EP2115128A2 - Method for modulating the immune response by activating or inactivating the notch and/or stat4 signal path - Google Patents

Abstract

The invention relates to a method for modulating the inflammatory potential of T cells, particularly by regulating the IL-10 production in pro-inflammatory T cells by influencing the activity or the activation of the notch and STAT-4 signal path. The invention further relates to the use of said method for controlling inflammations or for immune suppression. The invention particularly relates to the use of said method for treating diseases associated with inflammations. The invention also relates to the use of said method for reducing the IL-10 production in pro-inflammatory T cells and thus reinforcing T cell-induced immune response, especially in case of infections, tumor diseases, or vaccinations against infections or tumors.

Description

 Method for modulating the immune response by activation or inactivation of the Notch and / or STAT4 signaling pathway

description

The invention relates to a method for the targeted modulation of anti-inflammatory functions of T cells by [influencing the Notch and the STAT-4 signaling pathway.

The invention comprises:

1. the generation of anti-inflammatory T cells, in particular of interleukin (IL) 10-producing T cells, by the combined activation of the Notch and / or STAT-4 signaling pathway. The invention further relates to the use of the method for the inhibition of inflammation or for immunosuppression, in particular the invention relates to the use of the method for the treatment of diseases associated with inflammation and / or

2. the generation of T cells with enhanced inflammatory function, in particular by shutting down IL-10 production in T cells by blocking the Notch and STAT-4 signaling pathway. In this regard, the invention further relates to the use of the method for enhancing immune defense, in particular the invention relates to the use of the method for the treatment of diseases associated with infections or tumors.

The immune system also allows effective defense against pathogens and tolerance to the body's own cells and harmless substances. T helper cells play a key role in maintaining this balance. Failure can lead to autoimmune diseases or allergies.

The various tasks of T helper cells according Subpopulat there are different ^'ones of these cells. They are characterized by the differential expression of soluble messengers, the cytokines.

A subpopulation of T helper cells, the so-called Th1 cells, express cytokines that can direct the cellular immune response. These include interferon (IFN) gamma and tumor necrosis factor (TNF) alpha and beta. IFN-gamma is responsible for the activation of macrophages and is necessary for the fight against intracellular pathogens and viruses. Th1 cells are inflammatory through their cytokines and are therefore also called pro-inflammatory. Another subpopulation of T helper cells, the so-called Th17 cells, are characterized by the production of the cytokine IL-17 and also have a pro-inflammatory effect. Th17 cells seem to be particularly important for the development of autoimmune diseases.

While this pro-inflammatory function is necessary for successful immune defense, chronic inflammation or autoimmune disease can occur without appropriate counterregulation. To prevent an uncontrolled activation of immune cells, the immune system uses different mechanisms. One is the secretion of immunosuppressive cytokines. Of these, above all, the IL-10 is of crucial importance.

IL-10 was originally identified as a factor that inhibits cytokine secretion of Th1 cells and is produced by so-called Th2 cells. It is also produced by various populations of regulatory T cells (CD25 + FoxP3 + and tri-cells). IL-10 has anti-inflammatory and suppressive effects on most hematopoietic cells. It indirectly inhibits cytokine product ion and proliferation of antigen-specific effector T cells by inhibiting antigen presentation by various APCs such as dendritic cells, Langerhans cells and macrophages.

It is now known that pro-inflammatory T cells can also produce IL-10 themselves in order to limit the inflammatory response triggered by them and thus prevent immunopathologies. However, it was not clear by which signals the IL-10 production in the pro-inflammatory T-cells can be regulated. In particular, no method was known to induce or shut down IL-10 production in already established pro-inflammatory Th1 / Th17 cells, so-called memory cells.

Methods have been described in which naive, ie antigen-inexperienced T cells, IL-10 producing T cells can be generated (Tri-cells). These usually produce no or few pro-inflammatory cytokines except IL-10 and therefore have an anti-inflammatory effect. Described are the in vitro culture with immunosuppressants such as IL-10 itself, dexamethasone, vitamin D (et al.). Furthermore, there are reports that by notch signals alone, the emergence of tri-cells can be achieved. Notch can also induce Th1 or Th2 T cells. The exact molecular mechanisms and possibly necessary co-factors for these different differentiation pathways are unknown. Recently, however, it has been shown that Notch signals via the ligand Delta-like 4 together with interleukin-12 are necessary to induce a strong pro-inflammatory Th1 response to viral infections. Whether and how Notch induces regulatory T cells is therefore unknown.

There are reports that Notch signals in general in combination with interferons and thereby mainly type I interferons lead to an increased IL-10 production and at the same time pro-inflammatory molecules are reduced. Interferon treatment is thought to activate STAT1 and STAT2, which mediate the intracellular effects of the interferon signal. In addition, these reports do not differentiate by awake ligands, delta or Jagged family that can induce Notch effects. At the same time, there are reports that plasmacytoid dendritic cells (pDC) can induce IL-10 in T cells. It is believed that this is due to ICOS ligand or type I interferons, both of which are highly expressed by pDC upon activation.

