Method for in-vitro detecting pathogenic T helper cells and pharmaceutical compositions for treating autoimmune diseases

The invention relates to a method for in-vitro detecting pathogenic T helper cells by incubating a sample of a body fluid or tissue taken from a mammal with substances specifically interacting with at least one gene product encoded by selected genes, which are differentially expressed compared to normal T helper cells, determining specific incubation products, correlating an amount of signal or change in signal with a concentration of the gene product in the sample, and detecting cell pathogenicity by comparing the concentration with another gene product concentration in a sample of non-pathogenic and/or pathogenic cells Another object of the invention concerns a pharmaceutical composition for the prophylaxis and therapy of chronic inflammation The invention also relates to a method for screening substances with the property to reduce the pathogenicity of T helper cells along with the symptoms of chronic inflammation

Autoimmune diseases concern an exaggerated, chronic immune reaction directed against the body's tissue which is aberrantly assigned as deleterious target Chronic inflammation finally results in the damage of the affected organs Chronic inflammatory diseases include autoimmune hepatitis, chronic gastritis, diabetes mellitus type I, Morbus Crohn, Multiple Sclerosis, arthritis, psoriasis or rheumatism Several therapeutic treatments are applied, which success has to be evaluated in each case An abatement of symptoms and a reduction of acute attacks are achieved by cortisone However, cortisone treatment is associated with a couple of adverse effects restricting its administration to a short period Furthermore, the weakening of the immune system is known by immune suppressive agents For instance, methotrexate is used to treat Multiple Sclerosis or rheumatism Changes in hemogram, gastrointestinal dysfunction, nausea, diarrhea, tumor induction, alopecia and weight reduction arising from methotrexate administration are of disadvantage

T helper 1 (Th1) lymphocytes control immune reactions and inflammation through the secretion of signal proteins The potential to induce inflammation has been demonstrated by adoptive transfer of Th1 lymphocytes in murine models of Th 1 -associated inflammatory diseases such as diabetes, inflammatory bowel disease, and rheumatoid arthritis In these models, the induction of inflammation in a particular tissue by Th1 lymphocytes is dependent on re-stimuiation by their cognate antigen presented in that tissue Induction of inflammation by Th1 cells is mediated by expression of the pro-inflammatory cytokines TNF-α and

interferon-γ (IFN-γ), the latter being a hallmark of Th1 differentiation. IFN-γ also induces expression of the chemokine receptor CXCR3 and its ligands CXCL9, -10, and -11 , attracting Th1 cells specifically to inflamed tissue. Th1 cells with the capacity to recall IFN-γ expression, i.e. Th1 memory cells, are detectable in chronically inflamed tissue.

The role of Th1 cells in chronification and maintenance of chronic inflammation is less clear. Initial studies aiming at a complete depletion of CD4+ T lymphocytes suggested a clinical benefit in respect of rheumatoid arthritis chronic and inflammatory arthritis, respectively, but studies had to be terminated due to the severe side effects of systemic depletion of Th lymphocytes (Choy et al. (1996) Arthritis Rheum 39:52-56; Emmrich et al. (1991) Agents Actions Suppl 32:165-170; Homeff et al. (1991) Arthritis Rheum 34:129-140). Skurkovich & Skurkovich (2006) Ernst Schering Res Found Workshop 1-27, as well as Hommes et al. (2006) Gut 55:1 131-1137, showed that anti-IFN-γ therapy is beneficial in various Th1- associated inflammatory diseases. However, for IFN-γ, also a regulatory role has been demonstrated, based on the induction of inducible nitric oxide synthase (iNOS) and of IL-12 in antigen-presenting cells, in turn increasing the IL-10 synthesis of TM cells. Targeting of CD4 and CD3-expressing cells with non-depleting antibodies, and neutralization of Th- related effector cytokines have demonstrated clinical efficacy (Nepom (2002) Curr Opin Immunol 14:812-815, Panaccione et al. (2005) Curr Opin Pharmacol 5:566-572, Baumgart & Dignass (2004) Curr Pharm Des 10:4127-4147), but all of them impair protective as well as pathogenic T cell memory.

It has been recently suggested that pathogenic memory T cells are key players in chronic inflammation. Naive Th lymphocytes are instructed by antigen and polarizing signals during their primary activation to express particular cytokines during this activation. Upon re- stimulation, the pre-activated Th cells recall the expression of those cytokines they had been instructed to express during their primary activation, with much faster kinetics of expression, and without the requirement of polarizing signals. As memory cells, they are stably imprinted for the expression of distinct cytokines, chemokines, adhesion molecules and other genes determining the function of Th cells. This memory is based on epigenetic modification of the cytokine genes and the presence of particular transcription factors. Genes specifically expressed in repeatedly activated Th cells may qualify as diagnostic and therapeutic targets. Consequently, the transcriptome analysis of repeatedly activated Th cells resulted in the identification of a marker for chronically activated Th1 cells.

Therefore, the technical problem forming the basis of the present invention is to provide further substances for the detection of pathogenic T helper, which makes a simple and fast

determination of an instant autoimmune disease possible. It is another problem of the invention to provide a pharmaceutical composition allowing an effective application in diagnosis, prevention or therapy of chronic inflammation, especially such compositions that improve the therapeutic efficacy and minimize adverse effects. It is still another problem to provide a method for screening substances that effectively alter the expression pattern of differently expressed genes in pathogenic T helper cells.

The present invention solves the problem by providing:

The use of a gene product encoded by a Hop gene sequence with the accession No. AK

009007, AF536202 or NM 032495 for detection of pathogenic Th1 cells.

According to the present invention pathogenic Th1 cells can be detected by determining the expression level of the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495. Preferably the occurrence and/or amount of a gene product encoded by a Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495 is determined. For this purpose a number of well known techniques can be applied, e.g. microarray analysis, Nothem blotting, Western blotting, a variety of different PCR techniques (e.g. quantitative PCR and/or semi-quantitative PCR), immunoblotting in various formats, ELISA, RIA and/or various different FACS techniques.

In a preferred embodiment, pathogenic Th1 cells are detected in-vitro.

The present invention also provides the use of a substance specifically interacting with at least one gene product encoded by the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495 in a method of in-vitro detection of pathogenic Th1 cells. Examples of such substances are nucleic acid molecules capable of specifically hybridizing to at least one gene product encoded by the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495 Another example are antibodies ore fragments thereof specifically binding to at least one gene product encoded by the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495.

The present invention is also directed to a use of a gene product encoded by the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495 for the diagnosis, prophylactic or therapeutic treatment and/or monitoring of chronic inflammatory diseases.

The use of a kit comprising substances specifically interacting with at least one gene product encoded by the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495 for detection of pathogenic Th1 cells.

In all these uses, the gene product can be e.g. an mRNA molecule or a protein.

In all these uses the Th1 cells can be Th1 memory effector cells.

A method is also provided for screening substances, which reduce pathogenicity of Th1 cells, comprising the steps of: providing a sample of pathogenic Th1 cells, which are capable of differentially expressing a gene product encoded by the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495, in comparison with non-pathogenic T helper cells, dividing the sample into portions, contacting at least one portion with substances to be screened, comparing the expression pattern and/or cell viability in the portion with another portion that is not incubated with the substances, and detecting the specific binding of substances to said gene or a regulatory associated gene or a regulator protein or a gene product thereof, or a component of a signal transduction pathway comprising said gene or a regulatory associated gene or a gene product thereof.

Furthermore a method is provided for in-vitro detecting pathogenic Th1 cells comprising the steps of: contacting a sample of a body fluid or tissue taken from a mammal with specific substances capable of interacting with at least one gene product encoded by the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495, determining specific incubation products, comprising specific substance bound to, associated with and/or interacting with the at least one gene product, correlating an amount of signal or change in signal with a concentration of the gene product in the sample, and detecting cell pathogenicity by comparing the concentration with another gene product concentration in a sample of non-pathogenic and/or pathogenic cells. In this method a number of well known techniques can be applied, e.g. microarray analysis, Nothern blotting, Western blotting, a variety of different PCR techniques (quantitative PCR and/or semi-quantitative PCR), immunoblotting in various formats, ELISA, RIA and/or various different FACS techniques.

The present invention is also directed to a method for treating chronic inflammatory diseases, wherein an effective amount of at least one substance specifically interacting with at least one gene product encoded by the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495 is administered to a mammal in need of such treatment. In the following the invention will be described in more general terms. All definitions and/or explanations given below can also be applied mutatis mutandis to the more specific embodiments of the invention given above.

In more general terms the present invention solves the problem by providing a method for in- vitro detecting pathogenic T helper cells comprising the steps of incubating a sample of a body fluid or tissue taken from a mammal with substances specifically interacting with at least one gene product encoded by a gene that is selected from the group comprising the genes of Table 1 and Table 2, determining specific incubation products, correlating an amount of signal or change in signal with a concentration of the gene product in the sample, and detecting cell pathogenicity by comparing the concentration with another gene product concentration in a sample of non-pathogenic and/or pathogenic cells.

In a preferred embodiment the at least one gene product is encoded by the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495.

It has been surprisingly demonstrated by the inventors that the aforementioned group of 248 genes (in particular the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495) is correlated with pathogenicity of T helper cells. Consequently, the gene products of theses genes represent well suited targets for differentiating the stage of pathogenicity. The term "gene product" denotes molecules that are formed from the substrate of said genes by biochemical, chemical or physical reactions, such as DNA synthesis, transcription, splicing, translation, fragmentation or methylation. Preferred gene products of the invention are mRNA and proteins. The underlying genes are selected as result of a differential expression analysis. In this experimental approach, the transcriptional profiles of naive, once and repeatedly stimulated Th cells are compared using high-density DNA microarrays. The identified genes are not inevitably associated by function or location in their entity as presently known, but it is not excluded that such relations appear between single or more members of the group. Instead of that, all genes are characterized by a distinct difference to normal Th cells, which is exhibited by either up-regulation or repression by at least a factor of 2. The genes are already described in the state of the art by sequence and other features, but lacking a linkage to Th helper cells and the control of inflammation.

The aforementioned genes may be named in another way, but are easily assigned by the accession number, which is generally accepted and fixed in numerous data bases, such as the GenBank, SwissProt and the like.

The linkage of T helper pathogenicity to distinct genes such as the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495 is utilized for the in-vitro detection of harmful Th cells, which promote inflammation, by means of a single marker. However, more than a single marker can be used for the utmost test reliability. That means the inventive principle underlying the present method comprises prospecting for a gene product that can be either detected on the genetic level or on the protein level. The gene product is chosen in respect of both its absolute and relative amount as well as the specificity for a certain T cell type. T helper cells can be distinguished into Th1 cells and Th2 cells, which fulfill different biological functions. Different differential expression pattern are recognized in Th1 and Th2 cells. Furthermore, the gene expression is widely restricted to a subset of memory cells. In an embodiment, the method of the invention relates to Th1 effector memory cells and/or Th2 effector memory cells.

In a preferred embodiment of the present invention, the gene product to be analyzed is encoded by a gene that is selected from the group comprising the genes of Table 1. In a particular preferred embodiment the at least one gene product is encoded by the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495. Said genes and the gene products thereof act as biomarkers for Th1 cells, preferably for Th1 effector memory cells.

In another preferred embodiment of the invention, the gene product to be analyzed is encoded by a gene that is selected from the group comprising the genes of Table 2, which genes and the gene products thereof act as biomarkers for Th2 effector memory cells only.

A sample of a body fluid or tissue is taken from a mammal to be tested. The sample is especially taken from a human or a mouse, preferably a human. The withdrawal of the body fluid or tissue sample follows good medical practice. In the present invention, the sample of body fluid preferably consists of blood, serum, plasma, saliva or urine. It is preferred to gather a tissue sample by biopsy, especially taken close to the location of ailment. The sample may be purified to remove disturbing substances, such as inhibitors for the formation of hydrogen bonds. Alternatively, the nucleic acid or protein material, respectively, can be concentrated in the sample. Downstream processing and/or concentrating are preferably performed by the methods of precipitation, dialysis, gel filtration, gel elution or

chromatography, such as HPLC or ion exchange chromatography. It is recommended to combine several methods for better yields.

The human cell sample is stored, such as frozen, cultivated for a certain period or immediately incubated with substances that are specific for at least one gene product encoded by a gene that is selected from the aforementioned groups. The term "incubation" denotes the contacting of specific substances with the gene products for a distinct period, which depends on the kind of substance and/or target. The incubation procedure can be realized without a chemical conversion or may involve a biochemical reaction. Adding chemical solutions and/or applying physical procedures, e.g. impact of heat, can improve the accessibility of the gene products proteins in the sample. Specific incubation products are formed as result of the incubation. In other words a specific incubation product comprises of a given gene product with its specific substance bound to, associated with or interacting with it.

The term "specific substances" as used herein comprises molecules with high affinity to at least one gene product encoded by the selected genes, in order to ensure a reliable binding. In a preferred embodiment the at least one gene product is encoded by the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495. The skilled person is aware of a number of classes of compounds from which he is able to derive substances specifically interacting with at least one gene product encoded by the selected genes. Examples of such substances or compounds are nucleic acids capable of hybridizing with/to a respective gene product or antibodies. Specificity and affinity of the specific substance for a given gene product strongly depends on the nature of the specific substance and the method used for detection of specific interaction between specific substance and gene product. As described in detail in the further course of the specification, the term "specific substances" also comprise molecules with high affinity to the selected genes themselves, or a regulator protein or a gene product thereof, or a component of a signal transduction pathway comprising said gene or gene products thereof. Consequently, the specific interaction may be limited to the mere targeting, or the induction of alterations in cell function, or may even include both effects simultaneously. It shall be understood that variants, mutants, parts or homologous sequences having the same function, are included in the scope of definition as well as protection. The degree of alteration between the original sequence and its derivatives is inevitably limited by the requirement of gene product recognition within the structural context by means of the specific substances. Preferably, the homology amounts to at least 85 %. Possible alterations comprise deletion, insertion, substitution, modification and addition of at least one nucleotide or amino acid, respectively, or the fusion with another nucleic acid

or protein. Preferably, the substances are mono-specific in order to guarantee an exclusive and directed interaction with the chosen single target.

