DE102005002110A1 - Regulatory T cells containing proteins for the therapy and diagnosis of diseases - Google Patents

Abstract

The present invention relates to novel regulatory T cells containing proteins together with corresponding amino acid sequences as such, in particular their use as markers and for the therapy and diagnosis of diseases, in particular allergies, autoimmune diseases, in particular rheumatoid arthritis, multiple sclerosis or Crohn's disease, chronic inflammation , Asthma, immunodeficiency disorders, AIDS, transplant rejection and cancer, as well as diabetes.

Description

The The present invention relates to regulatory T cells Proteins, in particular their use as markers and for therapy and diagnosis of diseases, especially allergies, autoimmune diseases, in particular rheumatoid arthritis, multiple sclerosis or morbus Crohn, Chronic Inflammation, Asthma, Immunodeficiency Diseases, AIDS, transplant rejection and cancers as well as diabetes.

Further the invention relates to suitable binders and a test system (diagnostic agent) based on one or a combination of said proteins.

The Immune system is capable of between foreign proteins and structures of your own body, but also to distinguish between harmless and pathogenic antigens and thus unnecessary and to avoid autoaggressive immune responses. The maintenance immunological tolerance to endogenous structures simultaneous development of protective immune responses against Pathogens, based essentially on the formation of antigen-specific Effector cells for immune defense and the formation of antigen-specific Suppressor cells to maintain immunological tolerance.

Sakaguchi et al. first described a subpopulation of CD4 + T helper cells, characterized by constitutive expression of the alpha chain of the IL-2 receptor (CD25), which is essential for the control of autoaggressive Immune responses in mice (Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M., and Toda, M. (1995) Immunologic selftolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single Mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155, 1151-1164). Meanwhile, these CD4 + CD25 + T cells have been detected in different species, including the Humans, identified as CD25 + regulatory T cells (short: Treg, mentioned below; characterized by co-expression the surface proteins CD4 + and CD25 +), which as a resident population 5-10% of human peripheral Represent CD4 + T cells. Freshly isolated, CD25 + Tregs are anergic, i. they proliferate not after allogeneic or polyclonal stimulation but the proliferation and cytokine production of conventional CD4 + and CD8 + T cells. This suppression is cell-contact and activation-dependent, but antigen-nonspecific [Jonuleit, H., Schmitt, E., Stassen, M., Tuettenberg, A., Knop, J., and Enk, A.H. (2001) Identification and functional characterization of human CD4 (+) CD25 (+) T cells with regulatory properties isolated from peripheral blood. J. Exp. Med. 193, 1285-1294; Dieckmann, D., Plottner, H., Berchtold, S., Berger, T., and Schuler, G. (2001) Ex vivo isolation and characterization of CD4 (+) CD25 (+) T cells with regulatory properties from human blood. J. Exp. Med. 193, 1303-1310; ng, W.F., Duggan, P.J., Ponchel, F., Matarese, G., Lombardi, G., Edwards, A.D., Isaacs, J.D., and Lechler, R.I. (2001) Human CD4 + CD25 + cells: a naturally occurring population of regulatory cells. Blood 98, 2736-2744; Seddon, B. and Mason, D. (2000) The third function of the thymus. Immunol. Today 21, 95-99; Seddon, B. and Mason, D. (1999) Peripheral autoantigen induces regulatory T cells that prevent autoimmunity. J. Exp. Med. 189, 877-882; Thornton, A. M, and Shevach, E.M. (1998) CD4 + CD25 + immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188, 287-296; Suri-Payer, E., Amar, A.Z., Thornton, A.M., and Shevach, E.M. (1998) CD4 + CD25 + T cells Inhibit both the induction and effector function of autoreactive T cells and represent a unique lineage of immunoregulatory cells. J. Immunol. 160, 1212-1218; Piccirillo, C.A., and Shevach, E.M. (2001) Cutting Edge: control of CD8 + T cell activation by CD4 + CD25 + immunoregulatory cells. J. Immunol. 167, 1137-1140].

The Depletion of Tregs in vivo results in a number of autoimmune diseases, but also in an improved tumor defense (Sakaguchi (supra)). This finding supports the thesis of an ambivalent function of the Tregs. On the one hand prevent On the other hand, they complicate the development of autoaggressive immune reactions but at the same time an effective tumor defense, since tumor cells i.a. represent immunological "self" and therefore their elimination by effector T cells from Tregs is suppressed. The Increasing the suppressive function of Tregs will be helpful for the Therapy in particular of autoimmune diseases viewed while a Transient inhibition of their suppressive properties, the tumor defense support can.

The Fact that the suppressive properties are cell-contact-dependent, makes it clear that Treg-specific molecules (markers, Target) have a decisive influence on the functionality of the cells have and form the basis for the targeted exploitation of these properties to therapeutic and diagnostic purposes in the field of allergies, autoimmune diseases, Chronic inflammation, immunodeficiency disorders, transplant rejection and Cancers as well as AIDS, diabetes.

The It is therefore an object of the present invention to provide new Treg provide.

The Task is solved by that the new Treg characteristically express one or more proteins.

Therefore The invention relates to Treg-containing proteins and their isolation. Furthermore, the proteins mentioned in Tregs are suitable markers or Targets.

With Help of the proteome analysis was according to the invention targeted the protein composition of the individual T cell subpopulations, in particular the Treg (ie CD4 + CD25 + and CD4 + CD25 + β7 + subpopulations), investigated and specifically Treg - own Proteins identified as an expression product (Table 1 and Table 2).

in the During the proteome analysis proteins were identified as an expression product, which differ significantly in their expression rate in Treg from those in differentiate conventional T cells and therefore to surprising Properties of Treg lead, namely either an increase in the suppressing function of Tregs or a transient inhibition of their suppressive properties, which can support the tumor defense.

Some These proteins have already been in their function or involvement described in the regulation of the immune response.

