NF-AT and c-Maf can act in concert to induce expression ofthe endogenous IL-4 gene in cells that do not normally express IL-4 (e.g., B cells.) (see Example 14).
  • the invention still further is based, at least in part, on the discovery that a small maf protein, pl8, that lacks an activation domain, can repress cytokine gene expression mediated by c-Maf.
  • a small maf protein, pl8 that lacks an activation domain
  • Differentiation of T helper cell precursors in vitro is associated with upregulation of c-maf gene expression and downregulation of pl 8 gene expression (see Example 15).
  • coexpression of pl 8 with c-Maf represses IL-4 promoter activity, as compared to IL-4 promoter activity in the presence of c-Maf alone (see Example 16).
  • the invention is still further based on the generation of c-maf transgenic animals that overexpress c-Maf protein in T cells (see Example 17). These animals exhibit a phenotype very similar to transgenic animals that overexpress IL-4, namely small thymus and spleen, dramatic decreases in numbers of CD + /CD8 + (double-positive) thymocytes, decreased CD4 + positive T cells and increased basal levels of serum IgE.
  • Th2-associated cytokine is intended to refer to a cytokine that is produced preferentially or exclusively by Th2 cells rather than by Thl cells.
  • Th2-associated cytokines include IL-4, IL-5, IL-6 and IL-13.
  • a preferred Th2-associated cytokine whose production is modulated according to the methods ofthe invention is interleukin-4.
  • transcription factor is intended to refer to a factor (e.g., a protein) that acts in the nucleus to regulate the transcriptional expression of a gene.
  • transcription factor is intended to include factors that directly regulate transcription (e.g., have instrinsic transcriptional activation or inhibitory activity) and factors that indirectly regulate transcription (e.g., through interaction with other factors that have intrinsic transcriptional activation or inhibitory activity).
  • a transcription factor that "cooperates with a Nuclear Factor of Activated T cells family protein to regulate expression of a Th2 -associated cytokine gene” is intended to refer to a transcription factor that synergizes or acts in concert with an NF-AT protein to regulate expression of a Th2-associated cytokine gene. That is, the expression ofthe Th2-associated cytokine gene (e.g.. IL-4) is greater in the presence of both NF-AT and the cooperative transcription factor than in the presence of either alone.
  • the cooperative transcription factor may or may not physically associate with NF-AT.
  • transcription factors that cooperate with an NF-AT family protein to regulate expression of a Th2-associated cytokine gene include maf family proteins (e.g. , c-Maf) and NF-AT-interacting proteins (e.g., NIP45).
  • a transcription factor that "contributes to the regulation of a Th2- associated cytokine gene” is intended to refer to a transcription factor that participates in the transcriptional regulation of a Th2-associated cytokine gene, regardless of whether it cooperates with an NF-AT family protein.
  • Transcription factors that cooperate with NF- AT to regulate the expression of a Th2-associated cytokine gene also are factors that contribute to the regulation ofthe Th2-associated cytokine gene.
  • other transcription factors that do not cooperate with NF-AT also can contribute to the regulation ofthe Th2-associated cytokine gene.
  • transcription factors that are thought to contribute to the regulation of Th2 -associated cytokine genes include NF- AT family proteins, NF-AT-interacting proteins, maf family proteins, AP-1 family proteins, and Stat ⁇ (Lederer, J. et al. (1996) J. Exp. Med. 184:397-406).
  • Th2-specific transcription factor is intended to refer to a transcription factor that is expressed preferentially or exclusively in Th2 cells rather than in Thl cells.
  • contacting i.e., contacting a cell with an agent
  • contacting is intended to include incubating the agent and the cell together in vitro (e.g., adding the agent to cells in culture) and administering the agent to a subject such that the agent and cells ofthe subject are contacted in vivo.
  • modulation are intended to include stimulation (e.g., increasing or upregulating a particular response or activity) and inhibition (e.g., decreasing or downregulating a particular response or activity).
  • maf family protein is intended to refer to a member of a sub-family of AP-1/CREB/ATF proteins that includes v-Maf, c-Maf, mafB, Nrl, mafK, mafF, mafG and pl8. See e.g., Nishizawa, M. et al. (1989) Proc. Nail. Acad. Sci. USA 86:7711-7715; Kataoka, K. et al. (1993) J. Virol. 67:2133-2141 ; Swaroop, A. et al. (1992) Proc. Natl. Acad. Sci. USA 89:266-270; Fujiwara, K.T.
  • small maf protein is intended to refer to a maf family protein that lacks a domain corresponding to the amino-terminal activation domain of c- Maf. Examples of small maf proteins include mafK, mafF, mafG and pl ⁇ .
  • NF-AT family protein (also referred to interchangeably as simple "NF-AT”) is intended to refer to a member ofthe family of Nuclear Factors of Activated T cell transcription factors, including NF-ATp, NF-ATc, NF-AT4/x/c3 and NF-AT3/c4.
  • RHD domain Rel Homology Domain of an NF-AT family protein
  • NF-AT-interacting protein (used interchangeably with "a protein that interacts with an NF-AT family protein”) is intended to refer to a factor that forms a physical association with an NF-AT family protein (e.g., co- immunoprecipitates with an NF-AT family protein).
  • the NF-AT-interacting protein interacts with the RHD of an NF-AT family protein.
  • An example of an NF-AT- interacting protein is NIP45.
  • NIP45 is intended to include proteins having the amino acid sequence shown in SEQ ID NO: 6 (or encoded by the nucleotide sequence shown in SEQ ID NO: 5), as well as mammalian homologues thereof (e.g., human NIP45) and modified forms thereof (e.g., mutated or truncated forms) that retain the ability to interact with the RHD of NF-AT.
  • AP-1 family protein is intended to refer to a protein that is a member ofthe AP-1 family of transcription factors, examples of which include c-Jun, c-Fos, Fra- 1 , Fra-2, Jun B and Jun D.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA).
  • the nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • isolated nucleic acid molecule refers to a nucleic acid molecule that is free of gene sequences which naturally flank the nucleic acid in the genomic DNA ofthe organism from which the nucleic acid is derived (i.e., gene sequences that are located adjacent to the isolated nucleic molecule in the genomic DNA of the organism from which the nucleic acid is derived).
  • the isolated NIP45 nucleic acid molecule may contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA ofthe cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, may be free of other cellular material.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60 % homologous to each other typically remain hybridized to each other.
  • the conditions are such that at least sequences at least 65 %, more preferably at least 70 %, and even more preferably at least 75 % homologous to each other typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • a preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C.
  • SSC sodium chloride/sodium citrate
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • an "antisense” nucleic acid comprises a nucleotide sequence which is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDN A molecule, complementary to an mRNA sequence or complementary to the coding strand of a gene. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • coding region refers to regions of a nucleotide sequence comprising codons which are translated into amino acid residues
  • noncoding region refers to regions of a nucleotide sequence that are not translated into amino acids (e.g., 5' and 3 1 untranslated regions).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • the term "host cell” is intended to refer to a cell into which a nucleic acid ofthe invention, such as a recombinant expression vector ofthe invention, has been introduced.
  • the terms "host cell” and “recombinant host cell” are used interchangeably herein. It should be understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope ofthe term as used herein.
  • nucleic acid molecule that is "in a form suitable for expression ofthe nucleic acid molecule in a cell” is intended to means that the nucleic acid molecule includes one or more regulatory sequences, selected on the basis ofthe host cells to be used for expression and the level of expression desired, which is operatively linked to the nucleic acid molecule to be expressed such that a protein encoded by the nucleic acid molecule is expressed in the host cell.
  • nucleic acid molecules include recombinant expression vectors containing nucleotide sequences encoding the protein to be expressed in the host cell.
  • transgenic animal refers to a non-human animal, preferably a mammal, more preferably a mouse, in which one or more ofthe cells ofthe animal includes a "transgene".
  • transgene refers to exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome ofthe mature animal, for example directing the expression of an encoded gene product in one or more cell types or tissues ofthe transgenic animal.
  • a "homologous recombinant animal” refers to a type of transgenic non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell ofthe animal, e.g., an embryonic cell ofthe animal, prior to development ofthe animal.
  • an "isolated protein” refers to a protein that is substantially free of cellular material or culture medium when isolated from cells or produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • antibody is intended to include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as Fab and F(ab') 2 fragments.
  • an agent that "acts intracellularly to modulate the expression or activity of a transcription factor” is intended to refer to an agent that functions in an intracellular region of a cell, e.g., the cytoplasm or nucleus, to modulate the expression or activity ofthe transcription factor.
  • an agent that binds to the cell surface is not intended to be encompassed by the term "an agent that acts intracellularly to modulate the expression or activity of a transcription factor.”
  • agents that act intracellularly to modulate the expression or activity of a transcription factor include nucleic acid molecules that encode the transcription factor, antisense nucleic acid molecules, intracellular antibodies, dominant negative inhibitors and chemical agents that enter a cell and modulate (i.e., stimulate or inhibit) transcription factor expression or activity.
  • intracellular binding molecule is intended to include agents that act intracellularly to inhibit the expression or activity of a target protein of interest (e.g., a transcription factor) by binding to the protein itself or to a nucleic acid (e.g., an mRNA molecule) that encodes the protein.
  • a target protein of interest e.g., a transcription factor
  • nucleic acid e.g., an mRNA molecule
  • intracellular binding molecules include antisense nucleic acids, intracellular antibodies and dominant negative inhibitors.
  • the transcription factor responsible for the Th2-specific expression ofthe interleukin-4 gene has now been identified as the c-Maf proto-oncogene. Modulation of the expression and/or activity of c-Maf, therefore, provides a means to regulate the production of interleukin-4. Since IL-4 itself serves an autoregulatory function in the development of Th2 cells (see e.g., Paul, W.E. and Seder, R.A. (1994) Cell 76:241-251 ; Seder, R.A. and Paul, W.E. (1994) Ann. Rev. Immunol.
  • Th2-associated cytokines such as IL-5, IL-6, IL-10 and IL-13
  • Th2 differentiation modulation of c- Maf expression and/or activity provides a general approach for modulating production of Th2-associated cytokines.
  • the maf family of proteins are a sub- family of AP-1/CREB/ATF proteins that includes v-Maf, c-Maf, mafB, Nrl, mafK, mafF, mafG and pl 8.
  • the v-r ⁇ tf/Oncogene was originally isolated from a spontaneous musculoaponeurotic fibrosarcoma of chicken and identified as the transforming gene ofthe avian retrovirus, AS42 (Nishizawa, M. et al. (1989) Proc. Natl. Acad. Sci. USA 86 771 1-7715).
  • V-maf encodes a 42 kd basic region/leucine zipper (b-zip) protein with homology to the c-fos and c-jun oncogenes. Its cellular homologue, the c-maf proto-oncogene has only two structural changes in the coding region from v-maf (Kataoka, K. et al. ( 1993) J. Virol. 67:2133-2141).
  • the maf family includes c-Maf, mafB, a human retina-specific protein Nrl (Swaroop, A. et al. (1992) Proc. Natl. Acad. Sci. USA 89:266-270), mafK, mafF, mafG and pl8.
  • mafK, mafF, mafG and pl8 each encode proteins that lack the amino terminal two thirds of c-Maf that contains the transactivating domain ("small maf proteins")
  • small maf proteins proteins that lack the amino terminal two thirds of c-Maf that contains the transactivating domain
  • C-maf and other maf family members form homodimers and heterodimers with each other and with Fos and Jun, consistent with the known ability ofthe AP-1 proteins to pair with each other (Kerppola, T.K. and Curran, T. ( 1994) Oncogene 9:675-684; Kataoka, K. et al. (1994) Mol. Cell. Biol. 14:700-712).
  • the DNA target sequence to which c-Maf homodimers bind termed the c-Maf response element (MARE), is a 13 or 14 bp element which contains a core TRE (T-MARE) or CRE (C-MARE) palindrome respectively.
  • the small mafs have been shown to function as repressors of ⁇ and ⁇ -globin transcription when bound as homodimers but are essential as heterodimeric partners with the erythroid-specific factor p45NF-E2 to activate globin gene transcription (Kataoka, K. el al. (1995) Mol. Cell. Biol. 15:2180-2190; Igarashi, K. et al. (1994) Nature 367:568-572). MafK overexpression has been shown to induce erythroleukemia cell differentiation (Igarashi, K. et al. (1995) Proc. Natl. Acad. Sci. USA 92:7445-7449).
  • the present invention provides evidence that small maf proteins (e.g., pl 8) can modulate the expression of Th2-associated cytokine genes. Accordingly, modulation of the expression and/or activity of a small maf protein also provides a means to regulate the production of Th2 -associated cytokine genes.
  • small maf proteins e.g., pl 8
  • modulation of the expression and/or activity of a small maf protein also provides a means to regulate the production of Th2 -associated cytokine genes.
  • the present invention further provides an NF-AT-interacting protein, NIP45, that binds to and synergizes with NF-AT to regulate expression of a Th2-associated cytokine gene.
  • NIP45 was identified based upon its interaction with the Rel Homology Domain of NF-ATp. NIP45 is described in further detail hereinbelow. Modulation ofthe expression and/or activity of an NF-AT-interacting protein, such as NIP45, thus also provides a means to regulate the production of Th2-associated cytokine genes.
  • this invention provides methods for modulating production of a Th2-associated cytokine by a cell by modulating the expression or activity of one or more transcription factors involved in Th2-associated cytokine gene expression.
  • a cell is contacted with an agent that modulates the expression or activity of a transcription factor such that such that production ofthe Th2-associated cytokine by a cell is modulated.
  • the transcription factor to be modulated is characterized as a transcription factor that cooperates with an NF-AT family protein to regulate expression of the Th2-associated cytokine gene (e.g. , c-Maf or NIP45).
  • the transcription factor to be modulated is a maf family protein (e.g., c-Maf or a small maf protein, such as pl8).
  • the transcription is an NF-AT-interacting protein (e.g., NIP45).
  • the modulatory agents of the invention are characterized by acting intracellularly to modulate the activity of a transcription factor.
  • production of a Th2-associated cytokine by a cell is stimulated by contacting the cell with a stimulatory agent that stimulates transcription factor expression and/or activity.
  • a Th2-associated cytokine by a cell is inhibited by contacting the cell with a inhibitory agent that inhibits transcription factor expression and/or activity.
  • a inhibitory agent that inhibits transcription factor expression and/or activity.
  • c-Maf is responsible for the tissue specificity of IL-4 gene expression
  • c-Maf acts synergistically with one or more additional transcription factors to activate IL-4 gene transcription.
  • c-Maf acts synergistically with an NF-AT protein to stimulate IL-4 gene expression.
  • NF-AT proteins and other members ofthe AP-1/CREB/ATF family of transcription factors have been demonstrated to be involved in regulating expression of both Thl- and Th2-associated cytokine genes.
  • the method of the invention for modulating Th2-associated cytokine production by a cell can comprises contacting the cell with multiple agents that modulate the expression or activity of transcription factors.
  • the methods can further comprise contacting the cell with one or more additional agents that modulate the activity of one or more additional transcription factors that contributes to the regulation ofthe Th2-associated cytokine gene.
  • the additonal agent(s) modulates the expression or activity or an additional transcription factor(s) selected from the group consisting of NF-AT family proteins, NF-AT-interacting proteins, maf family proteins and AP-1 family proteins.
  • a small maf protein e.g., pl8
  • the method ofthe invention for modulating Th2-associated cytokine production by a cell comprises contacting the cell with an agent that modulates (i.e., stimulates or inhibits) the expression or activity of a small maf protein, alone or in combination with agents that modulate the activity of other transcription factors, such as other maf family proteins, NF-AT family proteins or NF-AT-interacting proteins.
  • the small maf protein is pl8.
  • Other examples of small maf proteins include mafK, mafF and mafG.
  • Th2-associated cytokine production by a cell the cell is contacted with a stimulatory agent that stimulates expression and/or activity of a transcription factor (e.g., c-Maf, NIP45, pl8) that regulates expression of a Th2 -associated cytokine gene.
  • a transcription factor e.g., c-Maf, NIP45, pl8
  • Th2-associated cytokine production can be stimulated in cell types that do not normally express such cytokines, such as Thl cells, B cells or non-lymphoid cells.
  • Th2-associatcd cytokine production can be stimulated in helper precursor cells (Thp) to promote their differentiation along the Th2 pathway instead of the Thl pathway.
  • Thp helper precursor cells
  • a preferred stimulatory agent is a nucleic acid molecule encoding a transcription factor that regulates expression of a Th2-associated cytokine gene, wherein the nucleic acid molecule is introduced into the cell in a form suitable for expression of the transcription factor in the cell.
  • a c-Maf cDNA is cloned into a recombinant expression vector and the vector is transfected into the cell.
  • ectopic expression of a c-maf recombinant expression vector in Thl cells, B cells or non-lymphoid cells leads to activation of the IL-4 promoter.
  • transcription ofthe endogenous IL-4 gene is stimulated, leading to IL-4 production by cells that do not normally express this cytokine (see Example 4).
  • a maf family cDNA is first introduced into a recombinant expression vector using standard molecular biology techniques.
  • a maf family cDNA can be obtained, for example, by amplification using the polymerase chain reaction (PCR) or by screening an appropriate cDNA library.
  • the nucleotide sequences of maf family cDNAs are known in the art and can be used for the design of PCR primers that allow for amplification of a cDNA by standard PCR methods or for the design of a hybridization probe that can be used to screen a cDNA library using standard hybridization methods.
  • the maf family cDNA is that ofthe c-maf proto-oncogene.
  • nucleotide and predicted amino acid sequences of a mammalian (mouse) c-maf cDNA are disclosed in Kurscher C. and Morgan, J.I. (1995) Mol. Cell. Biol. L5_:246-254 and deposited in the GenBank database at accession number S74567.
  • This mammalian c-maf ' is highly homologous to the avian v-maf sequence (disclosed in Nishizawa, M.K. et al. ( 1989) Proc. Natl. Acad. Sci. USA 86:771 1 -7715 and GenBank accession numbers D28598 and D28596), indicating that c-maf is well conserved among species.