The recognition of the target according to the invention can be realized by a specific interaction with substances on the primary, secondary and/or tertiary structure level of a nucleic acid sequence bearing the gene sequence or an amino acid sequence expressed by the gene. The coding function of genetic information favors the primary structure recognition, Contrary to that, the three-dimensional structure is mainly to be considered for protein recognition. In the context of the present invention, the term "recognition" - without being limited thereto - relates to any type of interaction between the specific substances and the target, particularly covalent or non-covalent binding or association, such as a covalent bond, hydrophobic/ hydrophilic interactions, van der Waals forces, ion pairs, hydrogen bonds, ligand-receptor interactions, interactions between epitope and antibody binding site, nucleotide base pairing, and the like. Such association may also encompass the presence of other molecules such as peptides, proteins or other nucleotide sequences.

The specific substances are composed of biological and/or chemical structures capable to interact with the target molecule in such a manner that makes a recognition, binding and interaction possible. In particular, the substances are selected from the group of nucleic acids, peptides, carbohydrates, polymers, small molecules having a molecular weight between 50 and 1.000 Da and proteins. The specific substances express a sufficient sensitivity and specificity in order to ensure a reliable detection.

The proteins or peptides are preferably selected from the group consisting of antibodies, cytokines, lipocalins, receptors, lectins, avidins, lipoproteins, glycoproteins, oligopeptides, peptide ligands and peptide hormones. More preferably, antibodies are used as specific substance. "Antibody" denotes a polypeptide essentially encoded by an immunoglobulin gene or fragments thereof. According to the invention, antibodies are present as intact immunoglobulins or a number of well-characterized fragments. Fragments are preferably selected from the group consisting of F _ab fragments, F _c fragments, single chain antibodies (scFv), variable regions, constant regions, H chain (V _H ), and L chain (V _L ), more preferably F _ab fragments and scFv. Fragments, such as F _ab fragments and F ₀ fragments, can be produced by cleavage using various peptidases. Furthermore, fragments can be engineered and recombinant^ expressed, preferably scFv.

The term "nucleic acid" refers to a natural or synthetic polymer of single- or double-stranded DNA or RNA alternatively including synthetic, non-natural or modified nucleotides, which can

be incorporated in DNA or RNA polymers. Each nucleotide consists of a sugar moiety, a phosphate moiety, and either a purine or pyrimidine residue. The nucleic acids are preferably single or double stranded DNA or RNA, primers, antisense oligonucleotides, ribozymes, DNA enzymes, aptamers and/or siRNA, or parts thereof. The nucleic acids can be optionally modified as phosphorothioate DNA, locked nucleic acid (LNA), peptide nucleic acid (PNA) or spiegelmer.

The specific substances can be labeled, in doing so the labeling depends on their inherent features and the detection method to be applied. For the detection of the specific incubation products, the applied methods depend on the specific incubation products to be monitored and are well known to the skilled artisan. Examples of suitable detection methods according to the present invention are fluorescence, luminescence, VIS coloring, radioactive emission, electrochemical processes, magnetism, or mass spectrometry.

Along with the detection, the concentration of the specific incubation products and the concentration of the gene product are determined. The signal intensity of the incubation products is correlated with the signal intensity of a calibration curve enabling the meter- reading of a matching concentration of the incubation product. Preferably, the calibration curve is based on the Lambert-Beer equation if using UVλ/IS coloring or luminescence. The concentration of the gene product is subsequently calculated by considering the molar part of gene product within the product complex. Preferably, the molar ratio of specific substance and gene product is 1 :1 , which is present in antibody complexes for instance, so that the molar concentration of the incubation products corresponds to the molar concentration of the gene product.

Pathogenicity of Th cells is diagnosed by comparing the concentration of the gene product in the sample with known gene product concentration levels of either non-pathogenic cells and/or pathogenic cells. It shall be understood that the known concentrations are statistically proven, therefore representing a certain level or range, respectively. The direction and strength of gene expression have also been figured out by the differential expression analysis of the target genes of the invention such that either a distinct up-regulation or down- regulation with a certain factor has been recognized, which forms the basis of biomarker selection. Any measured concentration, which differs from the gene product concentration level of unstimulated Th cells, indicates an abnormality of the tested cell sample, whereas the cells are healthy at a gene product concentration which is comparable to the concentration level of unstimulated cells. It is preferred to measure concentration, which are higher than the gene product concentration level of unstimulated Th cells, for detecting

pathogenicity. Contrary, a gene product concentration being in the range of the matching concentration of re-stimulated cells indicates a potential pathogenicity of the tested cells, whereas lower gene product concentrations than the aforementioned range confirm the absence Th cell pathogenicity.

Pathogenicity is influenced by the number of stimulating events, which the cells had experienced. Both, unstimulated cells, i.e. without any exposure to an antigen, and cells that are stimulated once by an antigen are regarded as not pathogenic. After a certain number of re-stimulations, a maximum is level is reached and remained stable thereafter. Surprisingly, it has been found that the gene product concentration is clearly correlated to the progress of Th cell pathogenicity. That means each concentration measured can be exactly assigned to a reference magnitude of gene product concentration on the calibration curve, the reference magnitude being in the range of a certain stage of pathogenicity.

It is an embodiment of the present invention that in the case of cell pathogenicity the gene product concentration exceeds at least twice the gene product concentration in a sample of unstimulated cells, preferably at least 15 times, more preferably at least 25 times, most preferably at least 40 times.

It is another embodiment of the present invention that in the case of cell pathogenicity the gene product concentration exceeds at least twice the gene product concentration in a sample of once antigen-stimulated cells, preferably at least 15 times, more preferably at least 25 times, most preferably at least 40 times.

Preferably, a gene is selected, which gene product may form incubation product on the cell surface or in the extracellular surrounding. Although receptor or membrane proteins are preferred, intracellular gene products, such as mRNA and intracellular proteins, can be used as target structure. A preferred encoding gene of the invention is the twisti gene. Twisti and the closely related twist2 (also known as dermol) genes encode basic helix-loop-helix transcription factors involved in formation of mesoderm in Drosophila melanogaster, cranial neural tube and limb morphogenesis in mice, metastasis of tumor cells, control of apoptosis, and expression of cytokine genes in inflammation. Mice deficient for twist2 or haploinsufficient for both twisti and twist2 succumb to severe systemic inflammation, demonstrating the central role and dose-dependency of Twist proteins for regulation of inflammation. Twisti and twist2 are expressed by fibroblasts and macrophages, and expression is promoted by tumor necrosis factor-α (TNF-α) and type I interferons. The basic helix-loop-helix transcriptional repressor twisti , as an antagonist of NF-kB-dependent

cytokine expression, is involved in the regulation of inflammation-induced immunopathology. Induction of twisti in Th cells is dependent on nuclear factor kB (NF-kB), nuclear factor of activated T cells (NFAT), and interleukin 12 (IL-12) signaling via signal transducer and activator of transcription 4 (STAT4), and thus twisti is specifically expressed by activated T helper 1 effector memory cells (Th1 EM cells). Expression of twisti is transient, following T- cell receptor engagement and increments upon repeated stimulation of Th1 cells. Imprinting for enhanced twisti expression marks repeatedly re-stimulated effector memory Th1 cells and thus the pathogenic memory Th cells of chronic inflammation. The cells isolated form the inflamed joint or chronically inflamed gut tissue of patients with ulcerative colitis or Crohn's disease, and synovial fluid of patients with spondyloarthopathies or rheumatoid arthritis are imprinted to express high levels of twisti , indicative of a history of repeated re-stimulation and an involvement in the pathogenesis of the disease by endogenous regulation of proinflammatory effector functions. Twist 1 reduces the functionality of Th1 cells by attenuating expression of interleukin 2 (IL-2), tumor necrosis factor-α (TNF-α), and the cytokines interferon-γ (IFN-γ), and ameliorates Th1-mediated immunopathology in delayed-type hypersensitivity and antigen-induced arthritis.

The method of the invention may comprise a further step, wherein such Th cells identified to be pathogenic are treated to be transferred into a non-pathogenic status, while remaining cell viability, or to be transferred into a status of reduced cell viability or even killed, which comes along with a diminished or abolished harmful impact of pathogenic cells. Thus, the present invention also relates to a method for reducing pathogenicity of T helper cells, by incubating a mammal cell sample comprising pathogenic Th cells with at least one substance being specific to at least one gene that is selected from the group comprising the genes of Table 1 and Table 2, or a regulator protein or at least one gene product thereof, preferably the at least one gene product is encoded by the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495, or a component of a signal transduction pathway comprising said gene or gene products thereof. The specific interaction may comprise both the targeting of gene products to be detected as well as the functional effect. Alternatively, more than one substance can be used to fulfill a different function each. The in-vitro method is preferably applied to samples of mammals suffering from chronic inflammation. Testing of several specific substances makes the selection of that substance possible that is best suited for the treatment of the mammal subject. The in-vivo dose rate of the chosen substance is advantageously pre-adjusted to the pathogenicity of the specific cells with regard to their in- vitro data. Therefore, the therapeutic efficacy is remarkably enhanced. Preferably, a monospecific substance is selected. More preferably, the substance is Twisti , or parts thereof, or the DNA encoding said substance. The ongoing teaching of the present specification

concerning the use of Twisti for the diagnosis, production of a medicament for the prophylactic or therapeutic treatment and/or monitoring is considered as valid and applicable without restrictions to the method for reducing pathogenicity of T helper cells if expedient.

It is another object of the invention to use substances specifically interacting with at least one gene product encoded by a gene that is selected from the group comprising the genes of Table 1 and Table 2 for detecting pathogenic T helper cells in-vitro, preferably the at least one gene product is encoded by the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495. Ligand binding to gene products may be performed by substances selected from the group of nucleic acids, peptides, carbohydrates, polymers, small molecules having a molecular weight between 50 and 1.000 Da and proteins. Preferably, substances are used being specific to a gene product of the twisti gene, such as twist1-mRNA or Twisti protein for the diction of pathogenic T helper cells. The prior teaching of the present specification concerning the method for in-vitro detecting pathogenic T helper cells is considered as valid and applicable without restrictions to the in-vitro use of the substances for pathogenic T helper cell detection if expedient.

Further, the invention may be practiced as a kit comprising substances specifically interacting with at least one gene product encoded by a gene that is selected from the group comprising the genes of Table 1 and Table 2, preferably the at least one gene product is encoded by the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495, particularly in order to perform the inventive method of detecting pathogenic T helper cells. The kit favorably comprises substances being specific to a gene product of the twisti gene or of the Hop gene sequence with the accession No AK 009007, AF536202 or NM 032495. The kit of the invention may include an article that comprises written instructions or directs the user to written instructions for how to practice the method of the invention. In an embodiment, the kit further comprises a reporter moiety or a reporter apparatus, preferably a fluorophore or a field-effect transistor. Additionally, the kit may comprise an extracting reagent for isolating an mRNA or a protein, for example, preferably containing the trancripted or translated twisti gene. The prior teaching of the present specification concerning the detection method is considered as valid and applicable without restrictions to the kit if expedient.

Another object of the invention is a pharmaceutical composition comprising as active ingredient an effective amount of at least one protein encoded by a gene that is selected from the group comprising the genes of Table 1 and Table 2, or parts thereof, or the DNA encoding said ingredient, optionally together with pharmaceutically tolerable adjuvants. In a

preferred embodiment of the present invention, the pharmaceutical composition comprises the Twisti protein. Alternatively, the pharmaceutical composition of the invention may comprise at least one substance specifically interacting with at least one gene that is selected from the group comprising the genes of Table 1 and Table 2, preferably the gene is Hop, or a regulator protein or a gene product thereof, or a component of a signal transduction pathway comprising said genes or gene products thereof, optionally together with pharmaceutically tolerable adjuvants. Which kind of active ingredient is eventually used, i.e. either the protein or the specifically interacting substance, depends on the inherent function of the gene selected for implementing the inventive composition, and can be determined by those skilled in the art. Furthermore, a synergistic effect may be achieved by using more than one protein, or substance, respectively, and the compounds can be used either simultaneously or sequentially.

A "pharmaceutical composition" in the meaning of the invention is any agent in the field of medicine, which can be used in prophylaxis, diagnosis, therapy, follow-up or aftercare of patients who suffer from allergy, autoimmune diseases, gout, aberrance of the immune response, cancer and/or infection diseases in such a way that a pathogenic modification of their overall condition or of the condition of particular regions of the organism could establish at least temporarily.

The terms "effective amount" or "effective dose" or "dose" are interchangeably used herein and denote an amount of a pharmaceutical compound having a prophylactically or therapeutically relevant effect on a disease or pathological conditions. A prophylactic effect prevents the outbreak of a disease or even the infection with a pathogen after the infiltration of single representatives such that the subsequent propagation of the pathogen is strictly diminished, or it is even completely inactivated. A therapeutically relevant effect relieves to some extent one or more symptoms of a disease or returns to normal either partially or completely one or more physiological or biochemical parameters associated with or causative of the disease or pathological conditions. The respective dose or dosage range for administering the pharmaceutical composition according to the invention is sufficiently high in order to achieve the desired prophylactic or therapeutic effect of reducing symptoms of autoimmune diseases. It will be understood that the specific dose level, frequency and period of administration to any particular human will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet time of administration, route of administration, rate of excretion, drug combination and the severity of the specific therapy. Using well-known means and methods, the exact dose can be determined by one of skill in the art as a matter of routine experimentation.