Treg enthaltend Proteine oder deren entsprechenden Aminosäuresequenzen Tabelle 2: Murine exprimierte Proteine gemäß Proteomanalyse:

(Die letzte Spalte in Tab. 1 und 2 ist in den Beispielen erläutert)However, the specific suitability of these proteins for the manipulation and modification of Treg is not recognized, particularly with regard to the regulation of an immune response. Treg containing proteins or their corresponding amino acid sequences according to Table 1: Human expressed proteins according to proteome analysis:

Treg containing proteins or their corresponding amino acid sequences Table 2: Murine-expressed proteins according to proteome analysis:

(The last column in Tab. 1 and 2 is explained in the examples)

The The proteins mentioned in Table 1 or 2 are hereinafter referred to as proteins or their corresponding amino acid sequence »called.

Therefore, the invention relates to an isolated regulatory CD4 ⁺ CD25 ⁺ T-cell and / or CD4 ⁺ CD25 ⁺ β7 ⁺ containing at least one protein according to SEQ ID No. Aminosäursequenzen 1-16 and / or SEQ ID NO. 17-27 or with SEQ ID NO. 1-16 and / or SEQ ID NO. 17-27 have a sequence identity or homology of 70% or more.

Furthermore, it is preferred that the proteins or their corresponding amino acid sequence are expressed and have a modified expression rate compared to conventional T cells (see 1 , as well as Table 1 or 2), which sufficiently indicate an altered immunological response.

Also can these proteins of the invention or their corresponding amino acid sequences be modified, e.g. using post-translational modifications, like glycolization.

These modified proteins are included according to the invention.

in the For the purposes of this invention, "Treg" is understood to mean those T cell subpopulations which are of human origin or derived from mammals (e.g., mouse) can. According to the invention, however, the subpopulations Treg-CD4 + CD25 + are preferred. "Insulated Treg "are ex-vivo Cells (outside of the living body) and optionally separated from other T cells. By means of isolation is also an accumulation of Treg cells, which contain the mentioned proteins are possible (see examples).

Of the Term "native Treg "describes" in vivo "(within the living body) Treg to be found, e.g. in human blood or thymus or of Mammals.

In a further embodiment are the Treg invention containing one or more of the proteins according to SEQ ID no. 1-16 and SEQ ID no. 17-27, recombinantly altered in this regard, that they have an amino acid sequence according to the invention, contained or from a corresponding nucleic acid sequence can be obtained can namely to the amino acid sequences SEQ ID no. 1-16 the nucleic acid sequences SEQ ID no. 28-43 and to the amino acid sequences SEQ ID no. 17-27 the nucleic acid sequences SEQ ID no. 44-54.

Therefore The invention also relates to such amino acid sequences which have sequence identity or homology of 70% and more, preferably 80% and more, more preferably from 90-95% and more with SEQ ID NO. 1-16 and SEQ ID no. 17-27 exhibit. Also included are such analogs Amino acid sequences due to the exchange of one or more amino acids in them Sequences, yet the desired Ensuring the function of each protein.

In another embodiment, fusion proteins are also involved, containing an amino acid sequence of the invention or a named protein as a partial sequence. Examples of recombinant fusion proteins are given in EP 282 042 B1 (His-tag).

Of Furthermore, the invention relates to nucleic acids for one of the said proteins encode, preferably for one or more of the proteins are contained in Tregs or for the amino acid sequences of the invention encode, in particular SEQ ID no. 28-43 and nucleic acid sequences SEQ ID no. 44-54.

In a further preferred embodiment contains the nucleic acid according to the invention a or more non-coding Sequences and / or a poly (A) sequence, one or more recognition sequences and, if necessary, one or more potential N-glycosylation sites. The non-coding Sequences are regulatory sequences, such as promoter or enhancer sequences, for the controlled expression of the coding gene containing the Nucleic acids according to the invention. Of others are possible nucleic acids Subject of usual Expression vectors, usual Host cells or usual gene therapeutic vectors (e.g., J. Sambrook, E. F. Fritsch, T. Maniatis (1989), Molecular cloning: A laboratory manual, 2nd Edition, Cold Spring Laboratory Press, Cold Spring Habor, USA or Ausubel, "Current Protocols in Molecular Biology ", Green Publishing Associates and Wiley Interscience, N.Y. (1989)).

Of the Term "nucleic acid" (synonym: polynucleotide) has the meaning in the sense of DNA or RNA or chemical analogues and the same.

The proteins of the invention or their corresponding amino acid sequences can secrete and membrane-bound Bind proteins to Treg or effector cells. Furthermore can they are such membranous Cross-link proteins and therefore influence their functions and regulate. This property can be used according to the invention to interact between Treg and T-effector cells, e.g. for the purpose of Treatment of diseases with Treg or effector cells keep in touch.

Further can the proteins of the invention or their corresponding amino acid sequences in Cytosol or other organelles of the Tregs, e.g. in the Mitochondria are present. Therefore, the invention relates to such Treg, wherein at least one of the proteins according to the invention or their corresponding amino acid sequences secreted, membranous or on the surface or presented in the cytosol is.

With Help of recombinant methods can be at least one protein according to the invention or its corresponding amino acid sequence in the Treg or on the surface the Treg be enriched. For this purpose, an amino acid sequence according to the invention or nucleic acid be introduced in Treg. For this purpose, the expert uses the usual Methods for transfection.

In a further embodiment are the "Treg invention containing Proteins or their corresponding amino acid sequences ", recombinant to that effect changed that they have an amino acid sequence according to the invention, preferably SEQ ID no. 1-16 or amino acid sequences SEQ ID no. 17-27 or Nucleic acid sequences according to the invention SEQ ID no. 28-43 and SEQ ID no. 44-54 contain.

The The invention further relates to binders for at least one isolated containing regulatory T cell or native regulatory T cell at least one of the proteins or their corresponding amino acid sequences.

The Binder can not final selected be from the group: inhibitor, agonist, antagonist, probe, antibody or Immunomodulator.

Of the Binder can also induce a signal, such as a color reaction, radioactive Mark, which is enough to identify and modify a Treg containing proteins. Therefore, the binder can be a "probe" Therefore, according to the invention, the binder is also an addressed one Molecule, which binds to a suitable signal-transmitting receptor to Treg containing proteins binds and due to containing the above Protein in Treg a feedback generated.