  • c-m ⁇ /cDNAs from other mammalian species, including humans can be isolated using standard molecular biology techniques (e.g. , PCR or cDNA library screening) and primers or probes designed based upon the mouse or avian sequences.
  • Human partial cDNA sequences homologous to the mouse c-maf cDNA are also deposited in the GenBank database at accession numbers H24189 and N75504.
  • the sequences of other maf family members are also known in the art, for example MafB (Kataoka, K. et al. (1994) Mol. Cell Biol. 14:7581-91 ; GenBank accession number D28600), MafG (Kataoka et al. (1994) Mol. Cell Biol.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno- associated viruses), which serve equivalent functions
  • the recombinant expression vectors ofthe invention comprise a nucleic acid in a form suitable for expression ofthe nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression and the level of expression desired, which is operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell, those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences) or those which direct expression of the nucleotide sequence only under certain conditions (e.g., inducible regulatory sequences).
  • the design ofthe expression vector may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma virus, adenovirus, cytomegalovirus and Simian Virus 40.
  • Non-limiting examples of mammalian expression vectors include pCDM8 (Seed, B., (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBO J. 6:187-195).
  • pCDM8 Seed, B., (1987) Nature 329:840
  • pMT2PC Kaufman et al. (1987), EMBO J. 6:187-195.
  • a variety of mammalian expression vectors carrying different regulatory sequences are commercially available.
  • a preferred regulatory element is the cytomegalovirus promoter/enhancer.
  • inducible regulatory systems for use in mammalian cells are known in the art, for example systems in which gene expression is regulated by heavy metal ions (see e.g., Mayo et al. (1982) Cell 29:99-108; Brinster et al. (1982) Nature 296:39-42; Searle et al. (1985) Mol. Cell. Biol. 5: 1480-1489), heat shock (see e.g., Nouer et al. (1991 ) in Heat Shock Response, e.d. Nouer, L.
  • tissue-specific regulatory sequences are known in the art, including the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268- 277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235- 275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO . I. 8 :729-733) and immunoglobulins (Banerji et al.
  • promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
  • Vector DNA can be introduced into mammalian cells via conventional transfection techniques.
  • transfection are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into mammalian host cells, including calcium phosphate co- precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker may be introduced into a host cell on a separate vector from that encoding a maf family protein or, more preferably, on the same vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g. , cells that have inco ⁇ orated the selectable marker gene will survive, while the other cells die).
  • Nucleic acid molecules encoding other transcription factors that regulate Th2- associated cytokine gene expression, in form suitable for expression of the transcription factor in a host cell can be prepared as described above using nucleotide sequences known in the art or disclosed herein.
  • the nucleotide sequences can be used for the design of PCR primers that allow for amplification of a cDNA by standard PCR methods or for the design of a hybridization probe that can be used to screen a cDNA library using standard hybridization methods.
  • the nucleotide and predicted amino acid sequence of NIP45 are disclosed in SEQ ID NOs: 5 and 6, respectively.
  • nucleotide and predicted amino acid sequences of small maf proteins are known in the art (see e.g., Fujiwara, K.T. et al. (1993) Oncogene 8:2371- 2380; Igarashi, K. et al. (1995) J. Biol. Chem. 270:7615-7624; Andrews, N.C. et al. (1993) Proc. Natl. Acad. Sci. USA 90: 11488-11492; Kataoka, K. et al. (1995) Mol. Cell. Biol. 15:2180-2190).
  • NF-AT family proteins including NF-ATp, NF-ATc, NF-AT4/x/c3 and NF-AT3/c4, are known in the art.
  • Four NF-AT family members have been identified (see e.g., Emmel, E.A. et al. (1989) Science 246: 1617-1620; Flanagan, W.M. et al. (1991 ) Nature 352:803-807: Jain, J. et al. (1993) Nature 365:352-355; McCaffrey, P.G. et al. (1993) Science 262:750-754; Rao, A. (1994) Immunol.
  • the NF-AT cDNA is that of NF-ATp.
  • the nucleotide and predicted amino acid sequences of a mammalian NF-ATp cDNA are disclosed in McCaffrey, P.G. et al. (1993) Science 262:750-754.
  • the nucleotide and predicted amino acid sequences of a mammalian NF-ATc cDNA are disclosed in Northrop, J.P. el al. (1994) Nature 369:497 and deposited in the GenBank database at accession number U08015.
  • nucleotide and predicted amino acid sequences of mammalian NF-AT3 and NF-AT4 cDNAs are disclosed in Hoey, T. et al. (1995) Immunity 2:461-472.
  • the nucleotide and predicted amino acid sequences of AP-1 family proteins are known in the art.
  • the nucleotide and predicted amino acid sequences of human c-fos are disclosed in van Straaten, F. et al. (1983) Proc. Natl. Acad. Sci. USA 80:3183-3187.
  • the nucleotide and predicted amino acid sequences of human c-jun are disclosed in Bohmann, D. et al. (1987) Science 238:1386-1392.
  • nucleotide and predicted amino acid sequences of human jun-B and jun-D are disclosed in Nomura, N. et al. (1990) Nucl. Acids Res. 18:3047-3048.
  • nucleotide and predicted amino acid sequences of human fra-l and fra-2 are disclosed in Matsui, M. et al. (1990) Oncogene 5:249-255.
  • a stimulatory agent for stimulating expression of a Th2- associated cytokine in a cell is a chemical compound that stimulates the expression or activity of an endogenous transcription factor that regulates expression of Th2- associated cytokine genes in the cell (e.g., a maf family protein, such as c-Maf or pl 8, or a protein that interacts with NF-AT, such as NIP45).
  • an endogenous transcription factor that regulates expression of Th2- associated cytokine genes in the cell
  • Such compounds can be identified using screening assays that select for compounds that stimulate the expression or activity of the transcription factor. Examples of suitable screening assays are described in further detail in subsection V below.
  • the stimulatory methods ofthe invention can involve the use of one or more additional agents that stimulate the expression or activity of one or more additional transcription factors that contribute to regulating the expression of a Thl- or Th2-associated cytokine gene.
  • stimulation ofthe expression of endogenous IL-4 in Ml 2 B lymphoma cells required the introduction into the cells of both a c-Maf expression vector and an NF-AT expression vector, thereby demonstrating that c-Maf and NF-AT act synergistically to activate IL-4 transcription, with c-maf responsible for the tissue-specificity of expression.
  • Example 14 it further is shown that stimulation of the expression of endogenous IL-4 in M12 B lymphoma cells is potentiated by coexpression of c-Maf, NF-AT and NIP45. While the skilled artisan will appreciate that certain cells may express sufficient amounts of endogenous c-Maf, NF-AT and/or NIP45 such that use of a single agent alone may be sufficient to stimulate expression of a Th2- associated cytokine gene, in certain situations and with certain cell types it may be necessary to stimulate multiple transcription factors, such as both c-Maf and NF-AT, both c-Maf and NIP45, or all three proteins (c-Maf, NF-AT and NIP45), to achieve the desired stimulation of Th2-associated cytokine production.
  • c-Maf NF-AT
  • NIP45 all three proteins
  • the method in which a cell is contacted with a first agent that stimulates the expression or activity of a first transcription factor, the method can further comprise contacting the cell with at least one additional agent that stimulates the expression or activity of at least additional transcription factors that contribute to regulating the expression of a Thl- or Th2- associated cytokine gene.
  • the at least one additional transcription factor whose expression or activity is modulated is selected from the group consisting of NF- AT family proteins, NF-AT-interacting proteins, maf family proteins and AP-1 family proteins.
  • a stimulatory method ofthe invention can involve the use of a first agent that stimulates the expression or activity of c-Maf and a second agent that stimulates the expression or activity of either an NF-AT family protein or a protein that interacts with an NF-AT family protein (e.g., NIP45).
  • the stimulatory methods of the invention involve the use of a first agent that stimulates the expression or activity of c-Maf, a second agent that stimulates the expression or activity of an NF-AT family protein and a third agent that stimulates the expression or activity of a protein that interacts with an NF-AT family protein (e.g., NIP45).
  • a preferred agent for stimulating NF-AT or NIP45 activity in a cell is a recombinant expression encoding NF-AT or NIP45, respectively, wherein the recombinant expression vector is introduced into the cell and NF-AT or NIP45 is expressed in the cell.
  • NF-AT- and NIP45-encoding expression vectors can be prepared and introduced into cells as described above for c- Maf expression vectors.
  • one or more chemical compounds that stimulate NF-AT or NIP45 activity in a cell can be used as a second (or additional) agent in a stimulatory method of the invention.
  • Compounds that stimulate NF-AT activity in cells are known in the art (for a review see Rao, A. ( 1994) Immunol. Today J_5 :274-281 ).
  • PMA phorbol ester phorbol myristate acetate
  • a calcium ionophore e.g., ionomycin
  • TcR T cell receptor
  • AP-1 family members including c-Jun, c-Fos,
  • Fra-1 , Fra-2, Jun B and Jun D have been shown to be involved in regulating the expression of both Thl- and Th2-associated cytokine genes (e.g., IL-2 and IL-4) (see e.g., Rao, A. et al. (1994) Immunol. Today ⁇ 5_:214-2% ⁇ ; Jain, J. et al. (1993) Nature 365:352-355; Boise. L.H. et al. (1993) Mol. Cell. Biol. 13: 191 1-1919; Rooney, J. et al. (1995) Immunity 2:545-553; Rooney, J. et al. (1995) Mol. Cell. Biol. 15:6299-6310).
  • Thl- and Th2-associated cytokine genes e.g., IL-2 and IL-4
  • AP-1 family members have been shown to increase IL-4 expression in Th2 cells (see e.g., Rooney, J. et al. (1995) Immunity 2:545- 553). Accordingly, in certain circumstances it may be beneficial, in addition to stimulating c-Maf activity (and possibly NF-AT activity), also to stimulate the activity of an AP-1 family protein.
  • the stimulatory methods of the invention involve the use of a first agent that stimulates the expression or activity of c-Maf and a second agent that stimulates the expression or activity of an AP-1 protein.
  • the invention involves the use of a first agent that stimulates the expression or activity of c-Maf, a second agent that stimulates the expression or activity of an NF-AT protein and a third agent that stimulates the expression or activity of an AP-1 protein.
  • NIP45 activity also can be modulated in combination with maf, AP-1 and/or NF-AT family proteins.
  • a preferred agent for stimulating AP-1 activity in a cell is a recombinant expression encoding an AP- 1 protein, wherein the recombinant expression vector is introduced into the cell and the AP-1 protein is expressed in the cell.
  • AP-1 -encoding expression vectors can be prepared and introduced into cells as described above for c- Maf expression vectors.
  • one or more chemical compounds that stimulate AP-1 activity in a cell can be used as additional agents in a stimulatory method of the invention.
  • Compounds that stimulate AP-1 activity in cells are known in the art, including PMA/calcium ionophore (e.g., ionomycin) and anti-CD3 antibodies.
  • Th2-associated cytokine production by a cell is contacted with an inhibitory agent that inhibits expression and/or activity of a transcription factor (e.g., c-Maf, NIP45, pl 8) that regulates expression of a Th2-associated cytokine gene.
  • a transcription factor e.g., c-Maf, NIP45, pl 8
  • Th2- associated cytokine production by a cell is inhibited by contacting the cell with an agent that modulates the expression or activity of a transcription factor that cooperates with an NF-AT family protein to regulate expression of a Th2-associated cytokine gene.
  • Th2-associated cytokine production by a cell is inhibited by contacting the cell with an agent that modulates the expression or activity of a Th2- specific transcription factor, preferably c-Maf.
  • Th2-associated cytokine production by a cell is inhibited by contacting the cell with an agent that modulates the expression or activity of a protein that interacts with an NF-AT family protein, preferably NIP45.
  • Th2 -associated cytokine production by a cell is inhibited by contacting the cell with an agent that modulates the expression or activity of a small maf protein.
  • the inhibitory methods of the invention can comprise contacting the cell with two or more agents that modulate the expression or activity of two or more transcription factors that regulate Th2-associated cytokine gene expression, including maf family proteins, NF-AT family proteins, NF-AT-interacting proteins and AP-1 family proteins.
  • Th2-associated cytokine production can be inhibited in, for example, Th2 cells or in helper precursor cells (Thp) to promote their differentiation along the Thl pathway instead ofthe Th2 pathway.
  • Inhibitory agents ofthe invention can be, for example, intracellular binding molecules that act to inhibit the expression or activity of the transcription factor.
  • intracellular binding molecule is intended to include molecules that act intracellularly to inhibit the expression or activity of a protein by binding to the protein or to a nucleic acid (e.g., an mRNA molecule) that encodes the protein.
  • nucleic acid e.g., an mRNA molecule
  • intracellular binding molecules include antisense nucleic acids, intracellular antibodies and dominant negative inhibitors.
  • an inhibitory agent ofthe invention is an antisense nucleic acid molecule that is complementary to a gene encoding a transcription factor (e.g., a maf family protein, such as c-Maf or pl8, or an NF-AT-interacting protein, such as NIP45), or to a portion of said gene, or a recombinant expression vector encoding said antisense nucleic acid molecule.
  • a transcription factor e.g., a maf family protein, such as c-Maf or pl8, or an NF-AT-interacting protein, such as NIP45
  • the use of antisense nucleic acids to downregulatc the expression of a particular protein in a cell is well known in the art (see e.g., Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol.
  • An antisense nucleic acid molecule comprises a nucleotide sequence that is complementary to the coding strand of another nucleic acid molecule (e.g., an mR ⁇ A sequence) and accordingly is capable of hydrogen bonding to the coding strand ofthe other nucleic acid molecule.
  • Antisense sequences complementary to a sequence of an mR ⁇ A can be complementary to a sequence found in the coding region ofthe mR ⁇ A, the 5' or 3' untranslated region of the mR ⁇ A or a region bridging the coding region and an untranslated region (e.g., at the junction of the 5' untranslated region and the coding region).
  • an antisense nucleic acid can be complementary in sequence to a regulatory region of the gene encoding the mR ⁇ A, for instance a transcription initiation sequence or regulatory element.
  • an antisense nucleic acid is designed so as to be complementary to a region preceding or spanning the initiation codon on the coding strand or in the 3' untranslated region of an mR ⁇ A.
  • An antisense nucleic acid for inhibiting in a cell the expression of a transcription factor discussed herein can be designed based upon the nucleotide sequence of the transcription factor, as disclosed herein or known in the art, constructed according to the rules of Watson and Crick base pairing.
  • an antisense nucleic acid can exist in a variety of different forms.
  • the antisense nucleic acid can be an oligonucleotide that is complementary to only a portion of a maf family gene.
  • An antisense oligonucleotides can be constructed using chemical synthesis procedures known in the art.
  • An antisense oligonucleotide can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability ofthe molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g. phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • one or more antisense oligonucleotides can be added to cells in culture media, typically at 200 ⁇ g oligonucleotide/ml .
  • an antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., nucleic acid transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).
  • Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the expression of the antisense RNA molecule in a cell of interest, for instance promoters and/or enhancers or other regulatory sequences can be chosen which direct constitutive, tissue specific or inducible expression of antisense RNA.
  • the antisense expression vector is prepared as described above for recombinant expression vectors, except that the cDNA (or portion thereof) is cloned into the vector in the antisense orientation.
  • the antisense expression vector can be in the form of, for example, a recombinant plasmid, phagemid or attenuated virus.
  • the antisense expression vector is introduced into cells using a standard transfection technique, as described above for recombinant expression vectors.
  • an antisense nucleic acid for use as an inhibitory agent is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region (for reviews on ribozymes see e.g., Ohkawa, J. et al. (1995) J. Biochem. 118:251-258; NASAdsson, S.T. and Eckstein, F. (1995) Trends Bioiechnol. 13:286-289; Rossi, J.J. (1995) 7ren.fr Bioiechnol. 13:301-306; Kiehntopf, M. et al. (1995) J. Mol. Med. 73:65-71).
  • a ribozyme having specificity for mRNA encoding a transcription factor discussed herein can be designed based upon the nucleotide sequence of the transcription factor.
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the base sequence ofthe active site is complementary to the base sequence to be cleaved in a c-maf mRNA or other transcription factor mRNA. See for example U.S. Patent Nos. 4,987,071 and 5,116,742, both by Cech et al.
  • c- m ⁇ /mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See for example Bartel, D. and Szostak, J.W. (1993) Science 261 : 141 1-1418.
  • Another type of inhibitory agent that can be used to inhibit the expression and/or activity of a Maf protein in a cell is an intracellular antibody specific for a transcription factor discussed herein.
  • intracellular antibodies to inhibit protein function in a cell is known in the art (see e.g., Carlson, J. R. (1988) Mol. Cell. Biol. 8:2638-2646; Biocca, S. et al.
  • a recombinant expression vector which encodes the antibody chains in a form such that, upon introduction of the vector into a cell, the antibody chains are expressed as a functional antibody in an intracellular compartment ofthe cell.
  • an intracellular antibody that specifically binds the transcription factor is expressed within the nucleus ofthe cell.
  • Nuclear expression of an intracellular antibody can be accomplished by removing from the antibody light and heavy chain genes those nucleotide sequences that encode the N- terminal hydrophobic leader sequences and adding nucleotide sequences encoding a nuclear localization signal at either the N- or C-terminus ofthe light and heavy chain genes (see e.g., Biocca, S. et al. (1990) EMBO J. 9:101 -108; Mhashilkar, A. M. et al (1995) EMBO J. 14: 1542- 1551).
  • a preferred nuclear localization signal to be used for nuclear targeting of the intracellular antibody chains is the nuclear localization signal of SV40 Large T antigen (see Biocca, S. et al. (1990) EMBOJ. 9: 101-108; Mhashilkar, A. M. et al. ( 1995) EMBO J. ⁇ 4: 1542- 1551).