The protein or the substance of the pharmaceutical composition can be conjugated to a chemotherapeutic, and/or fused or complexed with another molecule that promotes the directed transport to the destination, the incorporation into a pathogenic T helper cell, which is capable of differentially expressing said gene, and/or distribution within the pathogenic cells. It is especially preferred that the substance is conjugated to a chemotherapeutic if the substance does not exert any influence on the pathogenic Th cell by several reasons, such as the target is no receptor or regulator protein or component of a signal transduction pathway. While targeting a marker structure of pathogenic T helper cells by means of the substance, the chemotherapeutic moderates cell metabolism such that either differential gene expression is turned back or cell viability is shut down up to killing the cell. As a result of specific interaction on the surface, the construct of substance and chemotherapeutic may be subsequently incorporated into the desired pathogenic Th cells, which are clearly to be distinguished from non-pathogenic T helper cells and other cells types as well. The chemotherapeutic comprises a cytotoxin, a chemokine, a pro-apoptotic, an interferon, a radioactive moiety, or combinations thereof. Preferably, the chemotherapeutic moderates or alters nucleic acid metabolism, protein metabolism, cell division, DNA replication, purine biosynthesis, pyrimidine biosynthesis, amino acid biosynthesis, gene expression, mRNA processing, protein synthesis, apoptosis, or combinations thereof.

In particular, the pharmaceutical composition according to the invention comprises substances that are selected from the group of nucleic acids, peptides, carbohydrates, polymers, small molecules having a molecular weight between 50 and 1.000 Da and proteins. The substances are capable of discriminating between the background level if present and up-regulated concentrations of the gene product. That means, a minimum concentration of the gene product is required for binding and only concentrations exceeding the background threshold are measurable, or the gene product is exclusively expressed in pathogenic Th cells, the latter is preferred herein. The prior teaching of the present specification concerning the specific substances used in the method for in-vitro detecting pathogenic Th cells is considered as valid and applicable without restrictions to pharmaceutical compositions if these substances are expedient therein.

Preferably, small and/or compact nucleic acids are provided, which do not contact other structures within a tissue or organism, respectively, or which are not attacked by the pre- mentioned ones, but specifically interact with the target molecule. The nucleic acids may be part of complexes or formulations consisting of lipids, carbohydrates, proteins or peptides, such as in the shape of a nano-capsule. More preferably, endogenous expression of genes

can be silenced by RNA interference, which is based on dsRNA with 3' overlaps. Following the association of siRNA with specific proteins, a target RNA is recognized by the antisense siRNA strand and degraded by the intrinsic endonucleolytic activity of the ribonucleoprotein complex. The skilled artisan also knows theoretical models for the prediction of accessibility of mRNA regions. It is referred to the documents by Patzel et al. (Nucl. Acids. Res. 27, 4328- 4334, 1999) as well as Scherr et al. (Nucl. Acids Res. 28, 2455-2461 , 2000), which are incorporated as reference in the disclosure of the invention hereby. In doing so, the number of trial-and-error experiments is significantly reduced. Most preferably, cells are transfected with a small hairpin RNA (shRNA) of the sequence 5 -AAGCTGAGCAAGATTCAGACC-3'.

The composition of the invention is produced in a known way using common solid or liquid carriers, diluents and/or additives and usual adjuvants for pharmaceutical engineering and with an appropriate dosage depending on the intended mode of application. These pharmaceutically acceptable excipients comprise salts, buffers, fillers, chelating agents, antioxidants, solvents, bonding agents, lubricants, tablet coatings, flavor additives, flavors, preservatives and suspending agents. In the meaning of the invention, an "adjuvant" denotes every substance that enables, intensifies or modifies a specific immune response against the protein of the invention if administered simultaneously, contemporarily or sequentially. Known adjuvants for injection solutions are for example aluminum compositions, such as aluminum hydroxide or aluminum phosphate, saponins, such as QS21 , muramyldipeptide or muramyltripeptide, proteins, such as gamma-interferon or TNF, M59, squalen or polyols. The amount of excipient material that is combined with the active substance to produce a single dosage form varies depending upon the host treated and the particular mode of administration.

The proteins are adapted in forms which are suitable for oral administration, such as tablets, film tablets, lozenges, capsules, pills, powders, solutions, dispersions, suspensions or depot forms thereof, for transdermal administration, such as solutions, suspensions, creams, ointments, gels, emulsions or band-aids, for parental administration, such as suppositories, and for intravenous infusion, subcutaneous injection or intramuscular administration, examples for the latter three are solutions and suspensions. The substances can also be adapted for topical, transmucosal, transurethal, vaginal, rectal or pulmonary administration in the appropriate formulations given above.

Depending upon the manner of introduction, the compounds may be formulated in a variety of ways. The concentration of therapeutically active ingredients in the formulation may vary from about 0.1 to 100 wt %. The solution may be administered alone or in combination with

other treatments. In a preferred embodiment, the CEACAM proteins to be injected are in a water-soluble form, such as a pharmaceutically acceptable salt, which is meant to include both acid and base addition salts. The injection solution may also include one or more of the following: carrier proteins, such as serum albumin, buffers, stabilizing agents, coloring agents, and the like. Additives are well known in the art, and they are used in a variety of formulations.

Still another object of the invention is a protein, which is encoded by a gene that is selected from the group comprising the genes of Table 1 and Table 2, preferably theprotein is encoded by the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495, for the diagnosis, prophylactic or therapeutic treatment and/or monitoring of chronic inflammation. The protein of the invention also comprises variants, mutants, parts of the proteins or homologous sequences having the same function. A couple of methods are known to the skilled artisan to generate equivalent proteins, i.e. proteins that are analog in function to those of the inventive teaching. Therefore, the invention also contains the aforementioned modifications. For example, mutants can be generated by substitution, deletion, insertion, translocation, inversion and/or addition of at least a single amino acid. It is known that certain amino acids exhibit similar physicochemical characteristics making the substitution among each other possible. Variants of the protein can arise from modifications, such as alkylation, arylation or acetylation of at least a single amino acid, from incorporation of enantiomers and/or from fusion of the protein with a single or multiple amino acids, a peptide or a protein. It is preferred in the meaning of the invention that the protein is fused to a purification tag for affinity chromatography. Parts of the protein relates to a restriction to those regions that are sufficient for the expression of a specific function. All alterations are inevitably limited by the requirement of preserving the function. However, the parts of the protein can be very small due to the characterization of the binding site, which also triggers the signal cascade. In the meaning of the invention, it is to be clearly distinguished between protein parts of any size and homologous sequences which homology is related to the entire protein. Preferably, the homology between a natural protein and a derivative thereof, having the same features amounts to at least 60 %, more preferably 75 %, most preferably 90 %. Similarly, the homology is to be considered if the aforementioned part of any size is altered to a variant or mutant. In addition, several techniques are described in prior art to generate nonhomologous peptides with the same function. The present teaching if solving the problem of the invention covers all peptide derivatives, which are developed on the basis of the present ingredients by such procedures.

The present invention also relates to DNA encoding said proteins of the invention for the diagnosis, prophylactic or therapeutic treatment and/or monitoring of autoimmune diseases. The prior teaching of the present specification concerning the amino acid sequences and derivatives thereof is valid and applicable without restrictions to the underlying DNA.

The protein and DNA of the invention are preferably used for the therapeutic treatment. A therapeutically relevant effect relieves to some extent one or more symptoms of an autoimmune disease, or returns to normality, either partially or completely, one or more physiological or biochemical parameters associated with or causative of the disease or pathological conditions. Monitoring is considered as a kind of treatment provided that the compounds are administered in distinct intervals, e.g. in order to booster the proliferation response and eradicate the symptoms of the disease completely. Either the identical compound or different compounds can be applied. In the meaning of the invention, prophylactic treatment is advisable if the subject possesses any preconditions for the outbreak of an autoimmune disease, such as a familial disposition, a genetic defect, or a previously passed disease.

The autoimmune diseases concerned by the invention are selected from the group comprising arthritis, autoimmune hepatitis, chronic gastritis, neurodermatitis, psoriasis, spondyloarthopathies, arthrosis, rheumatic diseases, rheumatoid arthritis, juvenile idiopathic arthritis, Crohn's disease, ulcerative colitis, diabetes, inflammatory bowel disease, Multiple Sclerosis, Systemic Lupus Erythematosus and/or allergic inflammations. Among the group, the inflammatory diseases and/or the allergic diseases are preferably addressed. Each of these disease subsets is clearly related to a defined type of pathogenic T helper cells. A protein, which is encoded by a gene that is selected from the group comprising the genes of Table 1 , preferably the protein is encoded by the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495, is exclusively used for the diagnosis, prophylactic or therapeutic treatment and/or monitoring of inflammatory diseases, preferably spondyloarthopathies, arthrosis, rheumatic diseases, rheumatoid arthritis, juvenile idiopathic arthritis, Crohn's disease, ulcerative colitis, diabetes, inflammatory bowel disease, Multiple Sclerosis and/or Systemic Lupus Erythematosus. A protein, which is encoded by a gene that is selected from the group comprising the genes of Table 2, is exclusively used for the diagnosis, prophylactic or therapeutic treatment and/or monitoring of allergic diseases, preferably allergic inflammations.

In a preferred embodiment of the invention, the Twisti protein concerns the diagnosis, prophylactic or therapeutic treatment and/or monitoring of inflammatory diseases, preferably

spondyloarthopathies, arthrosis, rheumatic diseases, rheumatoid arthritis, juvenile idiopathic arthritis, Crohn's disease, ulcerative colitis, diabetes, inflammatory bowel disease, Multiple Sclerosis and/or Systemic Lupus Erythematosus. The key role of twist 1 and Twisti , respectively, for the self-limitation of Th1 cells is evident from the analysis of models of inflammation. Ectopic overexpression of twisti in Th1 cells, yet having a low endogenous expression level, drastically reduces their pathogenic contribution to delayed-type hypersensitivity. The fact that Th cells isolated from inflamed tissue of patients with chronic inflammatory gastrointestinal or rheumatic diseases show a dramatic up-regulation of twisti expression, can be utilized by inducing or artificially supplying Twisti , e.g. administering an effective amount of Twisti itself.

The invention also teaches a substance, which specifically interacts with at least one gene that is selected from the group comprising the genes of Table 1 and Table 2, preferably the at least one gene is the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495, or a regulator protein or a gene product thereof, or a component of a signal transduction pathway comprising said gene or gene products thereof, for the diagnosis, prophylactic or therapeutic treatment and/or monitoring of autoimmune diseases. Such substances directed against the selected genes of the invention, derivatives or associates thereof alter the functional effects triggered by them, and their interception modulates the immune response and may eliminate the accumulation of T cells within a center of inflammation. For example, an over-reaction of the immune system in a sick knee, which is full of blood or water due to a trauma or gout, respectively, is diminished or even completely prevented. The teaching of the present specification concerning the aforementioned clinical pictures in the context of using proteins for the diagnosis, prophylactic or therapeutic treatment and/or monitoring of autoimmune diseases is valid and applicable without restrictions to the substances if expedient, wherein the different approach of the compounds is recognized be the skilled artisan.

The invention also relates to the use of proteins encoded by a gene that is selected from the group comprising the genes of Table 1 and Table 2, preferably by the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495, or substances specifically interacting with said genes, or a regulator protein or a gene product thereof, or a component of a signal transduction pathway comprising said genes or gene products thereof, for the diagnosis, production of a medicament for the prophylactic or therapeutic treatment and/or monitoring of autoimmune diseases. The compounds, i.e. proteins or substances, can be either administered to prevent the initiation of autoimmune diseases of am mammal, preferably a human individual, and the resulting trouble in advance, or to treat the arising and

continuing symptoms by tackling the molecular reasons. In an embodiment of the present invention, mono-specific compounds, preferably mono-specific substances, are used for the diagnosis, production of a medicament for the prophylactic or therapeutic treatment and/or monitoring of autoimmune diseases. The term "mono-specific" denotes a mode of binding which is characterized by the exclusive recognition of a single target. The mono-specific substances used in the present invention preferably recognize twisti or any derivative thereof. The receptor/ligand-interaction is characterized by high affinity, high selectivity and minimal or even lacking cross-reactivity to other target molecules to exclude unhealthy and harmful impacts to the treated subject.

It is another object of the invention to provide a method for treating autoimmune diseases, wherein an effective amount of at least one substance specifically interacting with at least one gene that is selected from the group comprising the genes of Table 1 and Table 2, preferably the at least one gene is the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495, or a regulator protein or a gene product thereof, or a component of a signal transduction pathway comprising said genes or gene products thereof, is administered to a mammal in need of such treatment. The prior teaching of the invention and its embodiments is valid and applicable without restrictions to the method of treatment if expedient.

In addition, object of the invention is a method for screening substances, which reduce pathogenicity of T helper cells, comprising the steps of providing a sample of pathogenic T helper cells, which are capable of differentially expressing a gene that is selected from the group comprising the genes of Table 1 and Table 2, in comparison with non-pathogenic T helper cells, dividing the sample into portions, incubating at least one portion with substances to be screened, comparing the expression pattern and/or cell viability in the portion with another portion that is not incubated with the substances, and detecting the specific binding of substances to said genes or regulatory associated genes or regulator proteins or gene products thereof, or a component of a signal transduction pathway comprising said genes or regulatory associated genes or gene products thereof. The basic principles of such a screening method are described in detail in EP 1 780 220 A1 , which is incorporated as reference in the disclosure of the invention hereby.

Briefly, the inventive method makes the identification and analysis of substances possible, which exert an influence on the signal cascade via the selected genes of the invention. The cell sample refers to primary cells or genetically engineered cells. The latter are capable of expressing these genes by transfection with appropriate vectors harboring them or parts

thereof. Preferably, the recombinant cells are of eukaryotic origin. In an embodiment of the screening method, the pathogenic cells are provided by re-stimulating with an antigen, and compared to non-pathogenic cells not being stimulated or less stimulated by an antigen.

The cell sample is divided into multiple portions. At least two portions are provided; one is used for screening while the other one serves as negative control. Preferably, the number of portions for screening exceeds the number of control portions. Usually, numerous portions are subjected to a high-throughput screening.