For example can by means of an inhibitor or modulator one or more of Proteins or their corresponding amino acid sequences in Treg advantageous be enriched. With the aid of a probe, e.g. Further Treg cells containing one of the proteins or their corresponding amino acid sequences be identified. Such a probe is, for example, an antibody which specifically one or more epitopes present on the amino acid sequences of the invention (Production, for example, pertinent after Köhler).

For example can by bivalent binders having multiple epitopes, wherein one against one of said proteins, the other against e.g. CD25, CD44, CD45, GITR, CTLA-4, Galectin, Fox P3 or one of the proteins according to the invention and corresponding Amininosäurensequenzen is used to the invention Treg containing proteins or their corresponding amino acid sequences via them surface proteins be cross-linked with other Treg or effector cells, with the inventive goal an influence on the activity these effector cells.

In From a functional point of view, the binders have the function of the isolated Treg or native Treg containing at least one activate the proteins or their corresponding amino acid sequences or disable.

Therefore are the Treg containing proteins or their corresponding amino acid sequences or binder suitable as a medicament, preferably for treatment and therapy of diseases, namely allergies, autoimmune diseases, in particular rheumatoid arthritis, multiple sclerosis or morbus Crohn, Chronic Inflammation, Asthma, Immunodeficiency Diseases, AIDS, transplant rejection and cancers as well as diabetes.

Especially such autoimmune diseases selected from the group: alopecia Areata, ankylosing spondylitis, antiphospholipid syndrome, Addison's disease, Behcet's disease, celiac Disease Sprue, chronic fatigue syndrome (Chronic Fatigue Immune Dysfunction Syndrome (CFIDS)), Polyneuropathy, Churg-Strauss syndrome (granulomatosis), CREST syndrome (Raynaud's syndrome), Cold Agglutinin Disease, Cryoglobulinemia, Fibromyalgia, Fibromyositis, Graves' disease, Guillain-Barré syndrome, idiopathic pulmonary Fibrosis, idiopathic thrombocytopenia, IgA nephropathy, lichen Planus, Ménière's disease, polyarteritis Nodosa, Polychondritis, Polyglandular Syndrome, Polymyalgia Rheumatica, Primary Agammaglobulinemia, biliary Cirrhosis, psoriasis, Reiter's disease, sarcoidosis, Sjogren's disease, Takayasu's arteritis, Vasculitis, vitiligo, Wegener's granulomatosis.

isolated Treg containing one or more of the proteins or their corresponding Amino acid sequences, modified according to the invention, can the body to be treated be applied. For another suitable binder administered to the patient in sufficient dosage become. The Treg-containing proteins or their corresponding amino acid sequences and / or binders are if necessary formulated with other excipients.

Of Furthermore, the invention relates to the use of said proteins or their corresponding amino acid sequences in Treg as markers or targets.

Especially can the proteins serve as a target for the manipulation or modulation of the suppressive properties the Tregs. This can, for example, by means of a binder or a Substance take place. Further, the binder or the substance may be Inhibitor, which is the expression of one or more of the mentioned Proteins inhibit, inhibit or promote.

Further can the Treg-specific proteins individually or in combination as markers serve to Treg with (increased) to identify suppressive properties.

Of Furthermore, the invention relates to a test system containing at least a binder and at least one Treg containing proteins or their corresponding amino acid sequences, for the identification of suitable binders or Tregs, preferably those with increased suppressing properties.

Therefore The invention also relates to a test system comprising at least a Treg containing one or more of the proteins or their corresponding amino acid sequence and at least one target cell, in particular T cell, B cell, macrophage, Prädendritische Cell, dendritic cell, embryonic cell and / or fibroblast, which are incubated with at least one Treg for in vitro detection suppressive properties, in particular cellular immune response of effector cells of the immune system, in particular B cells, NK cells, preferably T cells, T helper cells.

by virtue of the particular cell-contact-dependent suppressing properties of Treg containing one or more The proteins or their corresponding amino acid sequence can be found in Test system according to the invention the cellular Immune response of the target cells tested become.

A Immune response can be achieved, for example, through the synthesis of cytokines such as. gamma interferon, interleukins are detected. The corresponding cytokine accumulates intracellularly in this test system and can over fluorescein-coupled antibodies (e.g., ELISA). Furthermore by expression of Surface molecules, lysis the target cell or cell proliferation. In a FACS (fluorescent activated cell sorter), the proportion of immune cells can be determined which stimulate or not stimulate or activate or deactivate to let. Further detection methods are not conclusive cytokine assay, ELISPOT, proliferation assays or 51Cr release assays (see General: Current Protocols of Immunology (1999), Coligan J.E., Kruisbeek A.M., Margulies D.H., Shevach E.M. and Strober W., John Wiley & Sons).

In a further embodiment are the effector cells, mammalian cells, in particular human or murine cells or immune cell line and / or cultivated primary Immune cell.

In a further embodiment the test system is incubated at least one other substance that trigger an immune response can, such as proteins, epitopes, protein fragments, antigens.

Further is such a test system suitable for the identification of binders of the invention.

Of Furthermore, the invention relates to a diagnostic agent (synonym: array or assay) for execution the test systems according to the invention and optionally a pharmaceutically acceptable carrier.

Examples of pharmaceutically acceptable carriers are glass, polystyrene, Polypropylene, dextran, nylon, amylase, natural or modified cellulose, Polyacrylamide, agarose, alumium hydroxide or magnitide. Furthermore, can the carrier from 96 corrugated plates and higher consist.

The Diagnostic can be in solution be bound to a solid matrix and / or with an adjuvant be offset.

Further The diagnostic agent can be applied to a patient as desired in vivo (e.g., capsule, tablet).

One Inventive Diagnostic is therefore suitable for the diagnosis of diseases and that of allergies, Autoimmune diseases, especially rheumatoid arthritis, multiple Sclerosis or Crohn's Disease, Chronic Inflammation, Asthma, Immunodeficiency Diseases, AIDS, transplant rejection and cancers as well as diabetes.