  • antibody light and heavy chain cDNAs encoding antibody chains specific for the target protein of interest are isolated, typically from a hybridoma that secretes a monoclonal antibody specific for the maf protein.
  • target protein of interest e.g., a Maf family protein or other transcription factor discussed herein.
  • Preparation of antisera against Maf family proteins has been described in the art (see e.g., Fujiwara, K.T. et al. (1993) Oncogene 8:2371-2380; Kataoka, K. et al. (1993) J. Virol. 67:2133-2141; Ke ⁇ pola, T.K. and Curran, T.
  • Anii-Maf protein antibodies can be prepared by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with a Maf protein immunogen.
  • a suitable subject e.g., rabbit, goat, mouse or other mammal
  • An appropriate immunogenic preparation can contain, for examples, recombinantly expressed Maf protein or a chemically synthesized Maf peptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent.
  • Antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497) (see also, Brown et al. ( 1981 ) J. Immunol 127:539-46; Brown et al. ( 1980) J Biol Chem 255:4980-83; Yeh et al. (1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75).
  • the technology for producing monoclonal antibody hybridomas is well known (see generally R. H.
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the culture supernatants ofthe resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds specifically to the Maf protein.
  • the immortal cell line e.g., a myeloma cell line
  • the immortal cell line is derived from the same mammalian species as the lymphocytes.
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation ofthe present invention with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
  • HAT medium culture medium containing hypoxanthine, aminopterin and thymidine
  • Any of a number of myeloma cell lines may be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines. These myeloma lines are available from the American Type Culture Collection (ATCC), Rockville, Md.
  • ATCC American Type Culture Collection
  • HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG").
  • PEG polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridoma cells producing a monoclonal antibody that specifically binds the maf protein are identified by screening the hybridoma culture supernatants for such antibodies, e.g., using a standard ELISA assay.
  • a monoclonal antibody that binds to a transcription factor discussed herein can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the protein, or a peptide thereof, to thereby isolate immunoglobulin library members that bind specifically to the protein.
  • Kits for generating and screening phage display libraries are commercially available
  • DNAs encoding the light and heavy chains of the monoclonal antibody are isolated by standard molecular biology techniques.
  • hybridoma derived antibodies light and heavy chain cDNAs can be obtained, for example, by PCR amplification or cDNA library screening.
  • cDNA encoding the light and heavy chains can be recovered from the display package (e.g., phage) isolated during the library screening process.
  • Nucleotide sequences of antibody light and heavy chain genes from which PCR primers or cDNA library probes can be prepared are known in the art. For example, many such sequences are disclosed in Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 and in the "Vbase" human germline sequence database. Once obtained, the antibody light and heavy chain sequences are cloned into a recombinant expression vector using standard methods.
  • sequences encoding the hydrophobic leaders ofthe light and heavy chains are removed and sequences encoding a nuclear localization signal (e.g., from SV40 Large T antigen) are linked in-frame to sequences encoding either the amino- or carboxy terminus of both the light and heavy chains.
  • the expression vector can encode an intracellular antibody in one of several different forms. For example, in one embodiment, the vector encodes full-length antibody light and heavy chains such that a full-length antibody is expressed intracellularly. In another embodiment, the vector encodes a full-length light chain but only the VH/CH1 region ofthe heavy chain such that a Fab fragment is expressed intracellularly.
  • the vector encodes a single chain antibody (scFv) wherein the variable regions of the light and heavy chains are linked by a flexible peptide linker (e.g., (Gly4Ser) 3 ) and expressed as a single chain molecule.
  • a flexible peptide linker e.g., (Gly4Ser) 3
  • the expression vector encoding the transcription factor-specific intracellular antibody is introduced into the cell by standard transfection methods, as discussed hereinbefore.
  • Yet another form of an inhibitory agent ofthe invention is an inhibitory form of a transcription factor discussed herein (e.g., a maf protein), also referred to herein as a dominant negative inhibitor.
  • the maf family of proteins are known to homodimerize and to heterodimerize with other AP-1 family members, such as Fos and Jun (see e.g., Ke ⁇ pola, T.K. and Curran, T. (1994) Oncogene 9:675-684; Kataoka, K. et al (1994) Mol. Cell. Biol. 14:700-712).
  • One means to inhibit the activity of transcription factors that form dimers is through the use of a dominant negative inhibitor that has the ability to dimerize with functional transcription factors but that lacks the ability to activate transcription (see e.g., Petrak, D. et al. (1994) J. Immunol. 153:2046-2051).
  • MafB has been identified as an interaction partner of Ets-1 and shown to inhibit Ets-1 -mediated transactivation of the transferrin receptor and to inhibit erythroid differentiation (Sieweke, M.H. et al. (1996) Cell 85:49-60).
  • an inhibitory agent ofthe invention can be a form of a Maf protein that has the ability to dimerize with c-Maf but that lacks the ability to activate transcription.
  • This dominant negative form of a Maf protein may be, for example, a small Maf protein (e.g., MafK, MafF, MafG) that naturally lacks a transactivation domain, MafB or a mutated form of c-Maf in which the transactivation domain has been removed.
  • Such dominant negative Maf proteins can be expressed in cells using a recombinant expression vector encoding the Maf protein, which is introduced into the cell by standard transfection methods.
  • nucleotide sequences encoding the amino terminal transactivation domain of c-Maf are removed from the c-maf cDNA by standard recombinant DNA techniques.
  • at least amino acids 1 -122 are removed. More preferably, at least amino acids 1 -187, or amino acids 1-257, are removed.
  • Nucleotide sequences encoding the basic-leucine zipper region are maintained.
  • the truncated cDNA is inserted into a recombinant expression vector, which is then introduced into a cell to allow for expression ofthe truncated c-maf, lacking a transactivation domain, in the cell.
  • Such compounds can be identified using screening assays that select for compounds that inhibit the expression or activity of a maf family protein. Examples of suitable screening assays are described in further detail in subsection V below.
  • the inhibitory methods of the invention can involve the use of one or more additional inhibitory agents that inhibit the expression or activity of one or more additional transcription factors that contributes to regulating the expression of a Thl- or Th2-associated cytokine gene.
  • the inhibitory method ofthe invention comprises contacting a cell with a first agent that inhibits the expression or activity a maf family protein and a second agent that inhibits the expression or activity of an NF-AT family protein or an NF-AT- interacting protein (e.g., NIP45).
  • the inhibitory method ofthe invention comprises contacting a cell with a first agent that inhibits the expression or activity a maf family protein and a second agent that inhibits the expression or activity of an AP-1 family protein. In yet another embodiment, the inhibitory method of the invention comprises contacting a cell with a first agent that inhibits the expression or activity a maf family protein, a second agent that inhibits the expression or activity of an NF-AT family protein and a third agent that inhibits the expression or activity of an NF- AT-interacting protein (e.g., NIP45).
  • inhibitory agents for inhibiting NF-AT, NF-AT-interacting and AP-1 proteins include antisense nucleic acids, intracellular antibodies, dominant negative inhibitors and chemical compounds that inhibit the endogenous proteins, as described above. Regarding the latter, it is known in the art that the nuclear translocation of NF-ATp is inhibited by the immunosuppressive drugs cyclosporin A and FK506 (see e.g., Rao, A. (1994) Immunol. Today 15:274-281 ; Rao, A. (1995) J Leukoc. Biol. 57:536-542).
  • an immunosuppressive drug such as cyclosporin A or FK506 (or other related drug that inhibits the calcineurin pathway) is used in combination with an agent that inhibits the expression or activity of c-Maf.
  • the method of the invention for modulating production of Th2-associated cytokines by a cell can be practiced either in vitro or in vivo (the latter is discussed further in the following subsection).
  • cells can be obtained from a subject by standard methods and incubated (i.e., cultured) in vitro with a stimulatory or inhibitory agent ofthe invention to stimulate or inhibit, respectively, the production of Th2 -associated cytokines.
  • a stimulatory or inhibitory agent ofthe invention to stimulate or inhibit, respectively, the production of Th2 -associated cytokines.
  • PBMCs peripheral blood mononuclear cells
  • Specific cell populations can be depleted or enriched using standard methods.
  • monocytes/macrophages can be isolated by adherence on plastic.
  • T cells or B cells can be enriched or depleted, for example, by positive and/or negative selection using antibodies to T cell or B cell surface markers, for example by incubating cells with a specific primary monoclonal antibody (mAb), followed by isolation of cells that bind the mAb using magnetic beads coated with a secondary antibody that binds the primary mAb.
  • mAb primary monoclonal antibody
  • Peripheral blood or bone marrow derived hematopoietic stem cells can be isolated by similar techniques using stem cell- specific mAbs (e.g., anti-CD34 mAbs).
  • Specific cell populations can also be isolated by fluorescence activated cell sorting according to standard methods.
  • the cytokine(s) can be recovered from the culture supernatant for further use.
  • the culture supernatant, or a purified fraction thereof can be applied to T cells in culture to influence the development of Thl or Th2 cells in vitro.
  • the culture supernatant, or a purified fraction thereof can be administered to a subject to influence the development of Thl vs. Th2 responses in the subject.
  • the method ofthe invention for modulating the production of Th2-associated cytokines by a cell further comprises administering the cell to a subject to thereby modulate development of Thl or Th2 cells in a subject.
  • Preferred cell types for ex vivo modification and readministration include T cells, B cells and hematopoietic stem cells.
  • Another aspect ofthe invention pertains to a method for modulating development of Thl or Th2 cells in a subject.
  • subject is intended to include living organisms in which an immune response can be elicited.
  • Preferred subjects are mammals. Examples of subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, goats and sheep.
  • Thl/Th2 ratios are modulated in a subject by administering to the subject an agent that modulates the activity of a transcription factor that regulates expression of a Th2-associated cytokine gene such that development of Thl or Th2 cells in the subject is modulated.
  • the transcription factor is a maf family protein, preferably a c-Maf protein or a small maf protein (e.g., pl8).
  • the transcription factor is a protein that interacts with an NF-AT family protein, preferably NIP45.
  • the Th2- associated cytokine is IL-4. Development of a Th2 response in the subject can be promoted by administration of one or more stimulatory agents ofthe invention, whereas development of a Thl response in the subject can be promoted by administration of one or more inhibitory agents ofthe invention.
  • multiple transcription factors e.g., combinations of a maf family protein, an NF-AT family protein, an NF-AT- interacting protein and/or an AP-1 family protein.
  • nucleic acids including recombinant expression vectors encoding transcription factors, antisense RNA, intracellular antibodies or dominant negative inhibitors
  • the agents can be introduced into cells ofthe subject using methods known in the art for introducing nucleic acid (e.g., DNA) into cells in vivo. Examples of such methods include:
  • Naked DNA can be introduced into cells in vivo by directly injecting the DNA into the cells (see e.g., Acsadi et ⁇ /. (1991) Nature 332:815-818; Wolff et al. (1990) Science 247: 1465-1468).
  • a delivery apparatus e.g., a "gene gun” for injecting DNA into cells in vivo can be used.
  • Such an apparatus is commercially available (e.g., from BioRad).
  • Naked DNA can also be introduced into cells in vivo by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor (see for example Wu, G. and Wu, C.H. (1988) J. Biol. Chem. 263 : 14621 ; Wilson et al ( 1992) J. Biol. Chem. 267:963-967; and U.S. Patent No. 5,166,320). Binding ofthe DNA-ligand complex to the receptor facilitates uptake ofthe DNA by receptor-mediated endocytosis.
  • a cation such as polylysine
  • a DNA-ligand complex linked to adenovirus capsids which naturally disrupt endosomes, thereby releasing material into the cytoplasm can be used to avoid degradation ofthe complex by intracellular lysosomes (see for example Curiel et al. (1991) Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl Acad. Sci. USA 90:2122-2126).
  • Retroviruses Defective retroviruses are well characterized for use in gene transfer for gene therapy pu ⁇ oses (for a review see Miller, A.D. (1990) Blood 76:271).
  • a recombinant retrovirus can be constructed having a nucleotide sequences of interest inco ⁇ orated into the retroviral genome. Additionally, portions ofthe retroviral genome can be removed to render the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology. Ausubel, F.M.
  • retroviruses examples include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art.
  • suitable packaging virus lines include ⁇ Crip, ⁇ Cre, ⁇ 2 and ⁇ Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al.
  • Retroviral vectors require target cell division in order for the retroviral genome (and foreign nucleic acid inserted into it) to be integrated into the host genome to stably introduce nucleic acid into the cell. Thus, it may be necessary to stimulate replication ofthe target cell.
  • Adenoviruses The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al (1992) Cell 68: 143-155.
  • Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus are well known to those skilled in the art.
  • Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA 89:6482- 6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin et al. (1992) Proc. Natl Acad. Sci. USA 89:2581-2584).
  • introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA).
  • the carrying capacity ofthe adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al. cited supra; Haj- Ahmand and Graham (1986) J. Virol. 57:267).
  • Most replication-defective adenoviral vectors currently in use are deleted for all or parts ofthe viral El and E3 genes but retain as much as 80 % of the adenoviral genetic material.
  • Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a he ⁇ es virus, as a helper virus for efficient replication and a productive life cycle.
  • another virus such as an adenovirus or a he ⁇ es virus
  • helper virus for efficient replication and a productive life cycle.
  • AAV Adeno-associated virus
  • It is also one ofthe few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol.
  • AAV vector such as that described in Tratschin et al. ( 1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells.
  • a variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 8L6466-6470; Tratschin et al. (1985) Mol. Cell. Biol.
  • DNA introduced into a cell can be detected by a filter hybridization technique (e.g., Southern blotting) and RNA produced by transcription of introduced DNA can be detected, for example, by Northern blotting, RNase protection or reverse transcriptase-polymerase chain reaction (RT-PCR).
  • RNA produced by transcription of introduced DNA can be detected, for example, by Northern blotting, RNase protection or reverse transcriptase-polymerase chain reaction (RT-PCR).
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • the gene product can be detected by an appropriate assay, for example by immunological detection of a produced protein, such as with a specific antibody, or by a functional assay to detect a functional activity of the gene product, such as an enzymatic assay.
  • a modulatory agent such as a chemical compound that stimulates or inhibits endogenous transcription factor activity
  • Such compositions typically comprise the modulatory agent and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be inco ⁇ orated into the compositions.
  • solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use of surfactants.
  • Prevention ofthe action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged abso ⁇ tion ofthe injectable compositions can be brought about by including in the composition an agent which delays abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by inco ⁇ orating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by inco ⁇ orating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets.
  • the active compound can be inco ⁇ orated with excipients and used in the form of tablets, troches, or capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part ofthe composition.
  • the tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Co ⁇ oration and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,81 1.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the stimulatory methods of the invention i.e., methods that use a stimulatory agent
  • result in production of Th2-associated cytokines with concomitant promotion of a Th2 response and downregulation of a Thl response.
  • inhibitory methods of the invention i.e., methods that use an inhibitory agent
  • inhibit the production of Th2- associated cytokines with concomitant downregulation of a Th2 response and promotion of a Thl response.
  • a stimulatory method ofthe invention is selected such that Th2 responses are promoted while downregulating Thl responses.
  • an inhibitory method ofthe invention is selected such that Th2 responses are downregulated while promoting Thl responses.
  • Thl or Th2-type response Numerous disease conditions associated with a predominant Thl or Th2-type response have been identified and could benefit from modulation ofthe type of response mounted in the individual suffering from the disease condition. Application ofthe immunomodulatory methods of the invention to such diseases is described in further detail below.
  • IL-4 is produced by Th2 cells, which further stimulates production of IgE antibodies and activation of cells that mediate allergic reactions, i.e., mast cells and basophils. IL-4 also plays an important role in eosinophil mediated inflammatory reactions. Accordingly, the inhibitory methods of the invention can be used to inhibit the production of Th2-associated cytokines, and in particular IL-4, in allergic patients as a means to downregulate production of pathogenic IgE antibodies.
  • An inhibitory agent may be directly administered to the subject or cells (e.g.
  • Thp cells or Th2 cells may be obtained from the subject, contacted with an inhibitory agent ex vivo, and readministered to the subject. Moreover, in certain situations it may be beneficial to coadminister to the subject the allergen together with the inhibitory agent or cells treated with the inhibitory agent to inhibit (e.g., desensitize) the allergen-specific response.
  • the treatment may be further enhanced by administering other Thl -promoting agents, such as the cytokine IL- 12 or antibodies to Th2-associated cytokines (e.g., anti-IL-4 antibodies), to the allergic subject in amounts sufficient to further stimulate a Thl -type response.
  • Th2-promoting cytokines has been reported to be elevated in cancer patients (see e.g., Yamamura, M., et al. (1993) J. Clin. Invest. 91 :1005-1010; Pisa, P., et al. (1992) Proc. Natl Acad. Sci. USA 89:7708-7712) and malignant disease is often associated with a shift from Thl type responses to Th2 type responses along with a worsening ofthe course ofthe disease.
  • the inhibitory methods of the invention can be used to inhibit the production of Th2-associated cytokines in cancer patients, as a means to counteract the Thl to Th2 shift and thereby promote an ongoing Thl response in the patients to ameliorate the course of the disease.
  • the inhibitory method can involve either direct administration of an inhibitory agent to a subject with cancer or ex vivo treatment of cells obtained from the subject (e.g., Thp or Th2 cells) with an inhibitory agent followed by readministration ofthe cells to the subject.
  • the treatment may be further enhanced by administering other Thl -promoting agents, such as the cytokine IL-12 or antibodies to Th2-associated cytokines (e.g., anti-IL-4 antibodies), to the recipient in amounts sufficient to further stimulate a Thl -type response.
  • Thl -promoting agents such as the cytokine IL-12 or antibodies to Th2-associated cytokines (e.g., anti-IL-4 antibodies)
  • Th2-promoting cytokines also has been reported to increase during a variety of infectious diseases, including HIV infection, tuberculosis, leishmaniasis, schistosomiasis, filarial nematode infection and intestinal nematode infection (see e.g.; Shearer, G.M. and Clerici, M. (1992) Prog. Chem. Immunol. 54:21- 43; Clerici, M and Shearer, G.M. (1993) Immunology Today 14:107-1 1 1 ; Fauci, A.S. (1988) Science 239:617-623; Locksley, R. M. and Scott, P.