The substances to be screened in the inventive method are not restricted anyway. In an embodiment of the invention, the substances are selected from the group of nucleic acids, peptides, carbohydrates, polymers, small molecules having a molecular weight between 50 and 1.000 Da, and proteins, preferably antibodies, cytokines and lipocalins. These substances are often available in libraries. It is preferred to incubate a single compound within a distinct portion of the cell sample. However, it is also possible to investigate the cooperative effect of substances by incubating at least two substances within one portion. A further portion of cells is simultaneously incubated in the absence of the substances. The incubation process of cells depends on various parameters, e.g. the cell type and the sensitivity of detection, which optimization follows routine procedures known to those skilled in the art.

The identification of effective substances in the meaning of the invention is indirectly performed by determining the expression patterns, which are altered, preferably moderated, and/or the cell viability, which is moderated, preferably apoptotic. The determination is performed at a specified moment and correlated to the signal strength at the beginning of the experiment and the negative control. Suitable tests are known to those skilled in the art or can be easily designed as a matter of routine.

Among those substances being revealed to reduce pathogenicity of Th cells each or some representatives are selected for further analysis. Preferably, the substances showing the greatest discrepancy to the control are chosen. They are analyzed for specificity to exclude another signal transduction, which is not initiated the binding to the genes of the invention and associated molecules thereof, and additionally tested for such a cross-reactivity in order to prevent adverse reactions or other effects by linked pathways if simultaneous docking to further receptor structures occur. Several methods are known in the field of the art for detecting specific and/or mono-specific binding, such as gel shift experiments, Biacore measurements, X-ray structure analysis, competitive binding studies, and the like. In a

preferred embodiment of the screening method, the mono-specific binding of substances to the target structures of the invention is detected.

In another embodiment of the screening method, at least two subjects of a non-human organism suffering of an autoimmune disease are provided as sample, a subset of them the substances are administered, and the expression pattern and/or cell viability are indirectly compared via the symptoms of the disease in subjects to which substances have been administered and subjects to which no substances have been administered. The non-human organism is preferably a mammal, more preferably species such as mice or rats that may be genetically modified. It is possible to contact mice or rats, for example, with the substance candidates by injection, infusion, oral or rectal intake. It is preferred to incubate a single substance within a distinct portion of the non-human organisms.

In the scope of the present invention, a method for in-vitro detecting pathogenic T helper cells, which applies substances specifically interacting with at least one gene product encoded by a gene that is selected from the group comprising the genes of Table 1 and Table 2, preferably the at least one gene product is encoded by the Hop gene sequence with the accession No. AK 009007, AF536202 or NM 032495, is provided for the first time. The present invention teaches genes that are associated with pathogenicity of Th cells, and implies that the genes are involved in the pathogenic Th cell-induced aggravation of certain autoimmune diseases. Analysis of the differential expressed genes is very suitable for large- scale screening tests. In doing so, individuals are identified with an elevated risk for initiating or continuing autoimmune diseases. The detection method as well as arising diagnostic method of the invention can be performed in a simple and fast manner. In addition, the appropriate kit is cost-efficiently produced. The characterization of 248 genes critically involved in the persistence of Th cells in chronic or allergic inflammations also resulted in the provision of pharmaceutical compositions for the diagnosis, prophylactic or therapeutic treatment and/or monitoring of such human chronic inflammatory diseases. Their use is a promising, novel approach for a broad spectrum of therapies causing a direct and immediate reduction of symptoms. T cells, which trigger the autoimmune impulse, are advantageously influenced as pro-inflammatory imprinting is reversed and anti-inflammatory cytokine gene expression is induced to terminate inflammation. In chronic inflammatory diseases, particularly, twist 1 and the genes controlled by it are qualified as biomarkers for pathogenic effector memory cells. Targeting of twisti -expressing Th cells is highly specific for the pathogenic memory driving chronic inflammation. All proteins and substances are characterized by a high affinity, specificity and stability; low manufacturing costs and convenient handling. These features form the basis for a reproducible action, wherein the

lack of cross-reactivity is included, and for a reliable and safe interaction with their matching target structures.

The present invention is also directed to:

i) A method for in-vitro detecting pathogenic T helper cells comprising the steps of:

- incubating a sample of a body fluid or tissue taken from a mammal with substances specifically interacting with at least one gene product encoded by a gene that is selected from the group comprising the genes of Table 1 and Table 2,

- determining specific incubation products,

- correlating an amount of signal or change in signal with a concentration of the gene product in the sample, and

- detecting cell pathogenicity by comparing the concentration with another gene product concentration in a sample of non-pathogenic and/or pathogenic cells.

ii) A use of substances specifically interacting with at least one gene product encoded by a gene that is selected from the group comprising the genes of Table 1 and Table 2 for detecting pathogenic T helper cells in-vitro.

iii) A kit for use in detection of pathogenic T helper cells comprising substances specifically interacting with at least one gene product encoded by a gene that is selected from the group comprising the genes of Table 1 and Table 2.

iv) A pharmaceutical composition comprising as active ingredient an effective amount of at least one protein encoded by a gene that is selected from the group comprising the genes of Table 1 and Table 2, optionally together with pharmaceutically tolerable adjuvants.

v) A pharmaceutical composition comprising as active ingredient an effective amount of at least one substance specifically interacting with at least one gene that is selected from the group comprising the genes of Table 1 and Table 2, or a regulator protein or a gene product thereof, or a component of a signal transduction pathway comprising said gene or a gene product thereof, optionally together with pharmaceutically tolerable adjuvants.

vi) A protein, which is encoded by a gene that is selected from the group comprising the genes of Table 1 and Table 2, for the diagnosis, prophylactic or therapeutic treatment and/or monitoring of chronic inflammatory diseases.

vii) A protein, which is encoded by a gene that is selected from the group comprising the genes of Table 1 , preferably Twisti , for the diagnosis, prophylactic or therapeutic treatment and/or monitoring of chronic inflammatory diseases, preferably spondyloarthopathies, arthrosis, rheumatic diseases, rheumatoid arthritis, juvenile idiopathic arthritis, Crohn's disease, ulcerative colitis, diabetes, inflammatory bowel disease, Multiple Sclerosis and/or Systemic Lupus Erythematosus.

viii) A protein, which is encoded by a gene that is selected from the group comprising the genes of Table 2, for the diagnosis, prophylactic or therapeutic treatment and/or monitoring of allergic diseases, preferably allergic inflammations.

ix) A substance, which specifically interacts with at least one gene that is selected from the group comprising the genes of Table 1 and Table 2, or a regulator protein or a gene product thereof, or a component of a signal transduction pathway comprising said gene or a gene product thereof, for the diagnosis, prophylactic or therapeutic treatment and/or monitoring of chronic inflammatory diseases.

x) A method for screening substances, which reduce pathogenicity of T helper cells, comprising the steps of:

- providing a sample of pathogenic T helper cells, which are capable of differentially expressing a gene that is selected from the group comprising the genes of Table 1 and Table 2, in comparison with non-pathogenic T helper cells,

- dividing the sample into portions,

- incubating at least one portion with substances to be screened,

- comparing the expression pattern and/or cell viability in the portion with another portion that is not incubated with the substances, and

- detecting the specific binding of substances to said gene or a regulatory associated gene or a regulator protein or a gene product thereof, or a component of a signal transduction pathway comprising said gene or a regulatory associated gene or a gene product thereof.

xi) A method for treating chronic inflammatory diseases, wherein an effective amount of at least one substance specifically interacting with at least one gene that is selected from the group comprising the genes of Table 1 and Table 2, or a regulator protein or a gene product thereof, or a component of a signal transduction pathway comprising said gene or gene products thereof, is administered to a mammal in need of such treatment.

It is to be understood that this invention is not limited to the particular methods, pharmaceutical compositions, kit or uses described herein, as such matter may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is only defined by the appended claims. As used herein, including the appended claims, singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise. Thus, e.g., reference to "a substance" includes one or more different substances and reference to "a method" includes reference to equivalent steps and methods known to a person of ordinary skill in the art, and so forth. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which this invention belongs.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable examples are described below. The following examples are provided by way of illustration and not by way of limitation. Within the examples, standard reagents and buffers that are free from contaminating activities (whenever practical) are used.

Mice and reagents

MRLIpr/lpr, BALB/c, STAT4-deficient mice (Stat4t ^m1Gr ), and OVA-TCR ^t9/t9 D01 1.10 mice were purchased from The Jackson Laboratory or were bred under specific pathogen-free conditions in our animal facility. All animal experiments were performed in accordance with institutional, state, and federal guidelines. Reagents were purchased from Sigma unless stated otherwise. BTP1 was synthesized by M. Paetzel (Humboldt University of Berlin, Germany), Cyclosporin A was purchased from Calbiochem.

Patients

Endoscopic mucosal and surgical mucosal specimens were obtained from ulcerative colitis (UC, n = 9) and Crohn ^' s disease (CD, n = 7) patients. UC and CD were diagnosed according to established clinical, endoscopic, radiologic and pathologic criteria. All UC and CD patients displayed moderately to severely active disease according to the Truelove and Witts Severity Index and the Harvey Bradshaw Severity Index, respectively. Control samples were obtained from patients (n = 4) undergoing colonectomy due to colon cancer. Mucosal control specimens used in the study were from the macroscopically noninvolved tissue distant to any

detectable lesion Synovial fluid was obtained from patients suffering from rheumatic diseases who had active synovitis with effusion Patients with rheumatoid arthritis (RA, n = 4) fulfilled the American College of Rheumatology (ACR) 1987 classification criteria for RA, patients with ankylosing spondylitis (AS, n = 5) fulfilled the modified New York criteria (1984), and patients with psoriatic arthritis (PsA, n=3), reactive arthritis (ReA, n = 3) or undifferentiated spondylarthritis (undiff SpA, n = 2) fulfilled the European Spondyloarthropathy Study Group (ESSG) criteria for SpA Clinical characteristics of the patients are listed in Table S2 All experiments were approved by the local ethics committee, and all patients gave informed consent

Isolation of human lymphocytes

Peripheral blood mononuclear cells (PBMC) from buffy coats from healthy donors and synovial fluid mononuclear cells were isolated by density gradient centrifugation (Lymphocyte separation medium, PAA) Pooled intraepithelial leukocytes (IEL) and lamina propria leukocytes (LPL) were obtained from mucosal specimens by treatment with collagenase type IV, followed by passage through a sieve Mononuclear cells were collected from the 40% - 70% interphase of a discontinuous percoll (Pharmacia) gradient

Magnetic cell sorting

Anti-human CD4, anti-mouse CD4 and CD62L MACS MicroBeads were obtained from

Miltenyi Biotec and were used according to the manufacturer ^' s protocol

Flow cytometry

The following antibodies directed against murine antigens were either purified from hybridoma supernatants and conjugated in house or purchased as indicated antι-CD3 (145- 2C11), antι-CD4 (GK1 5), antι-CD8 (53-6 7), antι-CD11c (N418), antι-CD44 (IM7), anti- CD62L (MEL14), antι-DO11 10 OVA-TCR (KJ1 26), antι-B220 (RA3 6B2), antι-IL2 (JES6- 5H4, Caltag), antι-IL-4 (11 B11 , BD Biosciences), antι-IFN-γ (AN18 17 24), antι-TNF-α (MP6- XT22, Caltag) Antibodies recognizing human antigens were obtained from BD Biosciences unless stated otherwise antι-CD3 (OKT3, in house conjugate), antι-CD4 (TT1 , in house conjugate), antι-CD27 (L128), antι-CD45RA (HH 00), antι-CRTh2 (BM16), antι-CCR5 (2D7/CCR5) Cytometric analysis was performed with FACSCalibur using CellQuest (BD Biosciences) and FCS Express (De Novo) software Cells were separated by fluorescence- activated cell sorting (FACSAria, FACSDiVa, BD Biosciences)

Cell culture

Lymphocytes were cultured in complete RPM11640 (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin and 10 μM β-mercaptoethanol. Naive CD4+CD62L+ lymphocytes from 6-8 w old DO11.10 mice were isolated as described. Irradiated (30 Gy) BALB/c splenocytes were used as APCs at a ratio of 5:1. The cognate peptide ova ₃₂₃ - ₃₃₉ (R. Volkrner-Engert, Humboldt University of Berlin, Germany) was added at 0.5 μM Alternatively, plates were coated with 3 μg/ml anti-CD3 (145-2C1 1) in PBS, and CD4+ cells were plated at a density of 2x106 cells/ml in medium plus 1 μg/ml soluble anti-CD28 (37.51). For Th1 differentiation, cells were stimulated in the presence of 5 ng/ml recombinant murine IL-12 (R&D Systems) and 5 μg/ml anti-IL-4 antibody (11 B11). For Th2 differentiation, cells were stimulated in the presence of murine IL-4 (100 ng/ml, culture supernatant of HEK293T cells transfected with an expression plasmid, encoding murine IL-4), 5 μg/ml anti-IL-12 antibody (C17.8.6) and anti-IFN-γ antibody (AN18.17.24). Dead cells were removed by Ficoll-Histopaque separation. Every 6 d viable Th cells were harvested and restimulated under the original conditions, except that 10 ng/ml murine IL-2 (R&D Systems) was added.

Mitogenic re-stimulation and intracellular cytokine staining

Cells were re-stimulated with 10 ng/ml PMA, and 1 μg/ml ionomycin. Alternatively, cells were re-stimulated with plate-bound anti-CD3 (10μg/ml), and soluble anti-CD28 (1 μg/ml). For re- stimulation of murine T cells, 10ng/ml IL-2 was added. For intracellular staining of cytokines, T cells were stimulated for 2 h with PMA/ionomycin and additional 3 h with 5 μg/ml of brefeldin A to block the secretion of cytokines. Cells were fixed with 2% formaldehyde in PBS for 15 min at room temperature and permeabilized with 0,5% w/v saponin.