In particular, autoimmune diseases such as alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, Addison's disease, Behcet's disease, celiac sprue, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), polyneuropathy, Churg-Strauss syndrome (granulematosis), CREST syndrome (Raynaud's syndrome), cold agglutinin disease, cryoglobulinemia, fibromyalgia, fibromyositis, Graves' disease, Guillain-Barre syndrome, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia, IgA nephropathy, lichen planus, Ménière's disease, polyarteritis nodosa, polychondritis, polyg Landular Syndrome, Polymyalgia Rheumatica, Primary Agammaglobulinemia, Biliary Cirrhosis, Psoriasis, Reiter's Disease, Sarcoidosis, Sjögren's Disease, Takayasu's Arteritis, Vasculitis, Vitiligo, Wegener's Granulomatosis.

The The following examples serve to illustrate the invention without to limit the invention to these. In addition, figures and sequences explained.

Examples

Example 1: Isolation and stimulating human T cell populations

conventional CD4 + CD25-T effector cells (hereinafter referred to as CD4 + T cells) and CD4 + CD25 + T cells (hereinafter referred to as CD25 + Treg cells) became buffy coats and leukopherapy healthy human donors isolated.

Example 1a: CD4 + CD25 + regulatory T cells (CD25 + Tregs)

The starting material is the leukapheresis of voluntary, healthy donors, which is produced by the Mainz Transfusion Center and contains an average of 7-10 × 10 ⁹ leucocytes.

in the first step, the mononuclear cells by Ficoll gradient centrifugation isolated and then washed intensively with PBS + 1 mM EDTA.

Then be the isolated leukocytes in PBS + 0.5% HSA (human serum albumin) + 1 mM EDTA and with anti-CD25 microbeads (2 μl Microbeads / 107 Leukocytes, Microbeads: Miltenyi GmbH, Bergisch-Gladbach, FRG) for 15 min. at 4 ° C incubated. After incubation, the leukocytes become 2x with PBS + 1 mM EDTA.

to Isolation of the CD25 + leukocytes will subsequently disrupt the cells a separation column applied (LS Columns, Miltenyi) and in the permanent magnet (Miltenyi) separated. The average yield of CD25 + leukocytes is 1.2-2% (purity> 97%).

To deplete CD4-negative contaminations, the CD25 + leukocytes are subsequently incubated with CD8, CD19, CD14 dynabeads (Dynal, Hamburg, Germany, 3 beads / cell) and mouse IgG1 anti-human CD45RA monoclonal antibodies (Coulter / Immunotech. Hamburg, Germany, 1 μg mAb / 10 ⁶ leukocytes) 20 min. incubated in X-VIVO-15. The bound CD8 +, CD19 + and CD14 + contaminations can be removed directly using a permanent magnet (Dynal), the CD45RA + cells are removed with anti-mouse IgG Dynalbeads (Dynal) in the permanent magnet. This depletion step is then repeated again (purity of CD4 + CD25 + leukocytes> 95%).

Example 1b: α4β1 + and α4β7 + subpopulations human regulatory T cells

Human CD25 + Tregs contain two functionally distinct subpopulations that differ in the expression of integrins. Approximately 20% of the Tregs express the α4β7 integrin, 80% the α4β1 integrin. Isolation of these subpopulations requires the following changes to the isolation protocol:
In the first step, the isolated leukocytes are 15 min. incubated at 4 ° C with mouse IgG anti-human CD25-FITC mAb (2 μl mAb / 107 leukocytes, M-A251, BD PharMingen, San Diego, USA) and then washed extensively with PBS + 1 mM EDTA.

The FITC-positive cells are made using anti-FITC multisort microbeads Isolated (Miltenyi). The procedure becomes analogous to the direct isolation of CD25 + leukocytes with CD25 microbeads carried out. Subsequently are the microbeads by enzymatic digestion, according to from the manufacturer (Miltenyi), removed from the surface of the leukocytes.

The Depletion of the CD4-negative contaminations occurs as previously described with CD8, CD19 and CD14 dynabeads, CD45RA + cells will not depleted (purity CD4 + CD25 + T cells> 95%).

In the next step, the α4β7 + subpopulation is isolated. For this purpose, the CD4 + CD25 + T cells are incubated with a rat IgG anti-human β7 integrin PE mAb (BD-PharMingen, 2 μl / 10 7 cells) for 15 min. incubated at 4 ° C and then washed extensively with PBS + 1 mM EDTA. The procedures for isolating the β7 + T cells are analogous to the isolation CD25 + T cells using anti-PE microbeads (Miltenyi) resul in a purity of CD4 + CD25 + α4β7 + cells> 90%. The negative fraction expresses the integrin α4β1 (purity CD4 + CD25 + α4β1 + cells> 80%).

Example 2: Characterization human CD4 + CD25 + regulatory T cells

CD25 + Tregs are characterized by their inhibitory effect on activation characterized by CD4 + and CD8 + T cells in vitro.

The functional characterization CD25 + Tregs in vitro is used in co-culture assays analyzed with CD4 + T helper cells. For this, the T-cells are either with allogeneic, mature dendritic T cells or polyclonal with anti-CD3 + anti-CD28 mAb stimulated.

Example 3: Multisort positive selection of CD4 + CD25 + and CD4 + CD25 + β7 + T cells

The Isolation of Treg occurred in several steps. At first were CD4 + T cells using the CD4 MACS multisort kit (Miltenyi, Bergisch-Gladbach, Germany) and isolated therefrom with anti-CD25-FITC (M-A251, BD PharMingen, San Diego, USA) and anti-FITC multisort beads (Miltenyi, Bergisch-Gladbach, Germany) the CD4 + CD25 + T cells. Subsequently, B cells became macrophages and CD8 + T cells by means of CD19, CD14 and CD8 Dynabeads (Dynal, Hamburg, Germany) depletes. For the isolation of CD4 + CD25 + β7 + Treg (subpopulation of CD4 + CD25 + Treg) were β7-PE and anti-PE beads (Miltenyi, Bergisch-Gladbach, Germany) used. The purity of the isolated cells was with controlled by the FACS analysis.