  • the inhibitory methods ofthe invention can be used to inhibit the production of Th2-associated cytokines in subjects with infectious diseases, as a means to counteract the Thl to Th2 shift and thereby promote an ongoing Thl response in the patients to ameliorate the course of the infection.
  • the inhibitory method can involve either direct administration of an inhibitory agent to a subject with an infectious disease or ex vivo treatment of cells obtained from the subject (e.g., Thp or Th2 cells) with an inhibitory agent followed by readministration of the cells to the subject.
  • the treatment may be further enhanced by administering other Thl -promoting agents, such as the cytokine IL-12 or antibodies to Th2-associated cytokines (e.g., anti- IL-4 antibodies), to the recipient in amounts sufficient to further stimulate a Thl -type response.
  • the stimulatory methods of the invention can be used therapeutically in the treatment of autoimmune diseases that are associated with a Th2-type dysfunction.
  • Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and that promote the production of cytokines and autoantibodies involved in the pathology ofthe diseases.
  • Modulation of T helper-type responses can have an effect on the course ofthe autoimmune disease. For example, in experimental allergic encephalomyelitis (EAE), stimulation of a Th2-type response by administration of IL-4 at the time ofthe induction ofthe disease diminishes the intensity of the autoimmune disease (Paul, W.E., et al. (1994) Cell 76:241-251).
  • Th2-specific cytokines Koury, S. J., et al. (1992) J. Exp. Med. 176: 1355-1364.
  • T cells that can suppress EAE secrete Th2-specific cytokines (Chen, C, et al. (1994) Immunity V ⁇ 41- 154). Since stimulation of a Th2-type response in EAE has a protective effect against the disease, stimulation of a Th2 response in subjects with multiple sclerosis (for which EAE is a model) is likely to be beneficial therapeutically.
  • stimulation of a Th2-type response in type I diabetes in mice provides a protective effect against the disease. Indeed, treatment of NOD mice with IL-4 (which promotes a Th2 response) prevents or delays onset of type I diabetes that normally develops in these mice (Rapoport, M.J., et al. (1993) J. Exp. Med. 178:87-99).
  • stimulation of a Th2 response in a subject suffering from or susceptible to diabetes may ameliorate the effects ofthe disease or inhibit the onset of the disease.
  • RA rheumatoid arthritis
  • the detrimental Thl response can be concomitantly downmodulated to thereby ameliorate the effects of the disease.
  • the stimulatory methods ofthe invention can be used to stimulate production of Th2-associated cytokines in subjects suffering from, or susceptible to, an autoimmune disease in which a Th2-type response is beneficial to the course of the disease.
  • the stimulatory method can involve either direct administration of a stimulatory agent to the subject or ex vivo treatment of cells obtained from the subject (e.g., Thp, Thl cells, B cells, non-lymphoid cells) with a stimulatory agent followed by readministration ofthe cells to the subject.
  • the treatment may be further enhanced by administering other Th2-promoting agents, such as IL-4 itself or antibodies to Thl - associated cytokines, to the subject in amounts sufficient to further stimulate a Th2-type response.
  • Thl -type response In contrast to the autoimmune diseases described above in which a Th2 response is desirable, other autoimmune diseases may be ameliorated by a Thl -type response.
  • Such diseases can be treated using an inhibitory agent ofthe invention (as described above for cancer and infectious diseases).
  • the treatment may be further enhanced by administrating a Thl -promoting cytokine (e.g., IFN- ⁇ ) to the subject in amounts sufficient to further stimulate a Thl -type response.
  • a Thl -promoting cytokine e.g., IFN- ⁇
  • the efficacy of agents for treating autoimmune diseases can be tested in the above described animal models of human diseases (e.g., EAE as a model of multiple sclerosis and the NOD mice as a model for diabetes) or other well characterized animal models of human autoimmune diseases.
  • Such animal models include the mrl/lpr/lpr mouse as a model for lupus erythematosus, murine collagen-induced arthritis as a model for rheumatoid arthritis, and murine experimental myasthenia gravis (see Paul ed.,
  • a modulatory (i.e., stimulatory or inhibitory) agent of the invention is administered to test animals and the course ofthe disease in the test animals is then monitored by the standard methods for the particular model being used. Effectiveness ofthe modulatory agent is evidenced by amelioration ofthe disease condition in animals treated with the agent as compared to untreated animals (or animals treated with a control agent).
  • Non-limiting examples of autoimmune diseases and disorders having an autoimmune component that may be treated according to the invention include diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sj ⁇ gren's Syndrome, including keratoconjunctivitis sicca secondary to Sj ⁇ gren's Syndrome, alopecia areata, allergic responses due to arthropod bite reactions, Crohn's disease, aphthous ulcer, ulceris, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma.
  • diabetes mellitus
  • vaginitis vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens- Johnson syndrome, idiopathic sprue, lichen planus, Crohn's disease, Graves ophthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis.
  • graft rejection or graft acceptance may not be attributable exclusively to the action of a particular T cell subset (i.e., Thl or Th2 cells) in the graft recipient (for a discussion see Dallman, M.J. (1995) Curr. Opin. Immunol. J_:632-63S), numerous studies have implicated a predominant Th2 response in prolonged graft survival or a predominant Th2 response in graft rejection.
  • Th2 cytokine pattern has been associated with production of a Th2 cytokine pattern and/or graft rejection has been associated with production of a Thl cytokine pattern (see e.g., Takeuchi, T. et al.
  • the stimulatory methods ofthe invention can be used to stimulate production of Th2-associated cytokines in transplant recipients to prolong survival of the graft.
  • the stimulatory methods can be used both in solid organ transplantation and in bone marrow transplantation (e.g., to inhibit graft-versus-host disease).
  • the stimulatory method can involve either direct administration of a stimulatory agent to the transplant recipient or ex vivo treatment of cells obtained from the subject (e.g., Thp, Thl cells, B cells, non-lymphoid cells) with a stimulatory agent followed by readministration of the cells to the subject.
  • the treatment may be further enhanced by administering other Th2- promoting agents, such as
  • IL-4 itself or antibodies to Thl -associated cytokines, to the recipient in amounts sufficient to further stimulate a Th2-type response.
  • the modulatory methods ofthe invention also are useful for other pu ⁇ oses.
  • the stimulatory methods of the invention i.e., methods using a stimulatory agent
  • the modulatory methods of the invention can be applied to vaccinations to promote either a Th 1 or a Th2 response to an antigen of interest in a subject. That is, the agents ofthe invention can serve as adjuvants to direct an immune response to a vaccine either to a Thl response or a Th2 response.
  • the antigen and a stimulatory agent of the invention can be coadministered to a subject to promote a Th2 response to the antigen in the subject, since Th2 responses provide efficient B cell help and promote IgGl production.
  • the antigen and an inhibitory agent of the invention can be coadministered to a subject to promote a Thl response to the antigen in a subject, since Thl responses favor the development of cell-mediated immune responses (e.g., delayed hypersensitivity responses).
  • the antigen of interest and the modulatory agent can be formulated together into a single pharmaceutical composition or in separate compositions. In a preferred embodiment, the antigen of interest and the modulatory agent are administered simultaneously to the subject.
  • the antigen may be desirable to administer first and then the modulatory agent or vice versa (for example, in the case of an antigen that naturally evokes a Thl response, it may be beneficial to first administer the antigen alone to stimulate a Thl response and then administer a stimulatory agent, alone or together with a boost of antigen, to shift the immune response to a Th2 response).
  • compositions that can be used to modulate Th2-associated cytokine production by a cell or Thl/Th2 development in a subject in accordance with the methods ofthe invention.
  • the invention provides recombinant expression vectors comprising a nucleotide sequence encoding a maf family protein operatively linked to regulatory sequences that direct expression of the maf family protein specifically in certain cell types.
  • the regulatory sequences direct expression of the maf family protein specifically in lymphoid cells (e.g. , T cells or B cells).
  • the lymphoid cells are T cells.
  • T cell specific regulatory elements are known in the art, such as the promoter regulatory region of T cell receptor genes (see e.g., Winoto and Baltimore (1989) EMBO J. 8:729-733; Leiden, J.M. (1994) Annu. Rev. Immunol. 11:539-570; Hettman, T. and Cohen, A. (1994) Mol. Immunol. 3_1 :315-322; Redondo, J.M. et al. (1991) Mol Cell Biol 11:5671-5680).
  • Other examples of T cell specific regulatory elements are those derived from the CD3 gene (see e.g., Clevers, H. et al. (1988) Proc. Natl. Acad. Sci.
  • CD4 gene see e.g., Sawada, S. and Littman, D.R. (1991) Mol. Cell. Biol. 11:5506-5515; Salmon, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:7739-7743; Hanna, Z. et al. (1994) Mol. Cell. Biol.
  • a DNA fragment comprising one or more T cell specific regulatory elements can be obtained by standard molecular biology methods, such as by PCR using oligonucleotide primers corresponding to the 5' and 3' ends of the desired region and genomic DNA from T cells as the template.
  • the DNA fragment comprising T cell specific regulatory elements can be operatively linked to a cDNA encoding a maf protein (e.g., the two DNA fragments can be ligated together such that the regulatory elements are located 5' of the maf sequences) and introduced into vector, such as a plasmid vector, using standard molecular biology techniques.
  • the lymphoid cells are B cells (i.e., within the recombinant expression vector the nucleotide sequences encoding a maf family protein are operatively linked to regulatory sequences that direct expression of the maf family specifically in B cells).
  • B cell specific regulatory elements are known in the art, such as the promoter regulatory region of immunoglobulin genes (see e.g., Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748).
  • Other examples of B cell specific regulatory elements are those derived from the CD20 (Bl ) gene (see e.g., Thevenin, C. et al. (1993) J Biol. Chem. 268:5949-5956; Rieckmann, P. et al (1991 ) 7. Immunol. 147:3994-3999), the Fc epsilon Rlla gene (see e.g., Suter, U. et al. (1989) J. Immunol. 143:3087-3092) and major histocompatibility class II genes (see e.g.,
  • a DNA fragment comprising B cell specific regulatory elements can be obtained by standard molecular biology methods, such as by PCR using oligonucleotide primers corresponding to the 5' and 3' ends ofthe desired region and genomic DNA from B cells as the template.
  • the DNA fragment comprising B cell specific regulatory elements can be operatively linked to a cDNA encoding a maf protein (e.g., the two DNA fragments can be ligated together such that the regulatory elements are located 5' of the maf sequences) and introduced into vector, such as a plasmid vector, using standard molecular biology techniques.
  • the invention provides recombinant expression vectors comprising a nucleotide sequence encoding a maf family protein operatively linked to regulatory sequences that direct expression ofthe maf family protein specifically in hematopoietic stem cells.
  • Hematopoietic stem cell specific regulatory elements are known in the art.
  • regulatory elements derived from the CD34 gene are used (see e.g., Satterthwaite, A.B. et al. (1992) Genomics 12:788-794; Burn, T. C. et al. (1992) Blood 80:3051-3059).
  • Another aspect ofthe invention pertains to recombinant host cells that express a maf family protein.
  • Such host cells can be used to produce a Th2-associated cytokine (e.g. , IL-4).
  • Th2-associated cytokine e.g. , IL-4
  • Such host cells also can be administered to a subject to produce a Th2- associated cytokine in the subject as a means to manipulate Thl :Th2 ratios in the subject.
  • the terms "host cell” and "recombinant host cell” are used interchangeably herein to refer to a cell into which a recombinant expression vector has been introduced. It is understood that such terms refer not only to the particular subject cell but to the progeny of such a cell.
  • progeny may not, in fact, be identical to the parent cell, but as long as these progeny cells retain the recombinant expression vector, these progeny are still intended to be included within the scope ofthe term "host cell" as used herein.
  • the invention provides a host lymphoid cell into which a recombinant expression vector encoding a maf family protein has been introduced.
  • the host lymphoid cell can be a T cell or a B cell.
  • a host T cell of the invention can be, for example a T cell clone that is cultured in vitro (such as those described in the Examples) or, alternatively, a normal T cell that is isolated from a subject (e.g., a peripheral blood T cell or a splenic T cell).
  • Standard methods for preparing and culturing T cell clones in vitro, or isolating T cells are known in the art, for example through the use of mAbs that bind T cell specific cell surface markers (e.g., CD3) or surface markers for specific subsets of T cells (e.g., CD4 or CD8).
  • the recombinant expression vector can be introduced into the T cell by one of a variety of known transfection methods for introducing DNA into mammalian cells, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al.
  • the host lymphoid cell ofthe invention is a host B cell into which a recombinant expression vector encoding a maf family protein has been introduced.
  • the B cell can be, for example a B lymphoma cell that is cultured in vitro (such as Ml 2 cells as described in the Examples) or, alternatively, a normal B cell that is isolated from a subject (e.g., a peripheral blood B cell or a splenic B cell).
  • Various B lymphoma cell lines are available in the art and standard methods for culturing such cells in vitro are known.
  • standard methods for isolating normal B cells are known in the art, for example through the use of mAbs that bind B cell specific cell surface markers (e.g. , membrane immunoglobulin, B7-1 , CD20).
  • B cell specific cell surface markers e.g. , membrane immunoglobulin, B7-1 , CD20.
  • the recombinant expression vector can be introduced into the B cell by standard methods, as described above for T cells.
  • the invention provides a host hematopoietic stem cell into which a recombinant expression vector encoding a maf family protein has been introduced.
  • Hematopoietic stem cells can be isolated from a subject (e.g., from peripheral blood or bone marrow ofthe subject) using standard methods known in the art for isolating such stem cells, for example through the use of mAbs that bind hematopoietic stem cell specific cell surface markers, preferably CD34 (for further descriptions of isolation of stem cells, see e.g., Wagner, J.E. et al. (1995) Blood 86:512- 523; Murray, L. et al. (1995) Blood 85:368-378; Bernardi, A.C. et al. (1995) Science 267: 104-108; Bernstein, I.D. et al.
  • the recombinant expression vector can be introduced into the hematopoietic stem cell by standard methods, as described above for T cells.
  • compositions described above with regard to maf family proteins can be prepared for various different maf family proteins, such as c-Maf and small mafs (e.g., pl8).
  • the invention further provides transgenic animals carrying a transgene encoding a maf family protein.
  • the maf family transgene is expressed preferentially or exclusively in T cells ofthe animal.
  • Tissue-specific expression of maf family proteins can be achieved through linkage ofthe maf coding sequences in the transgene to regulatory sequences that direct expression ofthe encoded protein in a particular cell type.
  • tissue-specific regulatory elements are known in the art and are described further herein.
  • a preferred regulatory element for T cell-specific expression of a maf family transgene is the CD4 promoter/enhancer.
  • a preferred maf family protein for use in the transgene is c-maf.
  • a schematic diagram of a preferred c-maf transgenic construct is shown in Figure 20.
  • the CD4 promoter/enhancer is operatively linked to the first intron ofthe mouse c-maf gene, which in turn is operatively linked to the mouse c-maf cDNA, which in turn is operatively linked to an SV40 polyadenylation sequence.
  • Transgenic animals can be prepared by microinjecting a c-maf transgene construct into fertilized oocytes according to standard procedures for making transgenic animals, described in further detail herein. The phenotype of c-maf transgenic mice that overexpress c-Maf protein in T cells is described further in Example 17.
  • Transgenic animals expressing a maf family protein can be used, for example, to evaluate test compounds for their ability to modulate the activity ofthe maf family protein.
  • a test compound can be administered to transgenic animal expressing a maf family protein and the effect ofthe test compound in the transgenic animal can be determined by comparing the phenotype ofthe transgenic animal in the presence ofthe test compound to the phenotype ofthe transgenic animal in the absence ofthe test compound.
  • an isolated nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in SEQ ID NO: 5.
  • the sequence of SEQ ID NO: 5 corresponds to the mouse NIP45 cDN A.
  • This cDNA comprises sequences encoding the NIP45 protein (i.e., "the coding region", from nucleotides 13-1248), as well as 5' untranslated sequences (nucleotides 1-12) and 3' untranslated sequences (nucleotides 1249-1946).
  • the nucleic acid molecule may comprise only the coding region of SEQ ID NO: 5 (i.e., nucleotides 13- 1248).
  • nucleic acid molecule ofthe invention can comprise only a portion of the coding region of SEQ ID NO: 5, for example a fragment encoding a biologically active portion of NIP45.
  • biologically active portion of NIP45 is intended to include portions of NIP45 that retain the ability to interact with the RHD of NF-AT family proteins. The ability of portions of NIP45 to interact with an NF-AT RHD can be determined in standard in vitro interaction assays, for example using a NF- AT RHD fusion protein.
  • Nucleic acid fragments encoding biologically active portions of NIP45 can be prepared by isolating a portion of SEQ ID NO: 5, expressing the encoded portion of NIP45 protein or peptide (e.g., by recombinant expression in a host cell) and assessing the ability ofthe portion to interact with NF-AT, in particular the NF-AT RHD, for example using a glutathione-S-transferase (GST)-NF-AT RHD fusion protein.
  • GST glutathione-S-transferase
  • the invention further encompasses nucleic acid molecules that differ from SEQ ID NO: 5 (and fragments thereof) due to degeneracy ofthe genetic code and thus encode the same NIP45 protein as that encoded by SEQ ID NO: 5. Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO: 6. Moreover, the invention encompasses nucleic acid molecules that encode portions of SEQ ID NO: 6, such as biologically active portions thereof.
  • a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 5, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • a NIP45 cDNA can be isolated from a cDNA library using all or portion of SEQ ID NO: 5 as a hybridization probe and standard hybridization techniques (e.g., as described in Sambrook, J., et al. Molecular Cloning: A Laboratory Manual. 2nd, ed.. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989).
  • nucleic acid molecule encompassing all or a portion of SEQ ID NO: 5 can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon the sequence of SEQ ID NO: 5.