Chromatin lmmunoprecipitation Assay (ChIP)

Cells were fixed with 2mM dithiobis (succinimidyl propionate) (DSP) for 30 min at room temperature, washed with PBS and fixed with 1 % formaldehyde for 10 min. The chromatin was sheared to 200-1000 bp of length by sonification with five pulses of 10 s at 30% power (Bandelin, Berlin, Germany). The sheared chromatin was pre-cleared by incubation with Protein A MACS MicroBeads (Miltenyi Biotec) and incubated with anti-NFAT1 (AB1-209, ImmunoGlobe Antikoerpertechnik) or with anti-p65 (C-20, Santa Cruz) for 2 h at 4 ⁰ C followed by incubation with Protein A microbeads for 30 min. Washing steps were performed on μ- columns (Miltenyi Biotec). Washing was performed sequentially with high salt, low salt, LiCI and TE buffer. Chromatin precipitate was eluted with 1% w/v SDS, 0.1 M NaHCO3 and 0,1 % v/v β-Mercaptoethanol. Cross-links were reversed by incubation at 65°C overnight in the presence of 0.2 M NaCI, and the DNA was purified with Nucleospin Extract Il (Macherey- Nagel). The relative amount of DNA was calculated with _E ^δ c ^{p (ιnp} _U ^t - , ^mmunoprec , _P , ^tate) _{The fo} || _owing

primers were used to amplify the proximal twisti promoter: (-150 forward) 5'- GGGCTGGAAAGAGGAAACTT-3'; (+4 reverse) δ'-CGCGAGGTGTCTGAGAGTT-S'.

Retroviral Infection

Retroviral stocks were obtained by calcium phosphate co-transfection of HEK293 cells with the retrovirus packaging plasmids pECO and pCGP. The medium was replaced after 4 h, and viral supernatants were collected 48 h later. Th cells were infected 40 h after activation by 60 min centrifugation at 70Og at 30°C with viral supernatant and 8μg/ml polybrene, followed by 1 h at 37 ⁰ C, and replacement of the viral supernatant with the former culture supernatant.

Th1-mediated DTH model

The ova-specific DTh model was performed as previously described. Briefly, 5x10 ⁵ DO1 1.10 TM cells were injected i.v. into BALB/c mice, and 24 h later the DTh response was induced by s. c. injection of 250 ng ova ₃₂₃ - ₃₃₉ /IFA into the left footpad. PBS/IFA, injected in the right footpad, served as a control. The footpad thickness was measured using an Oditest micrometer gauge (Kroeplin Laengenmesstechnik). The footpad thickness measured before the injection of the antigen was subtracted from the footpad thickness measured during the DTH response.

Antigen-induced arthritis

2x10 ⁶ 2-w-old GFP+ Th1 cells were transferred i.v. into naive SCID mice. 1 d later arthritis was induced by intraarticular injection of 60 μg cationised ovalbumin into one knee joint. The contralateral knee joint was left untreated. 3 weeks after disease induction, mice were sacrificed and knee joints were fixed in 10% formaldehyde, decalcified in saturated EDTA- solution and embedded in paraffin. Knee joint sections were stained with hematoxilin/eosin and scored for exudates, granulocyte infiltration, hyperplasia, fibroblast proliferation/mononuclear cell infiltration, periarticular mononuclear cell infiltration (each scoring 0-3), bone/cartilage destruction (scoring 0-4) and additional score of 1 for visible fibrin deposition and periarticular granulocyte infiltration, resulting in a maximum score of 21.

Luciferase reporter assay

To monitor NF-κB activity in murine Th cells the pNFkB-Luc vector (Clonetech) was used. Experimental variation and transfection efficiency was monitored by cotransfecting pRL-TK (Promega), encoding a renilla luciferase gene, driven by the constitutively active Herpes simplex virus thymidine kinase promoter. Murine T cells were electroporated with the reporter constructs using Mouse T cell kit (AMAXA) and Nucleofector I (AMAXA) according to the

manufacturer's protocol Luciferase activity was quantified with Dual Luciferase Assay Kit (Promega) and a luminometer (Moonlight 3096, BD Biosciences)

In-silico genomic DNA analysis

The genomic sequences for the twisti locus of Mus musculus and Homo sapiens were obtained from LJCSC Genome Bioinformatics (http //genome ucsc edu) To identify putative binding site for DNA binding factors the conserved DNA sequences were submitted to Matinspector at http //www genomatix de/matinspector html

Construction of retroviral expression vectors

The twisti targeting shRNA vector was generated by PCR amplification of a fragment containing the EF1α-promotor and green fluorescent protein (gfp) from pLVTHM, generously provided by D Trono (Ecole Polytechnique Federale de Lausanne, Switzerland) Xbal and EcoRV sites were introduced to replace the corresponding fragment in the retroviral expression plasmid pQCXIX (Clonetech) The sequence targeting twisti mRNA corresponds to the sequence of Twιst-sιRNA3 (5'-AAGCTGAGCAAGATTCAGACC-S') A corresponding scrambled sequence was used as control The DNA oligonucleotides for shRNA expression were annealed, phosphorylated, and subcloned into the Hpai and Xhol sites of the lentiviral shRNA expression plasmid pLL3 7, generously provided by L Van Parijs (Massachusetts Institute of Technology, Cambridge, Massachusetts) The fragment of pLL3 7 containing the murine U6-promotor and the shRNA-encoding sequence was amplified by PCR, introducing an additional 5'-Xhol site and hgated into the Sail site of pQXIX-gfp that is located in the inactivated 3'-LTR of the plasmid For retroviral overexpression the vector GFP-RV(44) was used, kindly provided by K M Murphy (Howard Hughes Medical Institute, St Louis, Missouri) The complete coding sequences of murine T-bet (cDNA generated from Th1- cells), murine twist 1 (IMAGE cDNA clone, AccessionlD BC033434-NCBI), and constitutively active lκBαM, lacking phosphorylation sites (pCMV-lκBαM, Clonetech) were amplified by PCR BgIII and Xhol-compatible restriction sites were introduced by the PCR primers for cloning into the unique sites of the vector upstream of the internal ribosome entry site (IRES)-gfp cassette The accuracy of cloning steps was confirmed by DNA sequencing Sequences of primers and oligonucleotides for shRNA expression are listed below

lmmunoblot

Cells were lysed on ice with 1% w/v SDS, 1OmM EDTA, 5OmM Tπs, pH 8,1 Chromatin was sheared by sonification Equal amounts of protein were resolved by SDS PAGE, then transferred onto polyvinyldifluonde membranes Membranes were blocked with 5% w/v nonfat dry milk and 0,1 % v/v Tween20 in PBS, and probed with a monoclonal Twist-specific

antibody (αTwiMab-1)(45) conjugated in house to digoxygenin (Roche Diagnostics), or anti- tubulin-α (DM1A, Calbiochem), followed by incubation with horseradish peroxidase-coupled anti-digoxigenin FAB-fragments (Roche Diagnostics) or anti-mouse (Santa Cruz) secondary antibodies. Individual bands were visualized with enhanced chemiluminescence (Amersham Biosciences) and the Intelligent Dark Box System LAS-3000 (Fujifilm).

Microarray experiments

Total RNA was extracted using Trizol reagent (Invitrogen). RNA concentration, purity and integrity were assessed with the Agilent 2100 Bioanalyzer, and the RNA 6000 Nano LabChip. Ten micrograms of total RNA was reverse-transcribed, followed by cDNA extraction using a PhaseLock gel (Eppendorf), and precipitation with ethanol and ammonium acetate. Biotinylated cRNA was in vitro transcribed using the MEGAscript high yield transcription kit (Ambion) according to the manufacturer's recommendations. Biotinylated cRNA was fragmented, and the hybridization cocktail was prepared according to Affymetrix protocols (15 μg fragmented biotin-labeled cRNA spiked with Eukaryotic Hybridization control). The Murine Genome U74A version 2, and 430A version 2 GeneChip arrays (Affymetrix) were loaded with the hybridization cocktail, hybridized at 45 oC for 16 h in a rotisserie motor, washed, and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400. Arrays were scanned on a Hewlett-Packard Gene Array Scanner (MGU74Av2 arrays) or on an Affymetrix GeneChip Scanner 3000 (MG430Av2 arrays). Data were analyzed using the Microarray Suite 5.0 software (Affymetrix). Microarrays were globally normalized and scaled to a trimmed mean expression value of 200. Quality checks were performed according to the manufacturer's recommendations. All arrays were compared to each other, and a relational database was generated using Microsoft Access software, including the following parameters: expression heights, call for presence of transcripts, p value for presence or absence of transcripts, Iog2 value of fold change and 95% confidence intervals, call for the significance of differentially expression, and the p value for that call. For each transcript the significance of differential expression between the groups of arrays was calculated using strict Bonferroni corrected Welch t-tests. Significantly differentially expressed genes were filtered according to the following criteria: mean fold change >= 2 or 1 ,5; difference of means >= 200; p-value <= 0,05; and immunoglobulin genes were excluded.

Real-time PCR

Total RNA was prepared using NucleoSpin RNA Il (Macherey-Nagel) or RNeasy kit (Qiagen). Reverse transcription (TaqMan Reverse Transcription Reagent, Applied Biosystems) was performed in a conventional thermocycler (10 min at 25°C, 40 min at 48°C, and 5 min at 95°C) with 500 ng of total RNA and a 1 :1 mixture of oligo(dT) and random hexamer primers. Real-time PCR was performed with the LightCycler instrument using the FastStart DNA Master SYBR Green I kit (Roche Diagnostics). For the normalization of murine and human cDNA the transcripts for the housekeeping genes hypoxanthine guanine phosphoribosyl transferase (HPRT) and ubiquitin ligase H5 (UbcH5) were quantified, respectively. Data were evaluated using Lightcycler software. Relative expression was calculated as follows: E _t ^{δCp target 9ene (reference "} ∞W^ ^P housekeeping gene (reference - sample), where Cp represents the crossing point and E represents the reaction efficiency, determined by serial dilution of DNA. Primer sequences are listed in Table S5.

Figures and Tables

Fig. 1 shows that twisti expression is induced in Th1 but not Th2 cells. (A) CD4 ⁺ CD62L ^hl DO11.10 T lymphocytes were repeatedly stimulated in vitro under Th1 or under Th2- polarizing conditions. Functional polarization of Th1 and Th2 cells, i.e. IFN-γ and IL-4 expression, respectively, was confirmed by intracellular immunofluorescence (Fig. 8). Twisti mRNA in resting cells 6 d after the last stimulation (Th 1 ; circles) or after 3 h of restimulation with anti-CD3/CD28 and IL-2 (Th1 ; squares) or PMA/ionomycin and IL-2 (Th1 ; triangles; Th2; diamonds) was determined by RT PCR and normalized to HPRT. (B) Kinetics of twisti mRNA expression in 1 (open bars) and 4 (filled bars) w-old Th1 cells, after stimulation with anti-CD3/CD28 and IL-2 or IL-2 alone. (C) Twisti protein expression in resting (-) and 5 h PMA/ionomycin restimulated (+) 1 and 4-w-old Th1 and Th2 cells. Control: α-Tubulin immunoblot (bottom). (D) Kinetics of Twisti protein expression in 4-w-old Th1 cells, before and at the indicated time intervals of stimulation with anti-CD3/CD28 and IL-2. Data are representative of two experiments.

Fig. 2 shows that signaling through NFAT and NF-κB is required for induction of twisti expression. (A) Comparison of the genomic sequence of the murine and the human proximal twisti promoter (-150 to -100). The murine sequence is displayed with conserved bases in capital letters. Selected putative DNA-binding motifs are indicated. ISRE (interferon- stimulated response element) (B) Twisti mRNA in 4-w-old Th1 cells restimulated for 3 h with PMA/ionomycin and IL-2 in the presence of serial dilutions of the NF-κB inhibitor PDTC (circles; starting concentration 50 μM), the NFAT inhibitor BTP1 (squares; starting cone. 100 nM), or the NF-κB and NFAT inhibitor CsA (triangles; starting cone. 30 nM). (C) 4-w-old Th1 cells were restimulated in the presence of IL-2 for 3 h with IL-2 alone (unstimulated), 10

ng/ml TNF-α, PMA, or PMA/ionomycin The binding of NFAT1 (D) and the NF-κB subunit p65 (E) to the proximal promoter of twisti was analyzed by chromatin immunoprecipitation (ChiP) 3 to 4-w-old Th1 and Th2 cells either in the resting state (-) or following restimulation with PMA/ionomycin and IL-2 for 1 h (+) were used The immunoprecipitated DNA was quantified by RT PCR using primers specific for the proximal promoter The precipitated DNA was normalized to the amount of input DNA Data are representative of three independent experiments

Fig 3 shows that twisti induction requires IL-12 signaling via STAT4, but not IFN-γ or T-bet Twist 1 mRNA of Th cells activated for 3 h with PMA/ionomycin and IL-2 was quantified by RT PCR (A-D) (A) CD62L ^hl DO1 1 10 Th cells were stimulated for 5 d under Th1-polarιzιng conditions (5 ng/ml IL-12), reduced IL-12 (1/25, 200pg/ml IL-12, 1/625, 8pg/ml IL-12), in the absence of IL-12 (antι-IL-12, antι-IL4), in the presence of IFN-γ (10 ng/ml IFN-γ, antι-IL-12, antι-IL-4), or under Th2-polarιzιng conditions The amount of twisti transcripts induced under Th1-polaπzιng conditions was set to 1 Data are presented as average ± s d of at least three experiments (B) CD4 ⁺ cells of STAT4 ' and syngenic BALB/c mice were stimulated with anti- CD3/CD28, and irradiated BALB/c APCs under Th1 (IL-12 and IFN-γ), or under Th2- polaπzing conditions for 5 d Data represent the average ± s d of four individual mice each (C) Naive D011 10 Th cells were stimulated twice under Th 1 -polarizing conditions, or in the absence of IL-12 On d 2 the cells were infected with control virus, or a virus encoding t-bet Infected cells were isolated on d 12 according to expression of the viral marker gene gfp Data are representative of two experiments (D) Naive DO11 10 Th cells were stimulated for 5 d with splenic APCs and ova _{327 339} under Th1-polaπzιng conditions Cells were restimulated under the same conditions (Th1), or in the presence of antι-IL12 Twisti transcripts were quantified on d 11 The amount of twisti mRNA on d 5 was set to 1 Data represent average ± s.d of three experiments