Example 4: Functional Analysis of human freshly isolated CD4 + CD25 + T cells

CD25 is a typical surface molecule on Treg, however, it is not only expressed in this cell type. For this Reason before any analysis was a functional control of the suppressive Properties of the isolated cells performed.

Example 5: Polyclonal Stimulation with anti-CD3 and anti-CD28 monoclonal antibodies

A constant number of conventional CD4 + T cells (1x105 / well) can be activated polyclonally with anti-CD3 (1 μg / ml, OKT-3) and anti-CD28 monoclonal antibodies (2μg / ml CD28.2) in the presence of a varying number of CD4 + CD25 + T cells (ratio 1: 1 to 1: 4). T-cell proliferation was measured after three days Cultivation and a subsequent 16 hours pulsed treatment with 3HTdR (37 kBq / well). The so tested Cells were used for used the proteome analyzes.

Total cell from cultured cells for 2DE Extraction of proteins from cells after cell lysis followed a slightly modified method according to Klose (Klose, J. and Kobalz, U., Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome. Electrophoresis 16, 1034-1059 (1995) and Klose, J. Fractionated extraction of total tissue proteins from mouse and human for 2-D electrophoresis. Methods Mol Biol 112, 67-85 (1999)). The cells were stored in a phosphate buffer, the protease inhibitors against a variety of different proteases contained mechanically means Ultrasound and glass beads lysed. The 2D gel electrophoresis interfering nucleic acids was at room temperature within 20 min by adding the nuclease Benzonase digested. The proteins were in a urea and thiourea containing Buffer with addition of DTT solved. For the Isoelectric focusing of the proteins was added to Servalyte 2-4.

Example 6: Isolation and stimulating murine T cell populations

conventional CD4 + CD25-T effector cells (hereinafter referred to as CD4 + T cells) and CD4 + CD25 + T cells (hereinafter referred to as CD25 + Treg cells) mouse spleens became healthy male BRLB / C Mice won.

Example 6a: CD4 + CD25 + regulatory T cells (CD25 + Tregs)

The starting materials are mouse spleens. In the first step, the CD25 + Treg cells are positively selected using MACS. For this purpose, the CD25 + Treg cells are first labeled with a biotinylated anti-CD25 monoclonal antibody and then with a strepavidin-phycoerithrin (PE). To isolate the CD25 + Treg cells, the cells are incubated with anti-PE beads following to a MACS Separation column applied (LS Columns, Miltenyi) and separated in the permanent magnet. Contaminations by B cells and macrophages as well as CD8 + T cells were caused by negative. Selection removed. For this wound the CD25 + Treg fraction with Dynabeads against B220, CD8 and MAC-1 offset. These cells were removed with a Magnetic Particel Concentrator according to the manufacturer's instructions. The average purity of the obtained CD25 + Treg in this method is> 97%. To obtain a sufficient number of cells for functional characterization and proteome analysis of the cells. An average of 30 mouse sponges are pooled.

Example 6b: CD4 + T cells

The CD25 + depleted fraction is monoclonal with biotinylated anti-CD4 antibody and subsequently labeled with strepavidin beads and then applied to a MACS separation column (LS Columns, Miltenyi) and separated in permanent magnet.

Example 7: Characterization murine CD4 + CD25 + regulatory T cells

CD25 + Tregs are characterized by their inhibitory effect on activation characterized by CD4 + and CD8 + T cells in vitro.

The Functional characterization of murine CD25 + Tregs is analogous on the characterization of human CD25 + Tregs in vitro in coculture assays analyzed with CD4 + T helper cells.

The cells tested in this way were used for proteome analyzes. are prepared analogously to the human T cells for proteome analysis.

Example 8: Protein separation by means of 2DE

The Isoelectric focusing (IEF) of the proteins was performed after the Klose's method (Klose, J., Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to test for induced point mutations in mammals. Human genetics, 26, 231-243 (1975)) with carrier ampholytes in polyacrylamide round gels under reducing conditions. The separations were carried out in a pH range of 2 to 11, wherein the length IEF gels 40 cm was. Protein separation of IEF-separated proteins SDS-PAGE was carried out in 15% polyacrylamide gels. Before the Order on the gel for the SDS-PAGE were the IEF gel strand twice with running buffer (0.3 (w / v) Tris base, 1.44% (w / v) glycine, 0.1% (w / v) SDS) to excess DTT to remove. Subsequently The gel strand was placed bubble-free on the SDS gel and with a 1% agarose solution (fixed with bromophenol blue). The entry of proteins into the gel took place at 65 mA for 15 min and the separation within about 5 h at 100 mA for 0.75 mm thick analytical gels or at 75 and 200 mA for 1.0 and 1.5 mm thick preparative gels. The separation distance was 30 cm.

Around one possible To obtain high sensitivity in the protein detection was done the coloring analytical gels with silver according to a modified method of Heukeshoven and Dernick (Heukeshoven, J. and Dernick, R. Improved silver staining procedure for fast staining in PhastSystem Development Unit. I. Staining of sodium dodecyl sulfate gels. Electrophoresis 9, 28-32 (1988)) modified according to Klose and Kobalz (Klose, J. and Kobalz, U. Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome. Electrophoresis 16, 1034-1059 (1995)). Because this method is an addition of glutaraldehyde and formaldehyde uses to increase the sensitivity is a subsequent mass spectrometric Identification of the proteins hardly possible. That's why If necessary, a modified variant of the mass spectrometry compatible coloring according to Blum et al. (Blum, H., Beier, H. and Gross, H. J. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis, 8, 93-99 (1987)). Colloidal Coomassie staining according to Neuhoff et al. (Neuhoff V., Arold N., Dove D. and Ehrhardt W., Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9, 255-262. (1988)) with Coomassie Brilliant Blue G-250 was for the proteins in preparative 2DE gels that were examined by mass spectrometry. Alternatively, especially for Proteins that are not stained with colloidal Coomassie staining could have been using silver after a modified protocol without the addition of glutaric dialdehyde (Blum, H., Beier, H. and Gross, H.J. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis, 8, 93-99 (1987)).