  • mRNA can be isolated from cells (e.g., by the guanidinium-thiocyanate extraction procedure of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and cDNA can be prepared using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Russia, FL).
  • reverse transcriptase e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Russia, FL.
  • Synthetic oligonucleotide primers for PCR amplification can be designed based upon the nucleotide sequence shown in SEQ ID NO: 5.
  • a nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to a NIP45 nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • NIP45 nucleotide sequence shown in SEQ ID NO: 5
  • DNA sequence polymo ⁇ hisms that lead to changes in the amino acid sequences of NIP45 may exist within a population.
  • Such genetic polymo ⁇ hism in the NIP45 gene may exist among individuals within a population due to natural allelic variation.
  • natural allelic variations can typically result in 1-5 % variance in the nucleotide sequence ofthe a gene. Any and all such nucleotide variations and resulting amino acid polymo ⁇ hisms in NIP45 that are the result of natural allelic variation and that do not alter the functional activity of NIP45 are intended to be within the scope ofthe invention.
  • nucleic acid molecules encoding NIP45 proteins from other species are intended to be within the scope ofthe invention.
  • Nucleic acid molecules corresponding to natural allelic variants and human and other mammalian homologues of the mouse NIP45 cDNA ofthe invention can be isolated based on their homology to the mouse NIP45 nucleic acid molecule disclosed herein using the mouse cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • an isolated nucleic acid molecule ofthe invention hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 5.
  • the nucleic acid is at least 15, 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or 1500 nucleotides in length.
  • an isolated nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 5 corresponds to a naturally-occurring nucleic acid molecule.
  • the nucleic acid encodes natural human NIP45 protein.
  • the nucleic acid molecule encodes a murine NIP45 protein, such as mouse NIP45 protein.
  • allelic variants ofthe NIP45 sequence that may exist in the population, the skilled artisan will further appreciate that changes may be introduced by mutation into the nucleotide sequence of SEQ ID NO: 5, thereby leading to changes in the amino acid sequence of the encoded protein, without altering the functional activity ofthe NIP45 protein.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues may be made in the sequence of SEQ ID NO: 5.
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of NIP45 (e.g., the sequence of SEQ ID NO: 6) without altering the functional activity of NIP45, such as its ability to interact with an NF-AT RHD or its ability to synergize with NF-AT and c-Maf in stimulating gene transcription, whereas an "essential" amino acid residue is required for functional activity.
  • another aspect ofthe invention pertains to nucleic acid molecules encoding NIP45 proteins that contain changes in amino acid residues that are not essential for NIP45 activity. Such NIP45 proteins differ in amino acid sequence from SEQ ID NO: 6 yet retain NIP45 activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least 60 % homologous to the amino acid sequence of SEQ ID NO: 6 and interacts with the RHD of an NF-AT family protein.
  • the protein encoded by the nucleic acid molecule is at least 70 % homologous to SEQ ID NO: 6, more preferably at least 80 % homologous to SEQ ID NO: 6, even more preferably at least 90 % homologous to SEQ ID NO: 6, and most preferably at least 95 % homologous to SEQ ID NO: 6.
  • the sequences are aligned for optimal comparison pu ⁇ oses (e.g., gaps may be introduced in the sequence of one protein for optimal alignment with the other protein).
  • the amino acid residues at corresponding amino acid positions are then compared.
  • a position in one sequence e.g. , SEQ ID NO: 6
  • amino acid "homology” is equivalent to amino acid "identity”
  • An isolated nucleic acid molecule encoding a NIP45 protein homologous to the protein of SEQ ID NO: 6 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO: 5 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into SEQ ID NO: 5 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g. , lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g. , lysine, arginine, histidine
  • acidic side chains e.g., aspart
  • an amino acid residue in NIP45 protein is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a NIP45 coding sequence, such as by saturation mutagenesis. and the resultant mutants can be screened for their ability to interact with an NF-AT RHD (e.g., using a GST-NF-AT-RHD fusion protein) to identify mutants that retain NF-AT-interacting ability.
  • an NF-AT RHD e.g., using a GST-NF-AT-RHD fusion protein
  • the encoded mutant protein can be expressed recombinantly in a host cell and the ability ofthe mutant protein to interact with NF-AT can be determined using an in vitro interaction assay.
  • a recombinant NIP45 protein (e.g., a mutated or truncated form of SEQ ID NO: 6) can be radiolabeled and incubated with a GST-NF-AT RHD fusion protein. Glutathione-sepharose beads are then added to the mixture to precipitate the NIP45-GST-NF-AT RHD complex, if such a complex is formed. After washing the beads to remove non-specific binding, the amount of radioactive protein associated with the beads is determined and compared to the amount of radioactive protein remaining in the eluate to thereby determine whether the NIP45 protein is capable of interacting with the RHD of NF-AT.
  • an antisense nucleic acid ofthe invention can be complementary to an entire NIP45 coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a coding region of the coding strand of a nucleotide sequence encoding NIP45 (e.g., the entire coding region of SEQ ID NO: 5 comprises nucleotides 13-1248).
  • the antisense nucleic acid molecule is antisense to a noncoding region of the coding strand of a nucleotide sequence encoding NIP45.
  • an antisense nucleic acid ofthe invention is at least 15, 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or 1500 nucleotides in length.
  • antisense nucleic acids ofthe invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule may be complementary to the entire coding region of NIP45 mRNA, or alternatively can be an oligonucleotide which is antisense to only a portion ofthe coding or noncoding region of NIP45 mRNA.
  • the antisense oligonucleotide may be complementary to the region surrounding the translation start site of NIP45 mRNA.
  • An antisense oligonucleotide can be, for example, about 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid ofthe invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g. , an antisense oligonucleotide
  • an antisense nucleic acid e.g. , an antisense oligonucleotide
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • an antisense nucleic acid ofthe invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • a ribozyme having specificity for a NIP45-encoding nucleic acid can be designed based upon the nucleotide sequence of a NIP45 cDNA disclosed herein (i.e., SEQ ID NO: 5).
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the base sequence ofthe active site is complementary to the base sequence to be cleaved in a NIP45-encoding mRNA. See for example Cech et al. U.S. Patent No. 4,987,071 ; and Cech et al. U.S. Patent No. 5,1 16,742.
  • NIP45 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See for example Bartel, D. and Szostak, J.W. (1993) Science 261 : 141 1-1418.
  • nucleic acid molecules encoding NIP45 fusion proteins.
  • Such nucleic acid molecules comprising at least a first nucleotide sequence encoding a NIP45 protein, polypeptide or peptide operatively linked to a second nucleotide sequence encoding a non-NIP45 protein, polypeptide or peptide, can be prepared by standard recombinant DNA techniques.
  • NIP45 fusion proteins are described in further detail below in subsection C.
  • the expression vectors ofthe invention comprise a nucleic acid ofthe invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis ofthe host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression ofthe nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector may depend on such factors as the choice ofthe host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., NIP45 proteins, mutant forms of NIP45 proteins, NIP45 fusion proteins and the like).
  • the recombinant expression vectors ofthe invention can be designed for expression of NIP45 protein in prokaryotic or eukaryotic cells.
  • NIP45 can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the recombinant expression vector may be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus ofthe recombinant protein.
  • Such fusion vectors typically serve three pu ⁇ oses: 1) to increase expression of recombinant protein; 2) to increase the solubility ofthe recombinant protein; and 3) to aid in the purification ofthe recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent to purification ofthe fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S.
  • GST glutathione S-transferase
  • Suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al, (1988) Gene 69:301-315) and pET l id (Studier et al. Gene Expression Technology: Methods in Enzymology 185. Academic Press, San Diego, California (1990) 60-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid t ⁇ -lac fusion promoter.
  • Target gene expression from the pET 1 Id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl).
  • This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident ⁇ prophage harboring a T7 gnl gene under the transcriptional control ofthe lacUV 5 promoter.
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 1 19-128).
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E.
  • the NIP45 expression vector is a yeast expression vector.
  • NIP45 can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al, (1983) Mol.
  • a nucleic acid ofthe invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pMex-NeoI, pCDM8 (Seed, B., (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBOJ. 6: 187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • the recombinant mammalian expression vector is capable of directing expression ofthe nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBOJ. 8:729-733) and immunoglobulins (Banerji et al.
  • promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription ofthe DNA molecule) of an RNA molecule which is antisense to NIP45 mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • a host cell may be any prokaryotic or eukaryotic cell.
  • NIP45 protein may be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and transfection are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory manuals. For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome.
  • a gene that encodes a selectable marker is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker may be introduced into a host cell on the same vector as that encoding NIP45 or may be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have inco ⁇ orated the selectable marker gene will survive, while the other cells die).
  • a host cell ofthe invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) NIP45 protein. Accordingly, the invention further provides methods for producing NIP45 protein using the host cells ofthe invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding NIP45 has been introduced) in a suitable medium until NIP45 is produced. In another embodiment, the method further comprises isolating NIP45 from the medium or the host cell.
  • NIP45 protein In its native form NIP45 protein is an intracellular protein and, accordingly, recombinant NIP45 protein can be expressed intracellularly in a recombinant host cell and then isolated from the host cell, e.g., by lysing the host cell and recovering the recombinant NIP45 protein from the lysate.
  • recombinant NIP45 protein can be prepared as a extracellular protein by operatively linking a heterologous signal sequence to the amino-terminus ofthe protein such that the protein is secreted from the host cells. In this case, recombinant NIP45 protein can be recovered from the culture medium in which the cells are cultured.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which NIP45-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous NIP45 sequences have been introduced into their genome or homologous recombinant animals in which endogenous NIP45 sequences have been altered. Such animals are useful for studying the function and/or activity of NIP45 and for identifying and/or evaluating modulators of NIP45 activity.
  • another aspect ofthe invention pertains to nonhuman transgenic animals which contain cells carrying a transgene encoding a NIP45 protein or a portion of a NIP45 protein.
  • the transgene alters an endogenous gene encoding an endogenous NIP45 protein (e.g., homologous recombinant animals in which the endogenous NIP45 gene has been functionally disrupted or "knocked out", or the nucleotide sequence of the endogenous NIP45 gene has been mutated or the transcriptional regulatory region ofthe endogenous NIP45 gene has been altered).
  • a transgenic animal ofthe invention can be created by introducing NIP45- encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the mouse NIP45 cDNA sequence of SEQ ID NO: 5 can be introduced as a transgene into the genome of a non-human animal (e.g., a mouse).
  • a mammalian homologue ofthe mouse NIP45 gene such as a human NIP45 gene, can be isolated based on hybridization to the mouse NIP45 cDNA and used as a transgene.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression ofthe transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to the NIP45 transgene to direct expression of NIP45 protein to particular cells.
  • transgenic founder animal can be identified based upon the presence of the NIP45 transgene in its genome and/or expression of NIP45 mRNA in tissues or cells ofthe animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding NIP45 can further be bred to other transgenic animals carrying other transgenes.
  • a vector which contains at least a portion of a NIP45 gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the endogenous NIP45 gene.
  • the NIP45 gene preferably is a mouse NIP45 gene.
  • a mouse NIP45 gene can be isolated from a mouse genomic DNA library using the mouse NIP45 cDNA of SEQ ID NO: 5 as a probe. The mouse NIP45 gene then can be used to construct a homologous recombination vector suitable for altering an endogenous NIP45 gene in the mouse genome.
  • the vector is designed such that, upon homologous recombination, the endogenous NIP45 gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the vector can be designed such that, upon homologous recombination, the endogenous NIP45 gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous NIP45 protein).
  • the altered portion ofthe NIP45 gene is flanked at its 5' and 3' ends by additional nucleic acid ofthe NIP45 gene to allow for homologous recombination to occur between the exogenous NIP45 gene carried by the vector and an endogenous NIP45 gene in an embryonic stem cell.
  • the additional flanking NIP45 nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5' and 3' ends
  • are included in the vector see e.g., Thomas, K.R. and Capecchi, M. R.
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced NIP45 gene has homologously recombined with the endogenous NIP45 gene are selected (see e.g. , Li, E. et al. (1992) Cell 69:915).
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed.
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells ofthe animal contain the homologously recombined DNA by germline transmission ofthe transgene.
  • Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec et al; WO 91/01 140 by Smithies et al. ; WO 92/0968 by Zijlstra et al; and WO 93/04169 by Berns et al.
  • the invention provides an isolated preparation of NIP45 protein.
  • the NIP45 protein has an amino acid sequence shown in SEQ ID NO: 6.
  • the NIP45 protein is substantially homologous to SEQ ID NO: 6 and retains the functional activity ofthe protein of SEQ ID NO: 6 yet differs in amino acid sequence due to natural allelic variation or mutagenesis, or is a mammalian homologue of the protein of SEQ ID NO: 6 (e.g., a human homologue), as described in detail in subsection A above.
  • the NIP45 protein is a protein which comprises an amino acid sequence at least 60 % homologous to the amino acid sequence of SEQ ID NO: 6 and that interacts with the RHD of an NF-AT family protein.
  • the protein is at least 70 % homologous to SEQ ID NO: 6, more preferably at least 80 % homologous to SEQ ID NO: 6, even more preferably at least 90 % homologous to SEQ ID NO: 6, and most preferably at least 95 % homologous to SEQ ID NO: 6.
  • the invention provides isolated portions ofthe NIP45 protein.
  • the invention further encompasses a portion of a NIP45 protein that interacts with NF-AT.
  • NIP45 protein interacts with the RHD of NF-AT.
  • An in vitro interaction assay (such as that described above in subsection A utilizing a GST-NF-AT RHD fusion protein) can be used to determine the ability of NIP45 peptide fragments to interact with the NF-AT Rel Homology Domain to thereby identify peptide fragments that interact with NF-AT.
  • NIP45 proteins are preferably produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the protein is cloned into an expression vector (as described above), the expression vector is introduced into a host cell (as described above) and the NIP45 protein is expressed in the host cell. The NIP45 protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Alternative to recombinant expression, a NIP45 polypeptide can be synthesized chemically using standard peptide synthesis techniques. Moreover, native NIP45 protein can be isolated from cells (e.g., from T cells), for example by immunoprecipitation using an anti-NIP45 antibody.
  • NIP45 fusion protein comprises a NIP45 polypeptide operatively linked to a non-NIP45 polypeptide.
  • a “NIP45 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to NIP45 protein, or a peptide fragment thereof, whereas a “non-NIP45 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to another protein.
  • the term "operatively linked” is intended to indicate that the NIP45 polypeptide and the non-NIP45 polypeptide are fused in-frame to each other.
  • the non-NIP45 polypeptide may be fused to the N-terminus or C-terminus of the NIP45 polypeptide.
  • the fusion protein is a GST-NIP45 fusion protein in which the NIP45 sequences are fused to the C-terminus ofthe GST sequences.
  • the fusion protein is a NIP45-HA fusion protein in which the NIP45 nucleotide sequence is inserted in to the pCEP4-HA vector (Herrscher, R.F. et al. (1995) Genes Dev. 9:3067- 3082) such that the NIP45 sequences are fused in frame to an influenza hemagglutinin epitope tag.
  • fusion proteins can facilitate the purification of recombinant NIP45.
  • a NIP45 fusion protein ofthe invention is produced by standard recombinant DNA techniques. For example. DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide or an HA epitope tag).
  • a NIP45-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NIP45 protein.
  • An isolated NIP45 protein, or fragment thereof, can be used as an immunogen to generate antibodies that bind NIP45 using standard techniques for polyclonal and monoclonal antibody preparation.
  • the NIP45 protein can be used to generate antibodies or, alternatively, an antigenic peptide fragment of NIP45 can be used as the immunogen.
  • An antigenic peptide fragment of NIP45 typically comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO: 6 and encompasses an epitope of NIP45 such that an antibody raised against the peptide forms a specific immune complex with NIP45.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of NIP45 that are located on the surface ofthe protein, e.g., hydrophilic regions. A hydrophobicity analysis ofthe NIP45 protein sequence of SEQ ID NO: 6 is shown in Figure 12.
  • a NIP45 immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen.
  • An appropriate immunogenic preparation can contain, for examples, recombinantly expressed NIP45 protein or a chemically synthesized NIP45 peptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent.
  • Immunization of a suitable subject with an immunogenic NIP45 preparation induces a polyclonal anti-NIP45 antibody response.
  • another aspect ofthe invention pertains to anti-NIP45 antibodies.
  • Polyclonal anti-NIP45 antibodies can be prepared as described above by immunizing a suitable subject with a NIP45 immunogen.
  • the anti-NIP45 antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized NIP45.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against NIP45 can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497) (see also, Brown et al. ( 1981) J. Immunol 127:539- 46; Brown et al. (1980) J Biol Chem 255:4980-83; Yeh et al. (1976) PNAS ' 76:2927-31 ; and Yeh et al. (1982) Int. J.
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the culture supernatants ofthe resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds NIP45.
  • the immortal cell line e.g., a myeloma cell line
  • the immortal cell line is derived from the same mammalian species as the lymphocytes.
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a number of myeloma cell lines may be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or S ⁇ 2/O-Agl4 myeloma lines.
  • HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG").
  • PEG polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind NIP45, e.g., using a standard ELISA assay.
  • a monoclonal anti-NIP45 antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g. , an antibody phage display library) with NIP45 to thereby isolate immunoglobulin library members that bind NIP45.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27- 9400-01; and the Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al.
  • recombinant anti-NIP45 antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Patent Publication PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171 ,496; Morrison et al. European Patent Application 173,494; Neuberger et al.
  • An anti-NIP45 antibody (e.g. , monoclonal antibody) can be used to isolate
  • NIP45 by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti-NIP45 antibody can facilitate the purification of natural NIP45 from cells and of recombinantly produced NIP45 expressed in host cells.