Fig 4 shows that ex-vivo isolated memory Th cells express twisti Cells were sorted by flow cytometry and restimulated 3 h with PMA/ionomycin (A-C) (A) Cells were isolated from the spleen and lymph nodes of 8-12-w-old D011 10 mice (squares, each representing a pool of 15 individual mice) and the pooied inguinal and mesenteric lymph nodes of 4-6-month old nephritic MRL/lpr mice (circles, 1-2 mice each) Of note 90% of the CD4 ⁺ CD62L ^hl cells in MRL/lpr mice represented activated (CD44 ⁺ ) cells (data not shown) (B) Twisti mRNA in peripheral human Th lymphocytes The mean expression of twisti mRNA normalized to ubiquitin ligase H5 in total CD3 ⁺ CD4 ⁺ cells was set to 1 Subpopulations were defined according to expression of the following surface markers Naive (CD4 ⁺ CD45RA ⁺ CCR7 ⁺ ), central memory (CM, CD4 ⁺ CD45RA CCR7 ⁺ ), effector memory (EM, CD4 ⁺ CD45RA CCR7 )

with each data point representing one individual healthy donor. (C) Twisti transcripts in CD3 ⁺ CD4 ⁺ cells purified from patient material: Blood (total peripheral CD3 ⁺ CD4 ⁺ cells from healthy donors, see above), colon (non-inflamed colon tissue), UC and CD (endoscopic biopsies from ulcerative colitis and Crohn's disease patients, respectively), RA, ReA, PsA, and AS (synovial fluid from rheumatoid arthritis, reactive arthritis, psoriatic arthritis, and ankylosing spondylitis patients, respectively) with each dot representing one individual patient. Mean twisti mRNA expression is displayed from patients who were repeatedly sampled.

Fig. 5 shows that Twisti modulates Th1-effector functions. (A) DO1 1.10 Th cells were stimulated with ova ₃₂ 7-339 and APC for 5 d. On d 2, cells were infected with control virus (open bars) or twisti -encoding virus (filled bars). On d 6, the resting cells were re-stimulated and stained for intracellular cytokine expression. Frequencies of cytokine expressing cells among infected, i.e. GFP ⁺ CD4 ⁺ T cells relative to the non-infected CD4 ⁺ cells are displayed. Data represent the mean ± s.d. of four independent experiments. (B) DO1 1.10 Th cells were stimulated with ova ₃₂ 7-339, APCs, and 1 ng/ml IL-12. On d 2 cells were infected with control virus, twisti , or 1-κBαM-encoding virus. On d 3, cells were nucleoporated with a mixture of a plasmid encoding renilla luciferase, and a firefly luciferase reporter construct, driven by a NF- κB-responsive promoter (4xκB-luc). Cells were then re-stimulated with PMA/ionomycin for 6 h, sorted according to expression of the viral marker gene gfp, and luciferase signals were quantified in duplicates (mean ± s.d.).

Fig. 6 shows that ectopic twisti over-expression controls DTH. (A) Naive DO11.10 Th cells were stimulated with anti-CD3/CD28 under Th1 -polarizing conditions. On d 2, cells were infected with control virus (circles) or twisti -encoding virus (squares). On d 6, infected GFP+ were injected i.v. into BALB/c mice. The DTH response was induced by s.c ova ₃₂₇ - ₃₃ g/IFA injection into the left footpad (filled symbols), and δ footpad thickness (mean ± s.d.; n = 6) was determined thereafter. Injection of PBS/IFA served as control (open symbols). (B) IFN-γ mRNA expression in transferred GFP ⁺ Th1 cells 24 h after DTH induction isolated from the draining popliteal lymph node (left foot).

Fig. 7 shows that twisti knock-down increases inflammatory response in murine arthritis. (A) Experimental scheme. (B) Twisti mRNA in 3-w-old Th1 expressing twisti -targeting shRNA or control shRNA re-stimulated with PMA/ionomycin. (C) Transfer of Th1 cells expressing a twisti -targeting shRNA leads to a significantly higher histological score in murine arthritis compared to control Th1 cells (d 21) Data are representative of two experiments. (D) Representative hematoxilin/eosin staining of knee joint sections (d21).

Fig 8 shows representative cytokine profiles of ex vivo-polaπzed TH1 and TH2 cells Naive CD4 ⁺ CD62L ^hl DO11 10 T _H cells cultured for 6 d under T _H 1 or T _H 2-polarιzιng conditions were re-stimulated with PMA/ionomycin and brefeldin A for 5 h, fixed, and stained for intracellular IL-4 and IFN-γ For cytometric analysis, cells were gated for expression of CD4 Numbers in quadrants indicate percentages of gated cells in each

Fig 9 shows that ectopic twisti overexpression attenuates cytokine expression in Th cells Representative histograms of cytokine expression in T _H cells ectopically expressing twisti (black line) and control cells (gray filled) The cells displayed were gated for expression of CD4 and the viral marker gene gfp CD4 ⁺ DO11 10 lymphocytes were stimulated with ova _{327 339} and APC for 6 d (Neutral) or under T _H 1-polaπzιng conditions (T _H 1) On d 2 cells were infected with control virus or twisti -encoding virus On d 6 cells were re-stimulated with PMA/ionomycin in the presence of brefeldin A for 5 h and stained for intracellular cytokine expression

Fig 10 shows the relative expression level of hop in Th cells activated once (1w) or 4 times (4w) under Th1 or Th2 polarizing conditions "res" indicates 3h restimulation with PMA and ionomycin prior to analysis

Fig 11 shows the relative expression level of hop in naive Th cells were stimulated under neutral conditions and retrovirally transduced with either an empty vector (E) or with a T-bet containing vector (T-bet) Green fluorescent protein (GFP) was used as a marker to identify transduced cells Both GFP+ and GFP- cells from the same culture were analysed

Fig 12 shows the effect of Hop expression inhibition in a histological analysis of the intestine in a transfer colitis model

Fig 13 shows relative Hop mRNA expression in various subsets of Th cells isolated from human PBMC CM central memory Th cells, EM effector memory Th cells

Table 1 lists genes differentially expressed in pathogenic Th1 cells

Table 2 lists genes differentially expressed in pathogenic Th2 cells

Table 3 lists genes differentially expressed upon ectopic twisti overexpression Splenic DO11 10 cells were activated in vitro with the cognate peptide ova _{327 339} in the presence of 1 ng/ml IL-12 and 1ng/ml IL-2 On d 2 cells were infected with control retrovirus, or twisti- encoding virus On d 5 cells were sorted according to expression of the viral marker gene gfp Cells were restimulated with PMA/ionomycin The transcriptional profiles of duplicates of cultures were compared

Table S1 lists genes differentially expressed in once versus four times stimulated T _H 1 cells Naive DO11 10 cells were activated under T _H 1 -polarizing conditions The transcriptional profiles of resting one-week-old T _H 1 (T _H 1 1w) cells and resting four-week-old T _H 1 (T _H 1 4w) cells were compared using Affymetrix Murine Genome (MG) U74Av2 GeneChip arrays The Affymetπx probe set ID (Affymetr_No), mean fold change, significance of differential expression (t-test), and mean signal intensity (T _H 1 1w, T _H 1 4w) of three arrays per group representing three independent cultures are shown Genes were filtered according to the following criteria fold change >= 2, difference of means >= 200, p-value <= 0,05

Table S2 lists clinical characteristics of patients in the study Erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) Twist 1 transcripts in CD3 ⁺ CD4 ⁺ cells purified from patient material were quantified after 3 h of re-stimulation with PMA/ionomycin The mean expression of twisti mRNA in total peripheral CD3 ⁺ CD4 ⁺ cells from healthy donors was set to 1

Table S3 lists genes differentially expressed upon ectopic twisti overexpression Splenic DO1 1 10 cells were activated in vitro with the cognate peptide OVa _{327 339} in the presence of 1 ng/ml IL-12 and 1ng/ml IL-2 On d 2 cells were infected with control virus, or twist 1- encoding virus On d 5 cells were sorted according to expression of the viral marker gene gfp Cells were re-stimulated for 4 h with PMA/ionomycin The transcriptional profiles of duplicates of cultures were compared using Affymetrix Murine Genome (MG) 430A 2 0 GeneChip arrays The Affymetrix probe set ID (Affymetr_No), mean fold change, significance of differential expression (t-test), and mean signal intensity (Vector, Twisti) of two arrays per group are shown Genes were filtered according to the following criteria fold change >= 1 ,5, difference of means >= 200, p-value <= 0,05, and excluding immunoglobulin genes

Table S4 shows that twist 1 knock-down results in a higher inflammatory response in murine arthritis After ι v cell transfer of two-week-old GFP ⁺ DO11 10 T _H 1 cells expressing twisti- targeting shRNA (Twιst1-5) or control shRNA (scrT1-5), arthritis was induced in the recipient SCID mice by intraarticular injection of cationized ovalbumin into the knee joint 21 days

later, knee joint sections were stained for hematoxilin/ eosin and scored for exudates, granulocyte infiltration (gran. inf. SM), hyperplasia (hyperpl.), fibroblast proliferation/ mononuclear cell infiltration (mono. inf. SM), periarticular mononuclear cell infiltration (peri, mino.) (each scoring 0-3), bone/cartilage destruction (scoring 0-4) and additional score of 1 for fibrin deposition and periarticular granulocyte infiltration (periart. gran), resulting in a maximum score of 21.

Table S5 lists primer sequences for real-time PCR.

Example 1 : Twisti is transiently expressed in repeatedly activated Th1 cells To define transcriptional changes during Th1 memory cell differentiation we compared the global gene expression of murine Th1 cells, activated once or on a weekly basis repeatedly with antigen. Naive, CD4 ⁺ CD62L ^hl T lymphocytes, expressing the transgenic DO11.10 TCR specific for ovalbumin were activated in vitro with splenic APCs and the cognate peptide ova ₃₂₇ - ₃₃ g under conditions that induce functional differentiation into Th1 cells, i.e. addition of IL-12 and blocking antibodies specific for IL-4. The transcriptional profiles of once and four times stimulated Th1 cells were compared using high-density DNA microarrays. Among the 17 genes differentially expressed by a factor of two or more was twist! Expression of twisti was up-regulated 38-fold in four times versus once stimulated Th1 cells (Table S1).

Twisti expression in Th1 , but not Th2 cells was confirmed by real-time PCR and immunoblot analysis (Fig. 1). Twisti mRNA expression in resting Th1 cells correlated with their age in vitro and the number of re-stimulations they had experienced. Expression was further enhanced three hours after polyclonal stimulation either with PMA and the Ca ²⁺ ionophore ionomycin, or with CD3 and CD28-specific antibodies (Fig. 1 A). When determined three hours after re-stimulation with anti-CD3/CD28 antibodies, expression of twisti was close to the detection limit in naϊve Th cells. Expression increased about 15-fold during the first Th1- polarizing stimulation, another 10-fold during the second stimulation, then another 3-fold during the third stimulation, reaching the maximum level after the fourth stimulation, and remained stable thereafter. With PMA/ionomycin re-stimulation, maximum levels of twisti mRNA were reached already after two rounds of stimulation. In Th1 cells, upon anti- CD3/CD28 re-stimulation, twisti mRNA expression was up-regulated within the first hour, reaching maximum levels after three hours, and decreasing again thereafter (Fig. 1 B). Expression of twisti mRNA was also detectable in re-stimulated Th2 cells, but its level remained 30-fold lower than in Th1 cells. Twisti protein was detectable in 6-day old, re- stimulated Th1 cells. In 4-week old Th1 cells, expression was enhanced. In resting Th1 cells and in reactivated or resting Th2 cells, Twisti was not detectable by immunoblotting (Fig. 1

C) in accordance with expression of twisti mRNA, Twisti protein expression peaked already three hours after reactivation, then ceased, with detectable levels still expressed 48 hours after re-stimulation (Fig 1 D) but no longer six days after re-stimulation Twιst2 was not expressed in the Th1 and Th2 cells analyzed here, as determined by real-time PCR

Example 2: Control of twist 1 expression in Th1 lymphocytes

Phylogenetic comparison of the proximal promoter of twisti from man and mouse (Fig 2 A) revealed conserved sequence motifs which qualify as binding sites for the transcription factors NFAT, STAT, and NF-κB While NFAT and NF-κB could transmit TCR signals to the promoter, the STAT-binding site could be a target of Th1-polaπzιng signals, i e IL-12 and/or IFN-Y With respect to control of twisti expression by TCR signaling in Th1 cells, NF-κB and NFAT were identified as the relevant transcription factors by specific inhibition and chromatin immunoprecipitation (ChIP) The NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC), the NFAT-specific inhibitory 3,5-bιstπfluoromethyl pyrazole derivative BTP1 , and the calcineunn- inhibitor cyclosporine A (CsA), blocking both NFAT and NF-κB, all blocked the PMA/ιonomycιn mediated up-regulation of twisti mRNA expression in a dose-dependent fashion (Fig 2 B) This shows that both NFAT and NF-κB are required for induction of twisti expression in Th1 cells, a result that was confirmed by observing that stimulation with either TNF-α or PMA alone, both activating NF-κB but not NFAT ₁ did not induce twisti expression, which in addition required the Ca ²⁺ ionophore ionomycin, activating NFAT via calcineuπn (Fig 2 C) The requirement of NFAT for induction of twisti expression in Th1 cells distinguishes control of twisti expression in Th cells from its control in fibroblasts, where TNF-α-ιnduced activation of NF-κB is sufficient to induce expression

By ChIP, the specific binding of NFAT1 and the transactivating NF-κB subunit p65 to the twisti promoter of repeatedly stimulated Th1 cells was evident one hour after reactivation (Fig 2 D-E) In Th2 cells, no activation-induced binding of NFAT1 or NF-κB to the twisti promoter was detectable, emphasizing that NFAT1 and NF-κB are required, but not sufficient to induce transcription of twisti in Th cells

The specific expression of twisti by Th1 cells raised the question which of the Th1 -polarizing signals is the specific inductive signal in these cells, cooperating with NFAT1 and NF-κB IL- 12, the primordial signal for Th1 differentiation induced twisti expression in a dose- dependent fashion (Fig 3 A) This effect is direct, and not indirect through induction of IFN-γ and STAT1 signaling, since addition of IFN-γ in the absence of IL-12 did not induce twisti expression IL-12 induces twisti expression via STAT4 When CD4 ⁺ T lymphocytes of STAT4-defιcιent mice were stimulated in the presence of IL-12 and IFN-γ, no induction of

twisti expression was detectable (Fig. 3 B). Neither IFN-γ and its signal transducer STAT1 , nor T-bet, a T box transcription factor sufficient to induce Th1 differentiation are involved in expression control of twisti . Ectopically expressed T-bet did not induce twisti expression in the absence of IL-12 (Fig. 3 C). IL-12/STAT4 is primarily required for the initial induction of twisti expression upon Th1 polarization to imprint the gene for re-expression. Th1 cells polarized for one week with IL-12, and reactivated for another week in the absence of IL-12, showed a 12-fold increased TCR activation-dependent expression of twisti over one week stimulated Th1 cells, as compared to a 16-fold increase in the presence of IL-12 (Fig. 3 D).