Example 9: Differential proteome

The Digitization of the polyacrylamide gels was carried out on silver-stained gels for the Image analysis after drying the gels with a transmitted light scanner.

The quantitative evaluation of the relative protein intensities took place with a special, for These analyzes suitable image analysis software (ProteomWeaver Vers. 2.0, Definiens, Germany).

The With the help of image analysis found proteins were manually cut out of the gels. With the help of a washing robot were the gel pieces alternately alternating three times each with 10 .mu.l digestion buffer (10 mM NH4HCO3) or digestion buffer / acetonitrile 1: 1 washed to the dye and buffer additives to remove. For silver-colored Spots were silver before washing by adding 15 ul deinking solution (100 mM potassium hexacyanoferrate (III) / 30 mM sodium thiosulfate, 1: 1) Room temperature oxidized within about 1 min. Subsequently were the gel pieces dehydrated in the vacuum centrifuge and with 2 ul each of a trypsin solution (0.05 ug / ul trypsin in digestion buffer). The proteolytic cleavage took place at 37 ° C for at least 4 h or over Night. The resulting proteolysis products were within 30 min by adding 5 μl 0.1% TFA extracted at room temperature from the gel matrix.

Example 10: MALDI-TOF mass spectrometry

The Determination of the peptide masses of proteolytically cleaved proteins was carried out with a MALDI-TOF mass spectrometer of the type Ultraflex (Bruker Daltonik, Bremen, Germany). In this method will be the analyte molecules (Peptides) cocrystallized in a UV-active matrix. For the matrix solution was a saturated α-cyano-4-hydroxy-cinnamic acid solution in 50% acetonitrile / 0.1% TFA 1: 1 (solution A) with solution A in proportion Diluted 1: 1. Before the measurements, the peptides were accumulated on C18 material in ZipTipsTM (activated by 10 μl 0.1% TFA) by adsorbing the analyte solution several times, once with 10 μl Washed 0.1% TFA and then with 1.2 ul matrix solution eluted the sample tray. The so-called peptide mass fingerprint spectra (PMFs) of the samples dried on the sample plate were at the following Measure settings: Acquisition method: Reflector, Voltage polarity: positive, Acceleration voltage: 25 kV, reflector voltage: 26.3 kV, lens voltage: 6.2 kV, reflector detector voltage: 1.72 kV and deflection voltage: 0 kV

The calibration of the mass spectra was carried out automatically by a calibration algorithm of the Proteinscape ^® database (Bruker Daltonik) autoproteolysis products of trypsin and on known, recurring in the spectra peptides from contaminants such as keratin.

The peptide mass spectra were analyzed with the aid of a non-redundant NCBI protein database using the meta search engine of ProteinScape ^® and the search algorithms MASCOT and ProFound (version 3.1.2002).

Evaluation:

at The following human proteins were reduced in protein in activated Treg cells opposite conventional T cells found: tumor protein, translationally-controlled 1 ; Homoebox Prox 1; FK506-binding protein 4; stathmin 1; MO25 protein; Proteasome subunit beta type 7; Nucleoside diphosphate kinase A; ribonucleoside diphosphate reductase; Flap structure-specific endonuclease 1; dUTP pyrophosphatase and eukaryotic translation initiation factor 5A.

at The following human proteins were shown to increase the amount of protein activated Treg cells opposite conventional T cells found: tropomyosin 3; Actin, beta, cytoplasmic; EH domain-containing 1; Actin gamma and heterogeneous nuclear ribonucleoprotein A2 / B1 isoform 2.

These Results were found in four independent human donors.

at The following murine proteins were reduced in protein in activated Treg cells opposite conventional T cells found: elongation factor 1-delta, elongation factor 1-beta (EF-1-beta), peroxiredoxin 4, LSP1_MOUSE lymphocyte-specific protein 1 (protein pp52) (52 kDa phosphoprotein) (lymphocyte-specific antigen WP34), protein annexin A11, ferritin light chain, ferritin heavy chain and enolase 3

For the following murine proteins, an increase in the amount of protein in activated Treg cells was ge compared to conventional T cells found: J kappa recombination signal binding protein, serine (or cysteine) proteinase inhibitor, clade B, member 9b serine (or cysteine) proteinase inhibitor, clade B, member 1b