  • an anti-NIP45 antibody can be used to detect NIP45 protein (e.g., in a cellular lysate or cell supernatant). Detection may be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • an anti-NIP45 antibody ofthe invention is labeled with a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase. or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include 125 I, 131 I, 35 S or 3 H. D. Pharmaceutical Compositions
  • compositions suitable for administration can be inco ⁇ orated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the protein or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be inco ⁇ orated into the compositions.
  • solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents such as ethylene
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention ofthe action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged abso ⁇ tion of the injectable compositions can be brought about by including in the composition an agent which delays abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by inco ⁇ orating the active compound (e.g. , a NIP45 protein or anti-NIP45 antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g. , a NIP45 protein or anti-NIP45 antibody
  • dispersions are prepared by inco ⁇ orating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the pu ⁇ ose of oral therapeutic administration, the active compound can be inco ⁇ orated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part ofthe composition.
  • the tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Co ⁇ oration and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,81 1.
  • the invention provides a method for detecting the presence of NIP45 protein or mRNA in a biological sample.
  • the method involves contacting the biological sample with an agent capable of detecting NIP45 protein or mRNA such that the presence of NIP45 protein or mRNA is detected in the biological sample.
  • a preferred agent for detecting NIP45 mRNA is a labeled nucleic acid probe capable of hybridizing to NIP45 mRNA.
  • the nucleic acid probe can be, for example, the NIP45 cDNA of SEQ ID NO: 5, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NIP45 mRNA.
  • a preferred agent for detecting NIP45 protein is a labeled antibody capable of binding to NIP45 protein.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used.
  • labeled with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling ofthe probe or antibody by reactivity with another reagent that is directly labeled.
  • indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids.
  • techniques for detection of NIP45 mRNA include Northern hybridizations and in situ hybridizations.
  • Techniques for detection of NIP45 protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. VI.. Combination Compositions and Kits
  • compositions comprising combinations of modulatory agents are also provided by the invention.
  • two or more nucleotide sequences encoding transcription factors that regulate Th2 -associated cytokine gene expression can be inco ⁇ orated into a recombinant expression vector and introduced into a host cell.
  • the invention provides recombinant vectors, and host cells into which such vectors have been introduced, comprising a first nucleotide sequence encoding a first transcription factor that cooperates with an NF-AT family protein to regulate expression of the Th2- associated cytokine gene and a second nucleotide sequence encoding a second transcription factor that contributes to the regulation ofthe Th2 -associated cytokine gene.
  • the first nucleotide sequence encodes a maf family protein (e.g., c- Maf) or an NF-AT-interacting protein (e.g., NIP45).
  • the second nucleotide sequence encodes a transcription factor selected from the group consisting of NF-AT family proteins, NF-AT-interacting proteins, maf family proteins and AP-1 family proteins.
  • Kits for modulating Th2-associated cytokine production or Thl/Th2 subset development are also encompassed by the invention.
  • a kit ofthe invention comprises at least one modulatory agent of the invention packaged with instructions for using the modulatory agent to modulate Th2-associated cytokine production or Thl/Th2 subset development.
  • the kit comprises at least one stimulatory agent for use in stimulating Th2-associated cytokine production or upregulating Th2 subset development (or downregulating Thl subset development).
  • the kit comprises at least one inhibitory agent for use in inhibiting Th2-associated cytokine production or downregulating Th2 subset development (or upregulating Thl subset development).
  • Combination kits, comprising two or more of the modulatory (e.g., stimulatory or inhibitory) agents of the invention are also provided.
  • Another aspect ofthe invention pertains to screening assays for identifying compounds that modulate the activity of a transcription factor that regulates expression of a Th2-associated cytokine gene.
  • these screening assays can identify, for example, compounds that modulate the expression or functional activity of the transcription factor, proteins that interact with the transcription factor, as well as compounds that modulate these protein-protein interactions, and compounds that modulate the interaction ofthe transcription factor with a cw-acting target site (e.g., a MARE) within a Th2-associated cytokine gene.
  • a cw-acting target site e.g., a MARE
  • the invention provides a method for identifying a compound that modulates the activity of a transcription factor that cooperates with a Nuclear Factor of Activated T cells (NF-AT) family protein to regulate expression of a Th2-associated cytokine gene, comprising providing a indicator composition having activity of a transcription factor that cooperates with an NFAT family protein to regulate expression ofthe Th2-associated cytokine gene; contacting the indicator composition with a test compound; and determining the effect ofthe test compound on the activity ofthe transcription factor in the indicator composition to thereby identify a compound that modulates the activity of a transcription factor that cooperates with an NFAT family protein to regulate expression of a Th2-associated cytokine gene.
  • NF-AT Nuclear Factor of Activated T cells
  • the transcription factor can be, for example, a maf family protein, such as c-Maf or a small maf protein (e.g., pl8).
  • the transcription factor can be a factor interacts with an NF-AT family protein, such as NIP45.
  • the indicator composition can be a cell-free composition or it can be a cellular composition.
  • the indicator composition is a lymphoid cell, such as a Th2 cell.
  • the indicator composition is a yeast cell.
  • the indicator composition comprises an indicator cell, wherein said indicator cell comprises: (i) the transcription factor and (ii) a reporter gene responsive to the transcription factor.
  • the indicator cell contains: i) a recombinant expression vector encoding the transcription factor; and ii) a vector comprising regulatory sequences of a Th2 -associated cytokine gene operatively linked a reporter gene; and said method comprises: a) contacting the indicator cell with a test compound; b) determining the level of expression ofthe reporter gene in the indicator cell in the presence ofthe test compound; and c) comparing the level of expression ofthe reporter gene in the indicator cell in the presence ofthe test compound with the level of expression ofthe reporter gene in the indicator cell in the absence of the test compound to thereby identify a compound that modulates the activity ofthe transcription factor.
  • the indicator composition comprises a preparation of: (i) the transcription factor and (ii) a DNA molecule to which the transcription factor binds, and said method comprises: a) contacting the indicator composition with a test compound; b) determining the degree of interaction of the transcription factor and the DNA molecule in the presence ofthe test compound; and c) comparing the degree of interaction ofthe transcription factor and the DNA molecule in the presence ofthe test compound with the degree of interaction ofthe transcription factor and the DNA molecule in the absence of the test compound to thereby identify a compound that modulates the activity of the transcription factor.
  • the transcription factor is a maf family protein and the DNA molecule comprises a maf response element (MARE).
  • MARE maf response element
  • the method identifies a protein from Th2 cells that interacts with the transcription factor.
  • the indicator composition is an indicator cell, which indicator cell comprises: i) a reporter gene operably linked to a transcriptional regulatory sequence; and ii) a first chimeric gene which encodes a first fusion protein, said first fusion protein including the transcription factor that cooperates with an NFAT family protein to regulate expression of a Th2-associated cytokine gene;
  • the test compound comprises a library of second chimeric genes, which library encodes second fusion proteins, the second fusion proteins including proteins derived from Th2 cells; expression ofthe reporter gene being sensitive to interactions between the first fusion protein, the second fusion protein and the transcriptional regulatory sequence; and wherein the effect ofthe test compound on the transcription factor in the indicator composition is determined by determining the level of expression ofthe reporter gene in the indicator cell to thereby identify a test compound comprising a protein from Th2 cells that interacts with the transcription factor.
  • the invention further provides a method for identifying a compound that modulates an interaction between NIP45 and an NF-AT family protein, comprising: a) combining: (i) NIP45, or an NF-AT-interacting portion thereof; and (ii) an NF-AT family protein, or a NIP45-interacting portion thereof; in the presence and absence of a test compound; b) determining the degree of interaction between (i) and (ii) in the presence and absence of the test compound; and c) identifying a compound that modulates an interaction between NIP45 and an NF-AT family protein.
  • the NIP45-interacting portion ofthe NF-AT family protein comprises the Rel Homology Domain of the NF-AT family protein.
  • the degree of interaction between (i) and (ii) is determined by labeling (i) or (ii) with a detectable substance, isolating non-labeled (i) or (ii) and quantitating the amount of labeled (i) or (ii) that has become associated with non-labeled (i) or (ii).
  • the method can be used to identify compounds that either increase or decrease the interaction between NIP45 and an NF-AT family protein.
  • the screening methods ofthe invention can further comprise determining the effect ofthe compound on an immune response to thereby identify a compound that modulates an immune response.
  • the effect of the compound on an immune response is determined by determining the effect of the compound on expression of a Th2 -associated cytokine gene, such as an interleukin-4 gene.
  • the effect of the compound of interest on an immune response is determined by determining the effect of the compound on development of T helper type 1 (Thl ) or T helper type 2 (Th2) cells.
  • Recombinant expression vectors that can be used for expression of a transcription factor in the indicator cell are known in the art (see discussions above and also the Examples).
  • the transcription factor-coding sequences are operatively linked to regulatory sequences that allow for constitutive expression of the transcription factor in the indicator cell (e.g., viral regulatory sequences, such as a cytomegalovirus promoter/enhancer, can be used).
  • regulatory sequences that allow for constitutive expression of the transcription factor in the indicator cell
  • Use of a recombinant expression vector that allows for constitutive expression ofthe transcription factor in the indicator cell is preferred for identification of compounds that enhance or inhibit the activity ofthe transcription factor.
  • the transcription factor-coding sequences are operatively linked to regulatory sequences of the endogenous corresponding transcription factor gene (i.e., the promoter regulatory region derived from the endogenous gene).
  • regulatory sequences of the endogenous corresponding transcription factor gene i.e., the promoter regulatory region derived from the endogenous gene.
  • the Th2-associated cytokine is interleukin-4. It has previously shown that Th2-specific, inducible IL-4 expression can be directed by as little as 157 bp of the proximal IL-4 promoter in Th2 cells (Hodge, M. et al. (1995) J. Immunol. 154:6397-6405). Accordingly, in one embodiment, a method ofthe invention utilizes a reporter gene construct containing this region of the proximal IL-4 promoter, most preferably nucleotides -157 to +58 (relative to the start site of transcription at +1) of the IL-4 promoter.
  • reporter gene constructs are described in Todd, M. et al. (1993) J. Exp. Med. 177:1663-1674. See also the Examples for descriptions of IL-4 reporter gene constructs.
  • a variety of reporter genes are known in the art and are suitable for use in the screening assays ofthe invention. Examples of suitable reporter genes include those which encode chloramphenicol acetyltransferase, beta-galactosidase, alkaline phosphatase or luciferase. Standard methods for measuring the activity of these gene products are known in the art.
  • a variety of cell types are suitable for use as an indicator cell in the screening assay.
  • a cell line is used which does not normally express c-Maf, such as a B cell (e.g. , the Ml 2 B lymphoma cell line) or a Thl cell clone (e.g., AE7 cells).
  • Nonlymphoid cell lines can also be used as indicator cells, such as the HepG2 hepatoma cell line.
  • the level of expression of the reporter gene in the indicator cell in the presence ofthe test compound is higher than the level of expression ofthe reporter gene in the indicator cell in the absence ofthe test compound and the test compound is identified as a compound that stimulates the expression or activity ofthe transcription factor.
  • the level of expression ofthe reporter gene in the indicator cell in the presence of the test compound is lower than the level of expression ofthe reporter gene in the indicator cell in the absence of the test compound and the test compound is identified as a compound that inhibits the expression or activity of the transcription factor.
  • an indicator cell can be transfected with a transcription factor expression vector, incubated in the presence and in the absence of a test compound, and Th2-associated cytokine production can be assessed by detecting cytokine mRNA (e.g., IL-4 mRNA) in the indicator cell or cytokine secretion (i.e., IL-4 secretion) into the culture supernatant.
  • cytokine mRNA e.g., IL-4 mRNA
  • RT-PCR reverse transcription-polymerase chain reaction
  • Standard methods for detecting cytokine protein in culture supernatants such as enzyme linked immunosorbent assays (ELISA) are also known in the art.
  • ELISA enzyme linked immunosorbent assays
  • the invention provides a screening assay for identifying proteins (e.g., proteins in Th2 cells) that interact with a transcription factor of interest, e.g., c-Maf or NF-AT or NIP45.
  • proteins e.g., proteins in Th2 cells
  • a transcription factor of interest e.g., c-Maf or NF-AT or NIP45.
  • these assays can be designed based on the two-hybrid assay system (also referred to as an interaction trap assay) known in the art (see e.g., Field U.S. Patent No. 5,283,173; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al.
  • the two-hybrid assay is generally used for identifying proteins that interact with a particular target protein.
  • the assay employs gene fusions to identify proteins capable of interacting to reconstitute a functional transcriptional activator.
  • the transcriptional activator consists of a DN A- binding domain and a transcriptional activation domain, wherein both domains are required to activate transcription of genes downstream from a target sequence (such as an upstream activator sequence (UAS) for GAL4).
  • UAS upstream activator sequence
  • DNA sequences encoding a target "bait" protein are fused to either of these domains and a library of DNA sequences is fused to the other domain.
  • “Fish” fusion proteins (generated from the fusion library) capable of binding to the target-fusion protein (e.g., a target GAL4-fusion "bait") will generally bring the two domains (DNA-binding domain and transcriptional activation domain) into close enough proximity to activate the transcription of a reporter gene inserted downstream from the target sequence.
  • the "fish” proteins can be identified by their ability to reconstitute a functional transcriptional activator (e.g., a functional GAL4 transactivator).
  • This general two-hybrid system can be applied to the identification of proteins in Th2 cells that interact with c-Maf (or, using similar methods, with other transcription factors of interest) by construction of a target c-Maf fusion protein (e.g., a c-Maf/GAL4 binding domain fusion as the "bait") and a cDNA library offish" fusion proteins (e.g., a cDNA/GAL4 activation domain library), wherein the cDNA library is prepared from mRNA of Th2 cells, and introducing these constructs into a host cell that also contains a reporter gene construct linked to a regulatory sequence responsive to c-Maf (e.g., a MARE sequence, for example a region ofthe IL-4 promoter, as discussed above).
  • cDNAs encoding proteins from Th2 cells that interact with c-Maf can be identified based upon transactivation of the reporter gene construct.
  • a “single-hybrid” assay such as that described in Sieweke, M.H. et al. (1996) Cell 85:49-60, can be used to identify proteins from Th2 cells that interact with c-Maf.
  • This assay is a modification ofthe two-hybrid system discussed above.
  • the "bait” is a transcription factor from which the transactivation domain has been removed (e.g., c-Maf from which the amino-terminal transactivation domain has been removed)
  • the "fish” is a non-fusion cDNA library (e.g., a cDNA library prepared from Th2 cells).
  • constructs are introduced into host cells (e.g., yeast cells) that also contains a reporter gene construct linked to a regulatory sequence responsive to the transcription factor (e.g., a MARE sequence, for example a region of the IL-4 promoter, responsive to c-Maf).
  • a regulatory sequence responsive to the transcription factor e.g., a MARE sequence, for example a region of the IL-4 promoter, responsive to c-Maf.
  • cDNAs encoding proteins from Th2 cells that interact with c-Maf (or other transcription factor of interest) can be identified based upon transactivation ofthe reporter gene construct.
  • the invention provides a screening assay for identifying compounds that modulate the interaction of a transcription factor of the invention with a DNA molecule to which the transcription factor binds, such as c-Maf and a MARE in an IL-4 gene regulatory region, respectively.
  • Assays are known in the art that detect the interaction of a DNA binding protein with a target DNA sequence (e.g., electrophoretic mobility shift assays, DNAse I footprinting assays and the like; for further descriptions see the Examples). By performing such assays in the presence and absence of test compounds, these assays can be used to identify compounds that modulate (e.g., inhibit or enhance) the interaction ofthe DNA binding protein with its target DNA sequence.
  • the amount of binding ofthe transcription factor to the DNA fragment in the presence ofthe test compound is greater than the amount of binding of the transcription factor to the DNA fragment in the absence ofthe test compound, in which case the test compound is identified as a compound that enhances binding of the transcription factor. In another embodiment, the amount of binding ofthe transcription factor to the DNA fragment in the presence ofthe test compound is less than the amount of binding of the transcription factor to the DNA fragment in the absence ofthe test compound, in which case the test compound is identified as a compound that inhibits binding ofthe transcription factor.
  • isolated NIP45 and/or NF-AT family proteins may be used in the method, or, alternatively, only portions of NIP45 and/or an NF-AT family protein may be used.
  • an isolated NF-AT Rel an isolated NF-AT Rel
  • Homology Domain (or a larger subregion of NF-AT that includes the RHD) can be used as the NIP45-interacting portion of NF-AT.
  • a portion of NIP45 capable of binding to the NF-AT RHD may be used.
  • one or both of (i) and (ii) are fusion proteins, such as GST fusion proteins (e.g. , GST-NF-AT RHD can be used as the NIP45-interacting portion of NF-AT).
  • the degree of interaction between (i) and (ii) can be determined, for example, by labeling one ofthe proteins with a detectable substance (e.g.
  • the assay can be used to identify agents that either stimulate or inhibit the interaction between NIP45 and an NF-AT family protein.
  • An agent that stimulates the interaction between NIP45 and an NF-AT family protein is identified based upon its ability to increase the degree of interaction between (i) and (ii) as compared to the degree of interaction in the absence of the agent, whereas an agent that inhibits the interaction between NIP45 and an NF-AT family protein is identified based upon its ability to decrease the degree of interaction between (i) and (ii) as compared to the degree of interaction in the absence ofthe agent.
  • Assays systems for identifying agents that modulate SH2 domain-ligand interactions as described in U.S. Patent No. 5,352,660 by Pawson can be adapted to identifying agents that modulate the NIP45/NF-AT RHD interaction.
  • Tissue specificity can be achieved through the action of repressor or silencer proteins.
  • IL-2 and IL-4 genes were actively repressed in Th2 and Thl cells respectively.
  • somatic cell fusions were performed between a Thl (D 1.1) and a Th2 (D10) clone of differing MHC Class I haplotypes.
  • the Thl clone Dl .l (K d ) and the Th2 clone D10 (K k ) were fused according to the "suspension cell fusion" procedure (Lane, R.D. et al. (1986) Methods Enzymol. 121 : 183- 192).