Example 3: Th cell-specific twisti expression in-vivo

Expression of twisti mRNA was low in PMA/ionomycin re-stimulated CD4+CD62Lhi naϊve and CD4 ⁺ CD62L'° memory cells isolated from the spleen and lymph nodes of healthy DO1 1.10 mice, kept under specific pathogen-free conditions (Fig. 4 A). Weak twisti expression was also detected in PMA/ionomycin reactivated CD4 ⁺ T cells from peripheral blood of healthy donors (Fig. 4 B). As compared to the reference expression of twisti in total peripheral human Th lymphocytes, twisti transcripts were enhanced 3-fold in effector memory (EM, i.e. CD45RAOCR7 ^" ) Th cells, and 8-fold in 'terminally' differentiated CD27 ^" EM Th cells. Naϊve (CD45RA ⁺ CCR7 ⁺ ) and central memory (CM, i.e. CD45RAOCR7 ⁺ ) Th lymphocytes had a lower expression level compared to unseparated Th cells. In contrast to CCR5 ⁺ Th1 EM, CRTh2 ⁺ Th2 EM cells showed no significant expression of twist! Thus, the phenotype of human peripheral twisti expressing Th cells is that of repeatedly re-stimulated Th1 EM cells.

Since twisti expression increases in Th1 EM cells with the number of re-stimulations, we analyzed twisti expression of Th cells in a murine model of chronic inflammation with a proposed involvement of Th1 cells. In CD4 ⁺ CD62L ^l0 memory Th cells of lymph nodes of 4-6 month old nephritic lupus-prone MRL/lpr mice twisti mRNA was up-regulated about 3-fold, as compared to CD4 ⁺ CD62L ^|0 Th cells of DO11.10 spleen (Fig. 4 A). Twist 1 expression was even further enhanced in CD3 ⁺ B220 ⁺ T cells from MRL/lpr mice, which presumably represent chronically activated Th lymphocytes.

Th cells isolated from inflamed tissue of patients with various chronic inflammatory diseases showed high PMA/ionomycin-inducible twisti expression. CD3 ⁺ CD4 ⁺ Th cells were isolated from inflamed tissues of patients suffering from inflammatory bowel disease or rheumatic diseases, re-stimulated, and analyzed for twisti expression (Fig. 4 C, Table S2). Th cells isolated from non-inflamed surgical colon specimens of control patients did not show enhanced twisti expression, as compared to expression in peripheral Th cells. Although

highly variable, twisti transcripts were increased up to 400-fold in Th cells isolated from the synovial fluid of inflamed joints of patients with rheumatoid arthritis or spondyloarthropathies, and in Th cells isolated from mucosa! endoscopic biopsies and surgical specimens of patients suffering from Crohn's disease or ulcerative colitis. Twisti mRNA expression in patients with persistent inflammation of colon or synovia, who were repeatedly sampled, remained in the same range over up to 18 months (Table S2). Among T cells isolated from the inflamed tissue, only CD4+ Th cells showed enhanced twisti expression. CD3 ⁺ CD4 ^" cells, i.e. cytotoxic (Tc) lymphocytes, expressed twisti transcript levels lower than the reference value of total peripheral Th cells.

Example 4: Functional modulation of Th1 cells by twisti

The impact of twisti on the function of Th1 EM cells was analyzed by ectopic overexpression of twisti in murine DO11.10 Th cells. A global view on twisti -induced modulation of gene expression in Th1 cells is provided in Table 3. Of the 14.000 genes analyzed for transcription, 58 were differentially expressed by a factor of 1.5 or more, between activated Th1 cells expressing twisti ectopically or not. These genes fall into 4 groups, with respect to their presumptive function. The first group comprises 17 genes which are involved in cell activation and apoptosis, 11 genes are involved in cell adhesion and motility, 13 genes relate to the chemokine/cytokine repertoire of Th1 cells, and 17 genes are of metabolic or undefined relevance.

With respect to activation and proliferation, ectopic twisti primarily induced genes that have been reported to inhibit T cell receptor signaling (programmed cell death 4 (pdcd4) and tescalin) and the anti-proliferative cellular repressor of E1A-stimulated genes (cregi) whose product is secreted. Twisti inhibited expression of genes promoting cell activation and cytokine expression in T cells: growth factor independent 1(gfi1), SLAM family member 6 (slamfδ, also known as Ly-108), membrane-spanning 4-domains A, 4B (ms4a4b), proinflammatory cyclic AMP (cAMP)-specific phosphodiesterase 4b (pde4b), the regulator of G- protein signaling 2 (rgs2), and lymphocyte-activation gene 3 (Iag3), a gene blocking memory cell formation. Twisti negatively regulated transformation related protein 53 inducible nuclear protein 1 (trp53inp1 , also known as stress-induced protein) a gene reported to enhance p53- induced apoptosis, and caspase 6, involved in execution of apoptosis.

Twisti modulation of expression of genes involved in motility and adhesion of Th1 cells presumably results in immobilizing the Th1 cells in inflamed tissue. Ectopically expressed twisti reduced transcript levels of matrix metalloproteinase 13 (also known as collagenase-

1), a disintegrin and metallopeptidase domain 8 (adamδ), and the collagen-binding receptor discoidin domain 1 (ddr1), genes favoring tissue destruction.

Ectopic twisti expression also regulated genes determining effector functions of Th1 cells. Twisti reduced expression of the chemokine (C-X-C motif) receptor 6 (cxcrθ), and of the Th1-related pro-inflammatory chemokine genes ccl3, ccl4, and xcl1 (also known as MIP-1α, MIP-1β, and lymphotactin, respectively), but enhanced expression of ccl5 (RANTES). Twisti impaired cytokine signaling through enhanced expression of the decoy IL-1 receptor, type II, the suppressor of cytokine signaling (socs) 1 and 2, and repression of the IL-12 signal transmitter janus kinase 2 Gak2), the latter qualifying twisti as part of a negative feedback loop with respect to IL-12 signaling. Twisti also attenuated expression of the Th1 effector cytokine genes il-2, ifn-γ and tnf-α by a factor of up to 1.6 (Table 3). This moderate reduction of mRNA levels had a drastic effect on protein expression. Ectopic twisti expression reduced the frequencies of cells expressing TNF-α, IFN-γ, or IL-2 to 40-50% of the controls (Fig. 5 A, and Figure 9). The cytokines IL-4 and IL-10 were only marginally expressed by the Th cells analyzed, whether they expressed twisti or not. Twisti in Th1 cells thus acts as an endogenous regulator limiting the pro-inflammatory potential of Th1 cells in face of continuous presence of antigen, i.e. repeated activation of NFAT and NF-κB.

The molecular basis of the regulation of NF-κB-dependent cytokine genes by twisti in TM cells could be binding of Twisti to regulatory elements of target genes, or direct inhibition of NF-κB. In macrophages, inhibition of TNF-α expression has been shown to require the integrity of E-boxes, the DNA-binding motif of Twist proteins, which are located within the TNF-α promoter, while in COS cells, ectopically expressed Twist proteins directly interacted with the p65 subunit of NF-κB, and inhibited its function. In activated primary murine Th cells a constitutively active inhibitor of NF-κB (1-κBαM) but not ectopically expressed twisti or was able to repress activation-induced transcription of an NF-κB-reporter construct lacking E- boxes (Fig. 5 B), while the expression of endogenous cytokine genes was attenuated to the same degree. This result strongly suggests that twisti directly regulates gene expression of Th1 cells by binding to E-boxes. Functional NF-κB signaling for the generation and survival of Th memory cells is not inhibited by twisti .

Example 5: Twisti regulates Th1 mediated inflammation

In a murine transfer model of ovalbumin-specific delayed-type hypersensitivity the effect of twisti expression on the inflammation induced by transferred Th1 cells was analyzed. Ovalbumin-specific TM cells, ectopically overexpressing twisti or not, were transferred intravenously into naive BALB/c mice. After 1 day, ovalbumin/IFA was injected into one

footpad, and swelling of this footpad was measured thereafter. Th1 cells overexpressing twisti showed a significantly reduced induction of footpad swelling, starting from day 2, when compared to control Th1 cells (Fig. 6 A). Transferred Th cells overexpressing twisti , when re-isolated from the draining lymph nodes of the host, expressed 4-fold reduced levels of IFN-γ mRNA as compared to the control cells (Fig. 6 B).

Autoregulation of Th1-mediated inflammation by twisti was also analyzed in a murine model of antigen-induced arthritis (Fig. 7 A) ₁ in which endogenous expression of twisti in Th cells was silenced by RNA interference. Murine DO11.10 TM cells were infected with a retrovirus encoding a small hairpin RNA (shRNA) targeting twisti or a corresponding scrambled control shRNA. Twisti -specific shRNA reduced the level of activation-induced endogenous twisti transcripts in Th1 cells to approximately 30% of the control value (Fig. 7 B). Two-week-old TM cells expressing shRNAs were intravenously injected into SCID mice. One day after cell transfer, arthritis was induced by injection of cationized ovalbumin into the knee joint and histological analysis was performed 3 weeks after induction of arthritis, i.e. in the chronic phase of inflammation. Twisti knockdown in TM cells resulted in a significantly higher histological score of inflammation and tissue destruction (Fig. 7 C). In particular, infiltration of granulocytes and monocytes into the inflamed tissue of the knee joint was drastically enhanced (Fig. 7 D and Table S4).

Example 6: Differential expression of Hop in Th cells

We used an T helper (Th) cell culture system to in vitro model acutely and chronically activated T cells. In our system, we make use of mice transgenic for a T cell receptor (TCR) recognizing the ova ₃₂₃ - ₃₃₉ peptide. Through addition of the cytokines IL-12 or IL-4, the cells can be differentiated to either TM or Th2 lineage. T cells which are stimulated only once mimic Th cells as they occur during an infection providing protection, whereas T cells which we have stimulated weekly for 4 times mimic T cells as they occur in chronic inflammatory disorders, where pathogenic T cells are constantly activated by antigen. In an analysis of the transcriptional profile of once and repeatedly activated TM and Th2 cells we identified Hop to be specifically upregulated in 4 times restimulated TM cells (Fig. 10). In Th2 cells Hop was not expressed above the level found in naϊve Th cells. Fig. 1 also shows that hop is downregulated immediately after TCR signal triggering by PMA/ionomycin.