These results were found in five independent murine T cell pools. Sequences (see Sequence Listing) and Figures: PROTEIN SEQ ID NO: 1 Tropomyosin 3: Protein aliases: TPM3; alpha tropomyosin 3; alpha tropomyosin, slow skeletal Gene map locus: 1q22-q23 Protein sequence Accession: P06753 Organism: Homo sapiens Size: 284AA DNA sequence Accession: X04201.1 Open Reading Frame: 59 to 916 SEQ ID NO: 28 PROTEIN SEQ ID NO. 2 EH domain containing 1 Protein aliases: EHD1; Calcium binding protein; Calcium io binding; Signal transduction; Cell communication Gene Map Locus: 11q13 Protein sequence Accession: NP_006786.2 Organism: Homo sapiens Size: 534AA SEQ ID NO: 2 DNA sequence Accession: NM_006795.2 Open Reading Frame: 256 to 1860 SEQ ID NO: 29 PROTEIN SEQ ID No.3 actin, beta, cytoplasmic isoform A, B and C: Protein aliases: ACTB, beta-actin Gene map locus: 7p22-p12 Protein sequence Accession: NP_001092.1 Organism: Homo sapiens Size: 375AA SEQ ID NO: 3 DNA sequence Accession: NM_001101.2 Open Reading Frame: 74 to 1201 SEQ ID NO: 30 PROTEIN SEQ ID No. 4 Actin gamma: Protein aliases: cytoskeletal gamma-actin; actin, cytoplasmic, 2 Gene map locus: 17q25 Protein sequence Accession: NP_001605.1 Organism: Homo sapiens Size: 375AA SEQ ID NO: 4 DNA sequence Accession: NM_001614.2 Open Reading Frame: 75 to 1202 SEQ ID NO: 31 PROTEIN SEQ ID NO: 5 Heterogeneous nuclear ribonucleoprotein A2 / B1 - isoform 2: Protein aliases: heterogeneous nuclear ribonucleoprotein a2; hnrpa2; heterogeneous nuclear ribonucleoprotein b1; HNRPB1; nuclear ribonucleoprotein particle a2 protein Gene map locus: 7p15 Protein sequence Accession: NP_112533.1; gi: 4504447 Organism: Homo sapiens Size: 353AA SEQ ID NO: 5 DNA sequence Accession: NM_031243.1 Open Reading Frame: 170 to 1231 SEQ ID NO: 32 PROTEIN SEQ ID No. 6 Tumor protein, translationally-controlled 1: Protein aliases: fortilin; histamine-releasing factor; immunoglobulin e-dependent; HRF Gene map locus: 13q12-q14 Protein sequence Accession: NP_003286; gi: 4507669 Organism: Homo sapiens Size: 172 AA SEQ ID NO. 6 DNA sequence Accession: NM_003295; gi: 4507668 Open Reading Frame: 1 to 172 SEQ ID NO: 33 PROTEIN SEQ ID NO.7 Homeobox prox 1: Protein aliases: homeobox gene prox1 Gene map locus: 1q32.2-q32.3 Protein sequence Accession: NP_057123; gi: 7706322 Organism: Homo sapiens Size: 244 AA SEQ ID NO. 7 DNA sequence Accession: NM_016039; gi: 7706321 Open Reading Frame: SEQ ID NO. 34 PROTEIN SEQ ID NO: 8 FK506-binding protein 4: Protein aliases: t-cell fk506-binding protein, 59kd; FKBP59; FKBP52 Gene map locus: 12p13.33 Protein sequence Accession: NP_002005.1; gi: 4503729 Organism: Homo sapiens Size: 459 AA SEQ ID NO: 8 DNA sequence Accession: NM_002014.2; gi: 17149846 Open Reading Frame: 1 to 2251 SEQ ID NO: 35 PROTEIN SEQ ID NO. 9 Stathmin 1: Protein aliases: stathmin; smn; leukemia-associated phosphoprotein p18; LAP18 METABLASTIN Gene map locus: 1p36.1-p35 Protein sequence Accession: NP_005554 Organism: Homo sapiens Size: 149AA SEQ ID NO. 9 DNA sequence Accession: NM_005563.2 Open Reading Frame: 92 to 541 SEQ ID NO. 36 PROTEIN SEQ ID NO: 10 MO25 protein: Protein aliases: CGI-66 Gene map locus: 2q37.1 Protein sequence Accession: NP_057373 Organism: Homo sapiens Size: 341 AA SEQ ID NO: 10 DNA sequence Accession: NM_016289; gi: 19745179 Open Reading Frame: 325 to 1350 SEQ ID NO. 37 PROTEIN SEQ ID NO. 11 Proteasome subunit beta type 7: Protein aliases: beta-type, 7; PSMB7 Gene map locus: 9q34.11-q34.12 Protein sequence Accession: Organism: Homo sapiens Size: 277AA SEQ ID NO: 11 DNA sequence Accession: NM_002799.2 Open Reading Frame: Open Reading Frame: 18 to 851 SEQ ID NO: 38 PROTEIN SEQ ID NO: 12 Nucleoside diphosphate kinase A - isoform A and B: Protein aliases: metastasis inhibition factor nm23; nonmetastatic protein 23; NM23; nonmetastatic protein 23, homolog 1; nm23h1 nucleoside diphosphates kinase-a; ndpka gzma-activated dnase; gaad awd; drosophila, homologue of; awd nm23h1b, included Gene map locus: 17q21.3 Protein sequence Accession: NP_000260; gi: 4557797 Organism: Homo sapiens Size: 152 AA SEQ ID NO: 12 DNA sequence Accession: NM_000269.1 Open Reading Frame: 85 to 543 SEQ ID NO: 39 PROTEIN SEQ ID NO: 13 Ribonucleoside diphosphate reductase - isoform A and B: Protein aliases: ribonucleotide reductase, large subunit ribonucleotide reductase, r1 subunit; r1 Gene map locus: 11p15.5 Protein sequence Accession: NP_001024; gi: 4506749 Organism: Homo sapiens Size: 792 AA SEQ ID NO: 13 DNA sequence Accession: NM_001033.2; gi: 21071083 Open Reading Frame: 233 to 2611 SEQ ID NO. 40 PROTEIN SEQ ID NO. 14 Flap structure-specific endonuclease 1 Protein aliases: maturation factor-1; Nose IV; MF1 Gene map locus: 11g12 Protein sequence Accession: NP_004102; gi: 4758356 Organism: Homo sapiens Size: 380 AA SEQ ID NO: 14 DNA sequence Accession: NM_004111; gi: 19718776 Open Reading Frame: 1 to 2265 SEQ ID NO: 41 PROTEIN SEQ ID NO: 15 dUTP pyrophosphatase isoform A and B: Protein aliases: deoxyuridine triphosphates; nucleotidohydrolase; deoxyuridine triphosphatase Gene map locus: 15q15-q21.1 Protein sequence Accession: P33316 Organism: Homo sapiens Size: AA252AA SEQ ID NO: 15 DNA sequence Accession: U90223.1 Open Reading Frame: 63 to 821 SEQ ID NO: 42 PROTEIN SEQ ID NO.16 Eukaryotic translation initiation factor 5A: Protein aliases: EIF5A; EIF5A1; eIF 4D Gene map locus: 17p13-p12 Protein sequence Accession: NP_001961.1 Organism: Homo sapiens Size: 154AA SEQ ID NO: 16 DNA sequence Accession: NM_001970.3 Open Reading Frame: 125 to 589 SEQ ID NO: 43

1 shows altered proteins in regulatory T cells containing proteins of the invention or their amino acid sequences compared to conventional CD4 + T cells.

cutouts from 2D gels of human protein spots, which differ in their protein spot intensity at the Comparison of activated conventional CD4 + T cells with activated CD25 + Tregs distinguished.