  • Cells expressing both MHC class I K ⁇ and K k markers were heterokaryons while cells expressing only KP or K* represented homokaryons and served as controls.
  • the three populations were then stimulated in culture with antibodies to CD3 to activate cytokine gene expression and RNA prepared for RT-PCR and Northern blot analysis. Approximately 5x1 ⁇ 5 cells were obtained for each population. Routinely, 5- 10% ofthe cells had undergone fusion. Each of these three populations was then split in half, one half transferred to pre-rinsed anti-CD3 coated plates, the remaining half to uncoated plates. After four hours, the cells were harvested, and poly(A+) RNA isolated using the Micro-FastTrackTM kit (Stratagene, La Jolla, CA).
  • cDNA was made using the Superscript kit (Gibco/BRL, Bethesda, MD), and used for PCR analysis using commercially available primers specific for murine IL-2, IL-4 and ⁇ -actin according to the manufacturer's instructions (Stratagene, La Jolla, CA).
  • PCR reactions included 0.5 ⁇ Ci ⁇ - 32 P-dCTP (3000 Ci/mmol, NEN Dupont). PCR products were ethanol precipitated, separated by nondenaturing PAGE and dried and visualized by autoradiography.
  • cDNA which was repeatedly isolated (60 of 140) detected transcripts only in RNA prepared from Th2 clones (D10, CDC35) and not from either Thl clones (AR5, OS6, Dl) or from a B cell lymphoma, Ml 2, as illustrated in the Northern blot analysis depicted in Figure 2 A. Further, the levels of transcripts detected in D10 Th2 cells were substantially increased upon activation by ligation ofthe T cell receptor with anti-CD3 antibody. No induction of the transcript detected by this cDNA clone occurred in Thl clones upon anti-CD3 treatment. A control probe, GAPDH, demonstrated approximately equal loading of RNA in all lanes. Thus, the expression of this cDNA clone in the lymphoid lineage appeared to be Th2-specific and sensitive to signals transmitted through the T cell receptor. For these Northern blots, total RNA was prepared by using Trizol
  • RNA from each sample was fractionated on a formaldehyde agarose gel and transferred to a nylon membrane.
  • a 300 bp Dral fragment derived from the 3' untranslated region of the isolated clone was labeled with ⁇ - 32 P-dCTP using Random Primed DNA Labeling Kit (Boehringer Mannheim, Indianapolis, IN). Hybridization was performed using QuikHyb (Stratagene, La Jolla, CA) according to manufacturer's instructions.
  • Th precursor (Thp) cells Naive spleen cells
  • Thp Th precursor cells
  • cytokines and anti-IL-4 for Thl , IL-4 and anti-IFNg for Th2 cytokines and anti-IL-4 for Thl , IL-4 and anti-IFNg for Th2).
  • Splenic cell suspensions were prepared from 6-8 week-old Balb/c mice, cultured in RPMI 1640 supplemented with 10% FCS at a density of 10 cells/ml, and stimulated with plate bound anti-CD3 antibody in the presence of 5 ⁇ g/ml of anti-IL4 antibody (1 1 B1 1 ) for the Thl lineage, or 5 ⁇ g/ml of anti-IFN ⁇ antibody (XMG- 1) for the Th2 lineage. 24 hours after stimulation, 50 U/ml IL2 was added to all cultures, and 500 U/ml IL4 (Genzyme) was added to Th2 cultures. 7 days after the primary stimulation, all cells were harvested, washed and restimulated with plate bound anti-CD3 antibody.
  • ELISA for cytokine quantitation was performed as follows. All anti-cytokine antibodies were purchased from Pharmingen. ELISA was performed according to Pharmingen's instructions with the exception that Avidin- Alkaline Phosphatase (Sigma) at 1 :500 dilution in PBS/BSA was used in place of avidin-peroxidase. P-nitrophenyl phosphate (GIBCO BRL) at 4 mg/ml in substrate buffer (10% diethanolamine, 0.5 mM MgCl2, 0.02% sodium azide, pH 9.8) was used as substrate.
  • Example 2 The identification of the isolated cDNA described in Example 2 as a member of the AP- 1 /CREB/ATF gene family, together with its selective expression in Th2 cells raised the possibility that c-Maf controlled the tissue-specific transcription ofthe IL-4 gene. Additionally, the presence of transcripts encoding c-maf correlated well with IL-4 expression in Th2 cells and in three of four transformed mast cell lines examined. To test whether c-Maf could transactivate the IL-4 promoter, cotransfection experiments were performed.
  • Thl clones and the B lymphoma M12.4.C3 neither express c-maf nor transcribe the IL-4 gene. If c-Maf is the transcription factor critical for controlling IL-4 gene expression, then forced expression in these cells should permit IL-4 gene expression. To test this, the full-length (4.3 kb) c-maf cDNA clone was inserted into the Sail site of the pMex-NeoI mammalian expression vector, which utilizes the CMV enhancer to drive expression ofthe inserted sequence. The c-Maf expression vector was then cotransfected with an IL-4 promoter reporter construct into the Thl clone AE7 and the B lymphoma Ml 2.
  • the generation ofthe wild type IL4 CAT reporter construct, containing an IL4 promoter fragment from -157 to +68 operatively linked to a chloramphenicol acetyltransferase gene is described in Hodge, M. et al. (1995) J. Immunol. 154:6397-6405.
  • the Thl clone was cultured in RPMI 1640 supplemented with 10% FCS and 10% Con-A stimulated rat splenocyte supernatant, and maintained by bi-weekly stimulation with appropriate antigen and APCs.
  • Ml 2 cells were cultured in RPMI 1640 supplemented with 10% FCS.
  • the Thl clone AE7 or Ml 2 B lymphoma cells were transiently transfected by preincubating 0.4 ml of cells, containing 2x10' cells/ml AE7 or 3x10" cells/ml Ml 2 cells in serum-free RPMI 1640 with 20 ⁇ g (AE7) or 5 ⁇ g (Ml 2) of each plasmid for 10 minutes at room temperature.
  • the samples were then electroporated using a BIO-RAD Gene Pulser (BIO-RAD, Richmond, CA) set at 975 ⁇ F, 280 V, and immediately placed on ice for 10 minutes.
  • the transfected cells were allowed to recover overnight in complete media and stimulated with plate bound anti-CD3 antibody ( ⁇ Pharmingen, San Diego, CA ⁇ 1 ⁇ g/ml in 1XPBS overnight at 4 °C) or with 50 ng/ml PMA (Sigma, St. Louis, MO) and 1 ⁇ M Ionomycin (Calbiochem Co ⁇ ., La Jolla, California), for 24 hours.
  • Cell lysate was prepared by freeze-thaw lysis in 0.25 M Tris-Cl, pH 7.8. Equal amounts of protein (between 5-20 ⁇ g) were used for CAT assays.
  • CAT assays were performed as described in Todd, M. et al. (1993) J. Exp. Med. 177:1663-1674.
  • Th2-specific, inducible IL-4 expression can be directed by as little as 157 bp of the proximal IL-4 promoter in Th2 cells (Hodge, M. et al. (1995) J Immunol. 154:6397-6405).
  • ectopic expression of c-Maf in the Thl clone AE7 results in substantial activity ofthe IL-4 promoter reporter after stimulation through the T cell receptor. The fold induction observed was approximately 5 fold over that observed with the control empty vector alone.
  • c-Maf As a control for the specificity of c-Maf as opposed to other AP-1 family members, the c-Fos and c-Jun proteins were also overexpressed in Ml 2 cells utilizing murine full-length cDNAs encoding c-Fos and JunD in the mammalian expression vector of pMEX-NeoI together with the IL-4 reporter plasmid. No IL-4 promoter activity could be achieved by overexpression of either of these two AP-1 family members in M12 cells. Thus, c-Maf has a unique ability to drive IL-4 gene transcription in M12 B cells.
  • NF-AT proteins have been shown to be critically important in the regulation of both the IL-4 and IL-2 cytokines.
  • NF-ATp was the first member of this family to be isolated (McCaffrey, P.G. et al. (1993) Science 262:750-754). Both AE7 and M12 cells have endogenous NF-ATp protein, but nevertheless do not transcribe IL-4. Although NF-ATp could not therefore account for selective IL-4 gene transcription, it was of interest to test whether overexpression of NF-ATp in unstimulated or stimulated M12 cells would further increase the transactivation of the IL-4 promoter by c-maf.
  • M12 cells were cotransfected with the IL-4 reporter construct and either an NFAPp expression vector (pREP ⁇ NF-ATp, which also carries a hygromycin resistance gene) alone or the NFAPp expression vector together with the c-Maf expression vector.
  • NF-ATp an NFAPp expression vector
  • Overexpression of NF-ATp alone in Ml 2 cells resulted in some modest transactivation of the IL-4 promoter. This transactivation was markedly increased by ectopic expression of c-Maf, an increase which was not just additive but was synergistic (see Fig. 3B and Table 1).
  • c-Fos overexpression did not further increase the modest transactivation achieved by NF-ATp.
  • EXAMPLE 4 Ectopic Expression of C-Maf Activates Transcription of the Endogenous IL-4 Gene in a B Lymphoma
  • c-Maf transactivates the IL-4 promoter in transient transfection assays in Thl , B and non-lymphoid cells.
  • the B lymphoma Ml 2 was stably transfected with expression vectors encoding c-maf, NF-ATp or both, or junD with and without NF-ATp as a control.
  • Ml 2 cells were transfected as described above in Example 3.
  • the transfected cells were allowed to recover in complete media for 48 hours before the addition of Neomycin (GIBCO/BRL, Gaithersburg, MD) and Hygromycin (Calbiochem, Co ⁇ .) at a concentration of 400 ⁇ g/ml of each antibiotic.
  • Neomycin GEBCO/BRL, Gaithersburg, MD
  • Hygromycin Calbiochem, Co ⁇ .
  • Ml 2 cells were plated at equal density supernatants harvested 24 hours later to measure cytokines by ELISA.
  • ELISAs were performed as described in Example 2. The results, shown in Figure 4, demonstrate that in these experiments M12 cells transfected with c-maf, junD or NF-ATp alone did not produce measurable IL-4 by ELISA. However, Ml 2 cells stably transfected with both c-maf and NF-ATp did produce detectable, but low level, IL-4 by ELISA. These results were confirmed by RT- PCR on RNA from these transfected cells. In contrast, these cells did not produce detectable IL-2.
  • Th2 clones D10, CDC35
  • Thl clones AE7, S53
  • DNAsel footprinting analysis was then performed according to standard methods using a Klenow end-labeled IL-4 promoter fragment (-157 to +68). The results are shown in Figure 5 A. Stimulated extracts from both Thl and Th2 cells footprinted the two NF-AT sites and the AP- 1 site upstream of the distal NF-AT site as described previously (Rooney, J. et al.
  • Th2 footprint observed was subtle, but reproducible in two experiments and is located in a site that has previously been demonstrated to be critical for IL-4 promoter activation in Th2 cells (Hodge, M. et al. (1995) J. Immunol. 154:6397-6405).
  • a schematic summary of sites occupied in the IL-4 promoter as detected by footprint analysis is shown in Figure 5B. These results indicate that a site in the proximal IL-4 promoter, previously shown to be functionally important, is occupied in activated Th2 but not in activated Thl cells.
  • the Th2-specific footprint does not contain a c-maf response element (MARE).
  • MARE c-maf response element
  • a truncated c-Maf recombinant protein containing the b-zip domain(amino acids 171-371) was expressed from E. coli, purified on an S-Tag agarose column and used in electrophoretic mobility shift assays with radiolabeled MAR ⁇ oligonucleotide.
  • the expression vector for recombinant c-Maf was constructed by inserting a cDNA fragment encoding a.a. residues 171 to 371 of c-Maf (disclosed in Kurschner C. and Morgan, J.I. (1995) Mol. Cell. Biol. 15:246-254) into the Notl site of p ⁇ T29 (Novagen, Inc. Madison, WI).
  • the truncated c-Maf protein was expressed using T7 polymerase in the BL21(DE3)strain. Cells were induced by the addition of 1 mM IPTG and incubated at 37° C for 3 hours.
  • the induced cells were lysed in 1 X Bind/Wash buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Triton X-100) followed by sonication.
  • the c-Maf protein was then purified from the soluble fraction by using the S-Tag Purification Kit (Novagen) according to manufacturer's instructions. Two additional proteins, NF-ATp and c-Jun, were also used in EMSA assays.
  • the recombinant NF-ATp containing the Rel domain of murine NF-ATp, was expressed using an in vitro transcription/translation vector TP7-NF-ATp, which contains a cDNA fragment encoding the Rel domain of murine NF-ATp.
  • the c-Jun expression vector, pGEM-c-Jun was constructed by inserting a full-length cDNA of murine c-Jun into the Pstl site of pGEM4. 1 ⁇ g of each plasmid DNA was transcribed from the T7 promoter and translated in rabbit reticulocyte lysate by using the TnT Coupled Transcription/Translation Kit (Promega, Madison, WI).
  • Electrophoretic mobility shift assays were performed as follows. 100 ng of double-stranded oligonucleotides were end-labeled with ⁇ - ⁇ p.dATP (DuPont NEN Research Product, Wilmington, DE) using T4 polynucleotide kinase (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ). The labeled ds-oligonucleotides were fractionated on 15-20% polyacrylamide gels, eluted overnight at 37° C in IX TE and precipitated in ethanol.
  • Binding assays were performed at room temperature for 20 minutes using 0.5 ⁇ g of recombinant proteins or 4 ⁇ l of in vitro translated products, 500 ng poly(dl-dC), and 20,000 cpm of probe in a 15 ⁇ l volume of 20 mM HEPES (pH 7.9), 100 mM KCl, 5% glycerol, ImM EDTA, 5mM DTT, 0.1% NP-40, and 0.5 mg/ml BSA. The samples were then fractionated in 4% non-denaturing polyacrylamide gel containing 0.5X TBE at room temperature.
  • Oligonucleotides derived from the murine IL4 promoter used in EMSA were: -59 to -27: 5'-CTCATTTTCCCTTGGTTTCAGCAACTTTAACTC-3' (SEQ ID NO: i );
  • oligonucleotides were annealed with their respective reverse-complementary strands to form double-stranded oligonucleotides.
  • c-Maf The ability of c-Maf to bind to the IL-4 promoter probe was specific since in vitro translated c-Jun protein did not bind to this oligonucleotide. The c-Jun protein was functional since it could bind to the consensus MARE which contains a core TRE site. These results indicate that c-Maf, but not another AP-1 family member (c-Jun), can bind to the MARE site within the proximal IL-4 promoter.
  • NF-AT proteins interact cooperatively with AP-1 family member proteins to form higher mobility complexes on IL-2 and IL-4 promoter DNA on EMSA (Jain, J. (1993) Nature 365:353-355; Rooney, J. et al.
  • IL-4 promoter located immediately upstream of the TATA element has been characterized by high resolution mutagenesis (Hodge, M. et al (1995) J. Immunol. 154:6397-6405). Mutagenesis of this 33 bp region (-59 to -28) demonstrated multiple sites required for inducible IL-4 transcription in Th2 cells. These sites included an NF-AT target sequence, the region footprinted by Th2 extracts, and what is now recognized as a MARE. A series of IL-4 reporter gene constructs comprising 4 base pair linker-scanning mutants generated across this region were used to map the target sequence utilized by c-Maf in vivo in M12 cells.
  • Mutation ofthe MARE (muts 3 and 4), or the site defined by the Th2 footprint (mut 2), abrogated (muts 2 and 4) or partially abrogated (mut 3) the ability of transfected c-maf to drive IL-4 transcription.
  • Mutation ofthe MARE (muts 3 and 4), or the site defined by the Th2 footprint (mut 2), abrogated (muts 2 and 4) or partially abrogated (mut 3) the ability of transfected c-maf to drive IL-4 transcription.
  • mutant 8 which disrupts the NF-AT sequence, consistent with the presence in Ml 2 cells of endogenous NF-ATp and with the synergy between NF-ATp and c-maf demonstrated in the previous examples.
  • EXAMPLE 8 Isolation of a NIP45 cDNA Using a Yeast Two-Hybrid Interaction Trap Assay
  • a yeast two-hybrid interaction trap assay was used to isolate proteins that could directly bind to the RHD of NF-ATp.
  • An NF-ATp(RPID)-Gal4 fusion protein was prepared for use as the "bait" in the yeast two-hybrid assay by cloning a 900 bp fragment of murine NF-ATp (McCaffrey, P.G. et al. (1993) Science 262:750-754), spanning amino acids 228 to 520, into the BamHl site of vector pEG202 (Gyuris, J. et al. (1993) Cell 75:791 -803).
  • NIP45 NF-AT Interacting Protein 45.
  • Figure 8 shows a photograph of yeast colonies (three representatives for each plasmid combination), cotransformed with the NIP45 plasmid and either the NF-ATp-RHD bait or control baits (Max-Gal4, CDK2-Gal4 and the control vector pEG202, expressing only an epitope tagged Gal4 protein), together with the LacZ reporter plasmid pSH18.
  • yeast colonies had been selected on appropriate media and were spotted onto plates containing Xgal and the nonrepressing carbon source galactose.
  • Yeast colonies cotransformed with the NIP45 plasmid and the NF-ATp-RHD bait were blue in color, demonstrating expression of the LacZ reporter plasmid (indicative of NIP-45/NF-ATp-RHD interaction), whereas yeast colonies transformed with the NIP45 plasmid and the control baits were white in color, indicating no interaction of NIP45 with the control baits.
  • the ability ofthe NIP45 polypeptide to interact specifically with NF-ATp in vivo was tested in mammalian cells.
  • the 1.9 kb NIP45 cDNA insert selected in the yeast two-hybrid system (described in Example 8) was subcloned into a mammalian expression vector which fuses the coding region to an epitope tag from a influenza hemagglutinin (HA) peptide, vector pCEP4-HA (He ⁇ scher, R.F. et al. (1995) Genes Dev. 9:3067-3082), to create the expression vector NIP45-HA.