Example 7: Regulation of hop expression in Th cells by transcription factor T-bet

Since hop was specifically upregulated in TM cells, we analysed whether transcription factors specific for Th1 differentiation regulate the expression of hop T-bet is considered the lineage determining transcription factor for TM differentiation Ectopic overexpression of T- bet with a retroviral vector in Th cells cultured under neutral conditions (ThO) lead to the induction of Hop No Hop was induced when an empty control vector was used (Fig 11) In addition, no Hop induction was seen, when T-bet deficient Th cells were polarized towards TM (data not shown) Since IL-12 is a strong TM inducing signal we also analysed Hop induction in IL-12 receptor deficient cells Hop induction was normal in cells lacking the IL-12 receptor (data not shown), providing strong evidence that T-bet is indeed controlling Hop expression

[Example 8: Functional role of Hop

To assess the functional role of hop we used a T cell dependent model of inflammatory bowel disease In this model, T cells are transferred into Rag-deficient or SCID mice lacking T and B cells In this model, the transferred T cells react against the intestinal bacterial flora leading to inflammation in the intestine For the analysis of the function of Hop we inhibited the expression of Hop by shRNA-mediated knock-down Polyclonally activated TM cells transduced with a retroviral vector encoding a Hop-specific shRNA or a control shRNA were transferred into Rag-deficient mice Fig 12 shows the histological analysis of the intestine from the mice having either received no cells, control shRNA or Hop-specific shRNA transduced Th1 cells The transfer of control TM cells lead to severe inflammation of the intestine with strong infiltration of inflammatory cells and a complete loss of the intestinal architecture Knock-down of Hop resulted in strongly reduced clinical signs of inflammation with very little to no cellular infiltrate and maintenance of the intestinal architecture (Fig 12A) The histology is summarized in Fig 12B

[Example 9: Hop expression in different human Th cell subsets

We next analysed subsets of human Th cells isolated from peripheral blood for the expression of Hop High Hop expression can clearly be detected in effector memory Th cells (Fig 13) Among the effector memory cells Hop is particularly upregulated among the CD27- terminally differentiated effector memory cells When using the chemokine receptor CCR5 as a marker to identify Th 1 -like effector memory cells, high Hop expression was detected Virtually no Hop expression was detected when analysing Th2-lιke CRTH2 ⁺ effector memory cells or CD25 ^h 'CD127- regulatory T cells

Table 1

Gene name Accession No. hydroxyacyl-Coenzyme A dehydrogenase type Il NM_016763

2'-5' oligoadenylate synthetase 3 AB067534 a disintegπn and metalloprotease domain 8 NM_007403 activating transcription factor 3 BC019946 adipose differentiation related protein NM_007408 annexin A1 NM_010730

B-cell leukemia/lymphoma 2 related protein A1a L 16462 beta-site APP-cleaving enzyme 2 BB348062 bone marrow stromal cell antigen 1 AI647987 cathepsin D NM_009983 chemokine (C-C motif) Iigand 1 NM_011329 chemokine (C-C motif) Iigand 5 NM_013653 chemokine (C-C motif) receptor 1 AV231648 chemokine (C-X-C motif) receptor 6 NM_030712 clusteπn NM_013492 coagulation factor Il (thrombin) receptor AV024285 colony stimulating factor 1 (macrophage) M21149 colony stimulating factor 2 (granulocyte-macrophage) X03019 cyclin D3 NM_007632 cyclm-dependent kinase inhibitor 1A (P21) NM_007669 cysteinyl leukotπene receptor 1 BC027102 cytoplasmic FMR1 interacting protein 1 NM_011370 diphtheria toxin receptor L07264 ectonucleoside triphosphate diphosphohydrolase 1 B1151440 endothelial cell-specific molecule 1 BC020038 epithelial V-like antigen BC015076 fibrinogen-hke protein 2 BF136544

FK506 binding protein 5 U 16959

RIKEN full-length enriched, 7 days neonate cerebellum

Mus musculus cDNA clone A730098C13 3- similar to

L16904 Mouse zinc-finger protein (Mfg-2) mRNA, mRNA sequence BB261287 glypican 1 NM_016696 granulin M86736 granzyme A NM_010370 granzyme C NM_010371 granzyme E NM_010372 granzyme F NM_010374 granzyme G NM_010375 granzyme K AB032200 hepatitis A virus cellular receptor 2, Tιm3 AF450241 histone 1 , H4h NM_013550 homeobox only domain AK009007 homeobox only domain AF536202 huntingtin interacting protein 1 BB794880 interferon gamma K00083 interferon induced transmembrane protein 1 BC027285 interferon, alpha-inducible protein AK019325

interferon-induced protein with tetratricopeptide repeats 1 NM_008331 continuation of table 1

Gene name Accession No. interleukin 1 receptor, type Il NM_010555 interleukin 1 receptor-like 1 D 13695 interleukin 10 NM_010548 interleukin 18 receptor accessory protein N MJD 10553 killer cell lectin-like receptor subfamily C, member 1 AF 106008 killer cell lectin-like receptor subfamily G, member 1 NMJ ⁾ 16970 killer cell lectin-like receptor, subfamily A, member 3 U49865 killer cell lectin-like receptor, subfamily D, member 1 NM_010654 lectin, galactoside-binding, soluble, 3 binding protein NM_011150 lymphocyte antigen 6 complex, locus I AF232024 lymphotoxin A N MJ) 10735

MAD homolog 3 (Drosophila) B1150236 mannose-6-phosphate receptor, cation dependent NM_010749 matrix metalloproteinase 9 NM_013599 membrane-spanning 4-domaιns, subfamily A, member 4B NM_029499 membrane-spanning 4-domaιns, subfamily A, member 4C NMJD22429 musculin NM_010827 natural killer cell group 7 sequence NM_024253 neoplastic progression 3 BC011325 nuclear protein 1 NM_019738 olfactory receptor 672 NM_020292 osteoclast stimulating factor 1 U58888 oxidized low density lipoprotein (lectin-like) receptor 1 NM_138648 perform 1 (pore forming protein) M23182 phospholipase C, gamma 2 AW546508 pleckstrin AF181829 pleckstπn homology-like domain, family A, member 1 NMJD09344 potassium channel tetrameπsation domain containing 12 BM220945 potassium channel, subfamily K, member 5 AF319542 potassium inwardly-rectifying channel, subfamily J, member 8 NM_008428 preproenkephalin 1 M 13227 programmed cell death 1 NMJD08798 programmed cell death 1 ligand 2 NM_021396 protein kinase inhibitor beta, cAMP dependent, testis specific AV047342 protein tyrosine phosphatase, receptor type, E U35368

RAR-related orphan receptor alpha BI660199 retinoic acid induced 14 NM_030690 runt related transcription factor 2 D 14636

S100 calcium binding protein A4 D00208 secreted phosphoprotein 1 NMJ309263 serine (or cysteine) proteinase inhibitor, clade B, member

6a NM_009254 serine (or cysteine) proteinase inhibitor, clade E, member

2 NMJD09255 serine/threonine kinase 32C BB320288 serum amyloid A 3 NM_011315 solute carrier family 17 (sodium-dependent inorganic phosphate cotransporter), member 6 NM 080853

continuation of table 1

Gene name Accession No. solute carrier family 37 (glycerol-3-phosphate transporter), member 2 BC022752 solute carrier family 38, member 4 NM__027052 suppressor of cytokine signaling 3 NM_007707 syndecan binding protein (syntenin) 2 BC005556 transmembrane protein 4 NM_019953 tumor differentially expressed 1 BM239368 tumor necrosis factor NM_013693 tumor necrosis factor (ligand) superfamily, member 11 AB032771 tumor necrosis factor (ligand) superfamily, member 6 NM_010177 tumor necrosis factor (ligand) superfamily, member 7 NM_011617 twist gene homolog 1 (Drosophila) NM_011658

Table 2

Gene name Accession No. sprouty homolog 1 (Drosophila) NMJD11896 tumor necrosis factor (ligand) superfamily, member 11 AB032771 v-raf-1 leukemia viral oncogene 1 AB057663

2'-5' oligoadenylate synthetase 3 AB067534 stanniocalcin 2 AF031035 hepatocyte growth factor AF042856 leukemia inhibitory factor AF065917 aldo-keto reductase family 1, member C12 AF 177041

G protein-coupled receptor 105 AF177211 pleckstπn AF181829 calcitonin receptor-like AF209905 tripartite motif protein 30 AF220015 lymphocyte antigen 6 complex, locus I AF232024 potassium channel, subfamily K, member 5 AF319542 interleukin 24 AF333251 activating transcription factor 5 AF375476 hepatitis A virus cellular receptor 2 AF450241 growth arrest and DNA-damage-inducible 45 beta AI323528 amyloid beta (A4) precursor-like protein 1 AI848048 vitamin K epoxide reductase complex, subunit 1 AK003237 ring finger protein 128 AK004847 transmembrane 7 superfamily member 3 AK010720 solute carrier family 37 (glycerol-3-phosphate transporter), member 3 AKO 12071 inhibitor of DNA binding 2 AKO 13239 interferon, alpha-inducible protein AKO 19325 coagulation factor Il (thrombin) receptor AV024285 hydroxyprostaglandin dehydrogenase 15 (NAD) AV026552

FBJ osteosarcoma oncogene AV026617 protein kinase inhibitor beta, cAMP dependent, testis specific AV047342 granulm AV166504 chemokine (C-C motif) receptor 1 AV231648 alanine and arginine rich domain containing protein AV256613

G protein-coupled receptor 43 AV370830 selenoprotein M AY043488

H2A histone family, member Z AY074806

RIKEN cDNA 2310061 N23 gene AY090098 triple functional domain (PTPRF interacting) BB080177 regulator of G-protein signaling 16 BB100249

UDP-GaI betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 2 BB223909

ELOVL family member 5, elongation of long chain fatty acids (yeast) BB254141 serine/threonine kinase 32C BB320288 solute carrier family 35 (UDP-glucuronic acid/UDP-N-acetylgalactosamine dual transporter), member D1 BB409668

RIKEN cDNA C030046M14 gene BB622792

TCDD-inducible poly(ADP-rιbose) polymerase BB707122

ASF1 anti-silencing function 1 homolog B (S cerevisiae) BC003428

Lutheran blood group (Auberger b antigen included) BC004826 twisted gastrulation homolog 1 (Drosophila) BC004850

S100 calcium binding protein A1 BC005590

continuation of table 2

Gene name Accession No.

2'-5' oligoadenylate synthetase 1G BC018470 endothelial cell-specific molecule 1 BC020038 interferon -stimulated protein BC022751 solute carrier family 37 (glycerol-3-phosphate transporter), member 2 BC022752 schlafen 8 BC024709 cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4) BC027026 microtubule-associated protein, RP/EB family, member 2 BC027056 cysteinyl leukotπene receptor 1 BC027102 sulfite oxidase BC027197 insulin-like growth factor binding protein 7 BE446893 fibrinogen-like protein 2 BF 136544 sterol O-acyltransferase 1 BG064396

Kruppel-like factor 4 (gut) BG069413 retinol dehydrogenase 10 (all-trans) BG073496

Recombinant antineuraminidase single chain Ig VH and VL domains (LOC56304), mRNA BG966217 ectonucleoside triphosphate diphosphohydrolase 1 BI151440 neoplastic progression 3 BM210600 potassium channel tetrameπsation domain containing 12 BM220945 colony stimulating factor 1 (macrophage) BM233698 protein tyrosine phosphatase, non-receptor type 13 BM236743

S100 calcium binding protein A4 D00208 runt related transcription factor 2 D 14636 a disintegπn-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 1 D67076 heat shock protein 1B M 12573 preproenkephahn 1 M 13227

Wilms tumor homolog M55512 gastrin releasing peptide receptor M57922 a disintegrin and metalloprotease domain 8 NM_007403 suppressor of cytokine signaling 2 NM_007706 chemokine (C-C motif) receptor 8 NM_007720 colony stimulating factor 2 receptor, beta 2, low-affinity (granulocyte-macrophage) NM_007781 cathepsin H NM_007801 early growth response 1 NM_007913

GLI-Kruppel family member GLI3 NM_008130 interferon-induced protein with tetratπcopeptide repeats 1 NM_008331 lymphocyte-activation gene 3 NM_008479 melatonin receptor 1A NM_008639 phosphatιdylιnosιtol-4-phosphate 5-kιnase, type 1 alpha NM_008846 serine (or cysteine) proteinase inhibitor, clade E, member 1 NMJD08871

S100 calcium binding protein A13 NM_009113 chemokine (C-X-C motif) ligand 2 NM_009140 serine (or cysteine) proteinase inhibitor, clade E, member 2 NM_009255 secreted phosphoprotein 1 NM_009263 pleckstπn homology-like domain, family A, member 1 NM_009344

T-cell lymphoma invasion and metastasis 1 NM_009384 amphiregulin NM_009704 carbonic anhydrase 2 NM_009801 cyclin D2 NM 009829

continuation of table 2

Gene name Accession No. cathepsin D NM_009983 tumor necrosis factor (ligand) superfamily, member 6 NM_010177 ganglioside-induced differentiation-associated-protein 10 NM_010268

H2A histone family, member X NM_010436 hydroxysteroid (17-beta) dehydrogenase 7 NM_010476 interleukin 5 N MJD 10558 keratin complex 1, acidic, gene 18 NM_010664 annexin A1 NM_010730 lymphocyte antigen 6 complex, locus C N M JD 10741 interleukin 1 receptor-like 1 NM_010743 musculin N MJD 10827 serine (or cysteine) proteinase inhibitor, clade B, member 2 NM_011111 phospholipid transfer protein NM_011125

POU domain, class 2, associating factor 1 NM_011136 peroxisome proliferator activated receptor gamma NMJD11146 lectin, galactoside-binding, soluble, 3 binding protein NM_011150 serum amyloid A 3 NM_011315 chemokine (C-C motif) ligand 1 NMJD11329 serine (or cysteine) proteinase inhibitor, clade F, member 1 NM_011340 tumor necrosis factor (ligand) superfamily, member 7 NM_011617 ubiquitin specific protease 18 NM_011909 tumor necrosis factor NM_013693 histone 1, H1c NM_015786 hydroxyacyl-Coenzyme A dehydrogenase type Il N MJD 16763 interferon regulatory factor 7 NMJD16850 atonal homolog 7 (Drosophila) NM_016864 solute carrier family 7 (cationic amino acid transporter, y+ system), member 8 NM_016972 tubulin, alpha 8 NM_017379 activity regulated cytoskeletal-associated protein NMJD 18790 cytochrome P450, family 11, subfamily a, polypeptide 1 N MJD 19779 transmembrane protein 4 N MJD 19953 resistin like alpha NMJD20509 parathyroid hormone NMJD20623 interleukin 4 NMJD21283 programmed cell death 1 ligand 1 NMJD21893 peπcentπolar material 1 NMJD23662 natural killer cell group 7 sequence NMJD24253

ORM1-lιke 3 (S cerevisiae) NMJD25661 solute carrier family 39 (metal ion transporter), member 8 NMJD26228 solute carrier family 38, member 4 NMJD27052 chemokine (C-X-C motif) receptor 6 NMJD30712

Jun dimeπzation protein 2 NMJD30887 interleukin 6 NMJD31168 leucine zipper transcription factor-like 1 NMJD33322 tubby like protein 4 NMJD54040 solute carrier family 17 (sodium-dependent inorganic phosphate cotransporter), member 6 NMJD80853 dual specificity phosphatase 16 NMJ30447 immediate early response 3 NMJ33662 oxidized low density lipoprotein (lectin-like) receptor 1 NM 138648

continuation of table 2

Gene name Accession N©.

SET and MYND domain containing 1 U76372 histone i, H2ae W91024