Claims (23)

Isolated regulatory CD4 + CD25 + T cell containing at least one protein corresponding to the amino acid sequences SEQ ID no. 1-16 and / or SEQ ID no. 17-27 or With SEQ ID no. 1-16 and / or SEQ ID no. 17-27 a sequence identity or homology of 70% and more. Isolated T regulatory cell according to claim 1, consisting of the subpopulation CD4 ⁺ CD25 ⁺ β7 ⁺ . Isolated regulatory T cell according to any one of claims 1 to 2, characterized in that at least one expressed protein according to the claims 1 or 2 secreted, membranous or on the surface the T-regulatory Cell or presented in the cytosol is. Isolated regulatory T cell according to any one of claims 1 to 3, characterized in that at least one expressed protein according to the claims 1 or 2 in the regulatory T cell or on the surface of the enriched in regulatory T cell. Isolated regulatory T cell according to any one of claims 1 to 4, characterized in that at least one nucleic acid encoding for at least a protein according to the claims Containing 1 or 2 and optionally one or more non-coding Sequences and / or a poly (A) sequence and / or recognition sequences and / or regulatory sequences, such as promoter or enhancer sequences. Isolated T regulatory cell according to any one of claims 1 to 5, characterized in that the nucleic acid sequence is selected from SEQ ID no. 28-43 and / or SEQ ID no. 44-54. Isolated or native regulatory CD4 ⁺ CD25 ⁺ T cell containing at least one protein according to claims 1 or 2 as target or marker. Binder attached to at least one isolated regulatory T cell after one the claims 1-7 or native regulatory T cell according to claim 7. Binder according to claim 8, selected from the group inhibitor, Agonist, antagonist, probe, antibody or immunomodulator. A binder according to any one of claims 8 or 9, wherein the binder one or more epitopes against at least one protein according to claims 1 or 2 has. A binder according to claim 10, wherein the binder additionally comprises or more epitopes against a surface protein. The binder of claim 11, wherein the surface protein selected is from the group CD25, CD44, CD45, GITR, CTLA-4, Galectin, Fox P3. A binder according to any one of claims 8 to 12, wherein the isolated containing regulatory T cell or native regulatory T cell at least one protein according to claims 1 or 2 activated or is deactivated. Medicament containing at least one binder according to one of the claims 8 to 13 or isolated T-regulatory Cells according to one of the claims 1 to 7. Medicament according to claim 14 for the treatment and Therapy of illnesses of allergies, autoimmune diseases, in particular rheumatoid arthritis, multiple sclerosis or morbus Crohn, Chronic Inflammation, Asthma, Immunodeficiency Diseases, AIDS, transplant rejection and cancers as well as diabetes. The pharmaceutical composition of claim 15, wherein the autoimmune diseases selected is from the group: Alopecia Areata, Ankylosing Spondylitis, Antiphospholipid Syndrome, Addison's Disease, Behcet's Disease, Celiac Sprue, chronic fatigue syndrome (Chronic Fatigue Immune Dysfunction Syndrome (CFIDS)), Polyneuropathy, Churg-Strauss syndrome (Granulomatosis), CREST syndrome (Raynaud's syndrome), cold agglutinin disease, cryoglobulinemia, fibromyalgia, Fibromyositis, Graves' disease, Guillain-Barré syndrome, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia, IgA nephropathy, lichen planus, Ménière's disease, polyarteritis Nodosa, Polychondritis, Polyglandular Syndrome, Polymyalgia Rheumatica, Primary agammaglobulinemia, biliary Cirrhosis, psoriasis, Reiter's disease, sarcoidosis, Sjogren's disease, Takayasu's arteritis, Vasculitis, vitiligo, Wegener's granulomatosis. Test system containing at least one binder and at least one regulatory T cell containing protein according to the claims 1 or 2, for the identification of suitable binders or regulatory T cells, preferably those with increased suppressive properties. Test system comprising at least one regulatory Containing T cell a protein according to any one of claims 1 or 2 and at least one target cell, in particular T cell, B cell, macrophage, Prädendritische Cell, dendritic cell, embryonic cell and / or fibroblast, which are incubated with at least one regulatory T cell for in vitro Detection of suppressive properties, in particular cellular immune response of effector cells of the immune system, in particular B cells, NK cells, preferably T cells, T helper cells. A test system according to claim 18, wherein the effector cells mammalian cells are, in particular human or murine cells or immune cell line and / or cultured primary Immune cell. Test system according to claim 18 or 19, wherein at least Another substance is incubated that can trigger an immune response, such as Proteins, epitopes, protein fragments, antigens or binders. Diagnostic containing a test system after a the claims 18 to 20 and optionally a pharmaceutically acceptable Carrier. Diagnostic agent according to claim 21 for the diagnosis of diseases namely from allergies, autoimmune diseases, especially rheumatoid Arthritis, Multiple Sclerosis or Crohn's Disease, Chronic Inflammation, Asthma, immunodeficiency disorders, AIDS, transplant rejection and cancers as well as diabetes. Diagnostic agent according to claim 22 for the diagnosis of diseases and of autoimmune diseases selected from the group Alopecia Areata, ankylosing spondylitis, antiphospholipid syndrome, Addison's disease, Behcet's disease, celiac disease Sprue, chronic fatigue syndrome (Chronic Fatigue Immune Dysfunction Syndrome (CFIDS)), Polyneuropathy, Churg-Strauss syndrome (granulomatosis), CREST syndrome (Raynaud's syndrome), Cold Agglutinin Disease, Cryoglobulinemia, Fibromyalgia, Fibromyositis, Graves' disease, Guillain-Barre syndrome, idiopathic pulmonary Fibrosis, idiopathic thrombocytopenia, IgA nephropathy, lichen Planus, Ménière's disease, polyarteritis Nodosa, Polychondritis, Polyglandular Syndrome, Polymyalgia Rheumatica, Primary Agammaglobulinemia, biliary Cirrhosis, psoriasis, Reiter's disease, sarcoidosis, Sjogren's disease, Takayasu's arteritis, Vasculitis, vitiligo, Wegener's granulomatosis.