  • HA hemagglutinin
  • This tagged construct was then cotransfected with an NF-ATp expression plasmid into HepG2 cells (which express low levels of NF-ATp).
  • HepG2 cells also were cotransfected with NIP45-HA along with the parental expression vector for the NF-ATp construct (i.e. , the expression vector without the NF-ATp insert) or with the NF-ATp expression vector along with an out of frame fusion of NIP45 with the epitope tag. Lysates were prepared from the transfected cells and immunoprecipitated with anti-NF-ATp antibody. Western blot analysis was then performed on the immunprecipitated material using either anti- NF-ATp or anti-HA antibodies. The results of this experiment are shown in Figure 9.
  • the 1.9 kb NIP45 cDNA insert from the clone isolated using the two-hybrid assay (described in Example 1) was used to screen a D10.G4 T cell lambda zap II cDNA library (Stratagene) to identify full length clones. Screening of a library containing approximately 8x l ⁇ 5 clones yielded 7 hybridizing clones most of which did not extend as far towards the 5' end as the original isolate. Sequence analysis ofthe longest clone (2.8 kb), however, demonstrated identity to the original clone at the 5' end. The structures ofthe original 1.9 kb cDNA isolate and the longest 2.8 kb cDNA isolate are compared in Figure 10.
  • the 2.8 kb cDNA isolate contained an additional segment of 180 bp located 868 bp downstream from the 5' end of the original clone. Junction sequences at the ends of this 180 nucleotide segment indicate it to be an unspliced intron and conceptual translation ofthe nucleotide sequence within this region revealed an in- frame stop codon. Much ofthe additional sequence in this clone was at the 3' end and represented an extensive 3' untranslated region followed by a poly-A+ tail (see Figure 10). Such extensive 3' untranslated regions have been observed in many genes. Allowing for the splicing ofthe small intron and translation ofthe single large open reading frame, the 2.8 kb cDNA clone is predicted to encode an identical polypeptide to that ofthe original 1.9 kb isolate.
  • the nucleotide and predicted amino acid sequences of the 1.9 kb cDNA isolate are shown in Figure 1 1 (and in SEQ ID NOs: 5 and 6, respectively).
  • the coding region is shown from the first initiation codon through the first in frame stop codon.
  • the nucleotide and amino acid positions are indicated to the right of the primary sequence.
  • Conceptual translation of the 1.9 kb nucleotide sequence predicted a polypeptide of 412 amino acids with a molecular mass of 45 Kd, and hence the protein has been termed NF ⁇ AT Interacting Protein 45 (NIP45). Inspection ofthe amino acid sequence of NIP45 revealed a highly basic domain at the N-terminus, in which 13 of 32 amino acid are basic. This region is underlined in Figure 1 1. This basic region appears as a hydrophilic stretch in the hydrophobicity plot shown in Figure 12.
  • RNA from different murine tissues was performed to investigate the tissue expression of NIP45 mRNA. 10 ⁇ g of total RNA from various tissues was separated on denaturing agarose gels, blotted and hybridized with a radiolabelled 1.4 kb NIP45 cDNA fragment. Samples were controlled for equivalent loading of RNA by comparison of ethidium bromide fluorescence. The results of the Northern blot analysis are shown in Figure 13. The hybridizations revealed a transcript of approximately 3.1 kb, which is of comparable size to the longest cDNA clones. RNA from testis contained an additional 1.4 Kb hybridizing species. The highest levels of NIP45 transcripts were seen in spleen, thymus and testis.
  • the preferential expression in lymphoid organs may indicate a specific function for NIP45 in the immune system.
  • the low intensity hybridization signal and the rare occurrence of NIP45 cDNA clones in the T cell cDNA library indicate that the NIP45 RNA is a relatively rare message.
  • Subcellular localization of epitope tagged NIP45 protein was determined by indirect immunofluorescence.
  • BHK cells were transfected with 1 ⁇ g of an expression construct encoding an HA-epitope tagged NIP45 (pCEP4-HA), using methodologies known in the art (see Heald, R. et al. (1993) Cell 74:463-474). Transfected cells were incubated overnight, fixed, permeabilized as described (Heald, R.
  • NIP45 The effect of NIP45 on the nuclear translocation of NF-AT4 also was investigated.
  • HepG2 cells were transfected with either NF-AT4 or NF-AT4 plus NIP45 and stimulated the following day with 1 ⁇ M ionomycin for 0, 2, 4, 8 or 15 minutes.
  • the cells were stimulated for 15 minutes with ionomycin and then washed with fresh media and allowed to rest for an additional 15 minutes (indicated as " 15 min. + 15 min. rest” in Table 1).
  • This analysis is designed to examine the function of NIP45 as a nuclear retention factor. Fifteen minutes has been shown to be sufficient time for NF-AT4 to be exported to the cytoplasm (Shibasaki, F. et al.
  • NIP45 was expressed at high levels in HepG2 cells.
  • HepG2 cells were chosen because they have low levels of endogenous NF-AT, and ectopic expression of NF-AT family member proteins has been shown to transactivate NF-AT-driven transcription in this cell line in the absence of exogenous stimulation (Hoey, T. et al. (1995) Immunity 2:461 -472).
  • HepG2 cells were transfected with a 3X NF-AT-CAT reporter from the IL-2 gene
  • NF-AT proteins share approximately 70% identity within the RHD, raising the possibility that NIP45 could also interact with other NF-AT family members.
  • NIP45 was cotransfected as above with expression constructs encoding either NF- ATc, NF-AT3 or NF-AT4 plus the 3X NF-AT-CAT reporter plasmid. The results of these experiments are also shown in Figure 8. It has previously been demonstrated that all NF-AT family members can transactivate a reporter gene containing 3 copies of an NF-AT/APl site when overexpressed in HepG2 cells, although to different levels (Hoey, T. et al. (1995) supra).
  • NF-ATp was the most potent transactivator of the NF-AT-CAT reporter followed by NF-ATc and NF-AT3 with only weak transactivation by NF-AT4, consistent with previous data (McCaffrey, P.G. et al. (1993) Science 262:750-754).
  • NIP45 substantially potentiated both NF-ATc and NF-AT3-driven transactivation and weakly potentiated NF-AT4-mediated transactivation (Figure 15).
  • NIP45 interacts with an NF-ATc RHD bait in yeast cells.
  • NIP45 overexpression resulted in a 4-fold increase in transactivation by NF-ATc, a 3-fold increase in NF-AT3- driven transactivation and a 2-fold increase in NF-AT4-driven transcription.
  • the ability of NIP45 to potentiate the activity of all NF-AT family members is not su ⁇ rising given the high degree of sequence conservation ofthe RHD of the NF-AT family members.
  • a sequence comparison ofthe NF-AT RHD domains reveals a higher level of sequence identity in the amino terminal portion compared to that ofthe carboxyl terminus (Hoey, T. et al. (1995) supra). Thus it is likely that the NIP45/NF-AT interaction site is located in the 5' portion of the RHD.
  • IL-4 expression is highly tissue specific and restricted to the Th2 subset of T cells and to mast cells.
  • the IL-4 promoter contains multiple NF-AT binding sites which have been shown to be critical for expression of IL-4 (Rooney, J.W. et al. (1995) Immunity 2:473-483).
  • the proto-oncogene c-Maf has been shown to direct tissue specific expression of IL-4 (Examples 3 and 4).
  • the IL-4 promoter is not active in the HepG2 cell line but can be activated by the introduction of NF-ATp and c- Maf.
  • HepG2 cells were transfected with an IL-4-CAT reporter construct (extending to -732 bp ofthe IL-4 promoter) and expression vectors or controls for NIP45, NF-ATp and c-Maf.
  • the controls for NIP45 was a frame shift mutant at amino acid 13.
  • Controls for NF-ATp and c-Maf were the empty expression vectors pREP4 and pMEX respectively (Ho, LC. et al. (1996) Cell 85:973-983).
  • Ml 2 B lymphoma cells were transiently cotransfected with expression plasmids for NF-ATp and c-Maf together with NIP45 or pCI vector control.
  • M12 cells were transiently transfected by electroporation as previously described (Ho, LC. et al. (1996) Cell 85:973-983) by incubating 3 x 10 ⁇ cells in 0.4 ml of PBS with 5 ⁇ g of each plasmid for 10 minutes at room temperature prior to electroporation at 975 ⁇ F, 280 V.
  • EXAMPLE 15 Expression of pl8 mRNA is Downregulated During
  • the Maf family protein pl 8 is a member ofthe "small mafs" that lack the amino terminal two thirds of c-Maf that contains the transactivating domain.
  • Th precursor (Thp) cells Naive spleen cells (Th precursor (Thp) cells) were driven along a Thl or Th2 pathway by treatment with anti-CD3 in the presence of cytokines and anti-cytokine antibodies (IFN ⁇ and anti-IL-4 for Thl, IL-4 and anti-IFN ⁇ for Th2).
  • EXAMPLE 16 pl8 Represses IL-4 Promoter Activity
  • IL-4 promoter/CAT reporter gene construct was transfected into Ml 2 cells with either a c- Maf expression vector, a pl 8 expression vector or both c-Maf and pl 8 expression vectors. Representative results of CAT assays are shown in Figure 19. Expression of c- Maf alone (5 ⁇ g of plasmid) resulted in activation of the IL-4 promoter construct (see lane 2 of Figure 19), evidenced by detectable CAT activity in the M12 cells.
  • EXAMPLE 17 Transgenic Mice that Overexpress c-Maf
  • FIG. 20 A schematic diagram ofthe c-maf transgenic construct is shown in Figure 20.
  • This construct comprises the 4 kb mouse c-maf cDNA and the first intron of the mouse c-maf gene.
  • Expression ofthe c-maf cDNA is controlled by the CD4 promoter/enhancer regulatory region, which is operatively linked to the 5' end ofthe c-maf first intron, thereby conferring T cell-specific expression on the construct.
  • An SV40 polyadenylation site is linked to the 3' end of the c-maf cDNA.
  • This c-maf transgenic construct was microinjected into fertilized mouse oocytes and transgenic mice were prepared according to standard procedures.
  • the phenotype ofthe c-maf transgenic animals were compared to that of wild type animals using a number of different assays.
  • First, total numbers of cells in lymphoid organs (lymph nodes, spleen and thymus) were quantitated, the results of which are shown in Figure 21.
  • This experiment demonstrated that the c-maf transgenic mice exhibited decreased cell numbers in all three lymphoid organs examined compared to wild type mice.
  • the abundance of specific thymocyte populations was compared in c-maf transgenic and wild type mice, using flow cytometry.
  • the phenotype ofthe c-maf transgenic mice described above is very similar to the phenotype described for IL-4 overexpressor transgenic mice (see Tepper et al. (1990) Cell 62:457; and Lewis et al. (1991) J Exp. Med. 173:89).
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:2
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • SEQUENCE DESCRIPTION SEQ ID NO:4 :
  • GAG AAA CAC CAG ATG TTG GAG ATC TCA CTG TCT CCT GAT TCT CCT CTT 1104 Glu Lys His Gin Met Leu Glu lie Ser Leu Ser Pro Asp Ser Pro Leu 350 355 360

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Abstract

L'invention concerne des procédés de modulation de la production d'une cytokine associée à un lymphocyte T auxiliaire de type 2 (Th2), en particulier l'interleukine 4, par la modulation de l'activité d'un ou plusieurs facteurs de transcription qui coopèrent avec des protéines de la famille NF-AT pour réguler l'expression d'un gène de cytokine associé à Th2. Dans un mode de réalisation, l'activité d'une protéine de la famille maf (ex. protéine c-Maf ou une petite protéine maf telle que p18) est modulée. Dans un autre mode de réalisation, l'activité d'une protéine interagissant avec une protéine de la famille NF-AT (ex: NIP45) est modulée. L'invention porte également sur des méthodes de combinaison, par exemple dans lesquelles les activités d'une protéine de la famille maf et d'une protéine NF-AT sont modulées ou les activités d'une protéine maf et d'une protéine interagissant avec NF-AT sont modulées. Des procédés de modulation du développement de sous-ensembles de lymphocytes T de type 1 (Th1) ou de type 2 (Th2) chez un sujet, au moyen d'agents qui modulent l'activité du facteur de transcription sont également décrits. Des compositions de NIP45, comprenant des molécules d'acide nucléique isolées codant NIP45, des molécules d'acide nucléique antisens, des vecteurs d'expression de recombinaison contenant une molécule d'acide nucléique NIP 45, des cellules hôtes dans lesquelles lesdits vecteurs d'expression ont été introduits et des animaux transgénique non humains portant un transgène de NIP45 sont également décrits. L'invention se rapporte aussi à des peptides et des protéines NIP45 isolés, des protéines de fusion NIP45 et des anticorps anti-NIP45. Des procédés d'utilisation des compositions de NIP45 selon l'invention sont également décrits.
EP97926377A 1996-04-23 1997-04-23 Procedes et compositions pour la regulation de sous-ensembles de lymphocytes t par la modulation de l'activite du facteur de transcription Withdrawn EP0891425A2 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US636602 1990-12-31
US08/636,602 US5958671A (en) 1996-04-23 1996-04-23 Methods and compositions for regulating T cell subsets by modulating transcription factor activity
US755592 1996-11-25
US755584 1996-11-25
US08/755,584 US5858711A (en) 1996-11-25 1996-11-25 NF-AT-interacting protein NIP45 and methods of use therefor
US08/755,592 US6537810B1 (en) 1996-04-23 1996-11-25 Methods for regulating T cell subsets by modulating transcription factor activity
PCT/US1997/006708 WO1997039721A2 (fr) 1996-04-23 1997-04-23 Procedes et compositions pour la regulation de sous-ensembles de lymphocytes t par la modulation de l'activite du facteur de transcription

Publications (1)

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EP0891425A2 true EP0891425A2 (fr) 1999-01-20

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EP97926377A Withdrawn EP0891425A2 (fr) 1996-04-23 1997-04-23 Procedes et compositions pour la regulation de sous-ensembles de lymphocytes t par la modulation de l'activite du facteur de transcription

Country Status (8)

Country Link
EP (1) EP0891425A2 (fr)
JP (1) JP2000510333A (fr)
KR (1) KR20000010606A (fr)
CN (1) CN1225682A (fr)
AU (1) AU720455B2 (fr)
CA (1) CA2252643A1 (fr)
NZ (1) NZ332897A (fr)
WO (1) WO1997039721A2 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9722388D0 (en) * 1997-10-24 1997-12-24 Zeneca Ltd NIP45 human homolog
US6274338B1 (en) 1998-02-24 2001-08-14 President And Fellows Of Harvard College Human c-Maf compositions and methods of use thereof
AU4219899A (en) * 1998-05-28 1999-12-13 President And Fellows Of Harvard College Methods and compositions relating to modulation of cartilage cell growth and/or differentiation by modulation of nfatp activity
JP2003518942A (ja) * 1999-12-30 2003-06-17 プレジデント・アンド・フェローズ・オブ・ハーバード・カレッジ 肝細胞成長、形質細胞分化、又はｔ細胞サブセットの活性を、ｘｂｐ−１活性の変調により変調することに関する方法及び組成物
EP1259592A1 (fr) * 2000-01-11 2002-11-27 Maxygen, Inc. Sous-ensembles de cellules dendritiques d riv es de monocytes
EP1280897B1 (fr) * 2000-04-25 2006-09-20 Bayer HealthCare AG Regulation de variante de proteine nip 45 a interaction avec nf-at
WO2002029403A1 (fr) * 2000-10-02 2002-04-11 Mochida Pharmaceutical Co., Ltd. Inhibiteur de l'expression de gata-3 et procede de depistage dudit inhibiteur
EP1372664A1 (fr) 2001-03-01 2004-01-02 Hollis-Eden Pharmaceuticals Inc. Traitement de d ficiences affectant les globules sanguins
US20050079496A1 (en) * 2001-08-17 2005-04-14 Edgar Serfling Methods for diagnosing and treating neoplasias using nf-at transcriptions factors
IL147138A0 (en) 2001-12-17 2002-08-14 Yeda Res & Dev Methods of and pharmaceutical compositions for modulating cell adhesion, migration and extravasation
EP1989544B1 (fr) * 2006-03-02 2011-06-22 Antitope Limited Dosages de lymphocytes t
US8940479B2 (en) 2008-01-14 2015-01-27 Cornell University Methods for modulating de novo hepatic lipogenesis by modulating XBP-1 activity
KR101146335B1 (ko) * 2009-10-12 2012-05-21 한국원자력의학원 알파태아단백 인헨서 및 프로모터의 조절을 받아 리포터 단백질을 발현하는 형질 전환 마우스, 그의 제조 방법 및 이를 이용한 알파태아단백 발현의 증가 또는 감소 유도 물질을 스크리닝하는 방법
WO2012109238A2 (fr) 2011-02-07 2012-08-16 President And Fellows Of Harvard College Procédés d'augmentation des réponses immunitaires à l'aide d'agents qui se lient directement à ire-1 et activent ire-1
WO2013134774A1 (fr) 2012-03-09 2013-09-12 Cornell University Modulation de la croissance du cancer du sein par modulation de l'activité de la xbp1
WO2015019125A1 (fr) 2013-08-05 2015-02-12 Societe De Developpement Et De Recherche Industrielle Cibles moléculaires destinées au traitement des blessures, en particulier des blessures chroniques
ES2724300T3 (es) 2013-09-25 2019-09-10 Univ Cornell Compuestos para inducir inmunidad antitumoral y métodos de lo mismo

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
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See references of WO9739721A2 *

Also Published As

Publication number Publication date
WO1997039721A3 (fr) 1998-04-09
KR20000010606A (ko) 2000-02-15
CA2252643A1 (fr) 1997-10-30
CN1225682A (zh) 1999-08-11
AU3115597A (en) 1997-11-12
JP2000510333A (ja) 2000-08-15
NZ332897A (en) 2000-04-28
AU720455B2 (en) 2000-06-01
WO1997039721A2 (fr) 1997-10-30

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