These different methods are essentially also characterized in that naive T cells or not-specifically defined T cells (mixture of naive and memory T cells) which are relatively easy to differentiate by means of specific signals are used as the starting cells can be brought. On the other hand, it is not known how in already differentiated memory T cells, as they are usually present in already established immunopathologies, a modulation can be achieved. In memory cells, certain differentiation steps are already fixed, which generally makes the EB influence of memory T cells more difficult, which makes therapy of established inflammatory diseases more difficult.

The production of IL-10 by actually pro-inflammatory T cells is to be regarded as a regulatory function intended to prevent excessive immune responses and the resulting immunopathology. On the other hand, IL-10 production can also prevent effective pathogen defense. For example, in several models of infection, IL-10-producing Th1 cells are responsible for the inability to effectively eliminate the pathogens and to chronicle the infection. The molecular signals that could be responsible for this are not defined more precisely, but the mechanisms described above may be relevant. Targeted and selective blockade of IL-10 production means that immune responses directed against pathogens can be enhanced or vaccinations against pathogens or tumors can be improved.

The object of the invention was to provide a method by which inflammatory processes can be inhibited or enhanced by the modulation of inflammatory mediating cells in an organism or in vitro, in particular by enhancing IL-10 production directly in the inflammatory T cells or is switched off. In particular, a possibility should be created to effect the alteration of IL-10 production also in memory T cells and in T cells from chronic inflammatory situations. Particularly preferably, the method should be used or used independently of interferon.

The invention relates to the surprising teaching that a method for the modulation and generation of anti-inflammatory functions in T cells, in particular by the modulation of IL-10 production, in vitro or in vivo, preferably in inflammatory reactions can be provided, wherein note and STAT4 signals are modulated, whereby inhibition of the signals reduces IL-10 production and enhances the inflammatory response and activation of the signals enhances IL-10 production and decreases the inflammatory response.

It was completely surprising that the object according to the invention can be achieved with a method for generating modified T helper cells, whereby Th1 cells or Th17 cells are brought into contact with signal-active Notch molecules and signal-activated STAT4 molecules. The combined activation of Notch and STAT4, simultaneously or with a time delay, shuts off or reduces the proinflammatory effect of Th1 / 17 by inhibiting protective, in particular IL- 10-producing, T cells are obtained. Accordingly, the method according to the invention relates, on the one hand, to the modulation of anti-inflammatory functions in T cells and, on the other hand, to the generation of protective anti-inflammatory, in particular IL-10-producing cells, T cells having signal-active Notch molecules and signal-active STAT-4 Molecules are brought into contact, whereby the T cells are obtained and the modulation of the anti-inflammatory function of T cells is possible.

The invention also relates to a method for modulating anti-inflammatory functions in T cells, in particular for switching off IL-10 in these cells and generating highly reactive inflammatory T cells, wherein the contacting of the T cells with signal-active Notch or STAT-4 is prevented and thus the T-cells are obtained.

In particular, it was surprising that activation of STAT-4 (but not STAT-1 or STAT-2 or ICOS) with Notch was necessary for IL-10 induction, since STAT4 was mainly responsible for the development of pro-inflammatory Th1 / Th17 cells is done. Furthermore, it was surprising that the Notch ligands from the Delta-like family, in particular Delta-Iike4, cause the IL-10 induction, while Jagged has no or rather opposite effects. The process according to the invention can preferably be used or used independently of interferon.

It was also surprising that pDC, particularly after stimulation with ligands of the TolWike receptors (TLR 1-13 and in particular TLR9), express particularly large amounts of delta-like 4 and, under these conditions, particularly efficiently the induction of IL-10 in T cells cause.

Furthermore, it was also completely surprising that by blocking the Notch signaling pathway, amplification of the T-cell response can be achieved, in particular by reducing or shutting down IL-10 FToduction of the proinflammatory T-cells.

The application according to the method is characterized by the following advantages:

- departure from the technical usual

- new task

- There is a long unresolved urgent need for the solution of the problem solved by the invention

- Past futile efforts of the experts

- The simplicity of the solution speaks to inventive step, especially as it replaces more complicated teachings

- Development of scientific technology went in a different direction

- development-firming achievement - misconceptions of the experts about the solution of the corresponding problem (prejudice)

^■ technical progress, such as Improvement, performance increase, cheapening, saving time, material, work stages, costs or difficult-to-procure raw materials, increased reliability, elimination of errors, quality improvement, freedom from maintenance, greater efficiency, higher yield, increase in technical possibilities, provision of a further means, opening of a second path, opening of a new area, first solution of a task, reserve resources, alternatives, possibility of rationalization, automation or miniaturization or enrichment of the drug

- happy grip, because of a variety of possibilities was chosen a particular whose result could not be predicted, so it is a patentable lucky grip)

^■ Errors in the literature or very contradictory presentation of the subject of the invention

^■ young field of technology

Combination invention, i. several known elements are combined to a combination which has a surprising effect

- Licensing

- praise the experts and

- economic success.

In particular, the advantageous embodiments of the invention have at least one or more of the advantages mentioned.

It was surprising that the modification of naive and memory T helper cells by a combination of Notch and STAT-4 can be used simply, safely, and effectively to limit inflammation-promoting processes.

Furthermore, it was surprising that pro-inflammatory Th1 and Th17 cells, when brought into contact with signal-active Notch and signal-activated STAT-4, are converted to anti-inflammatory T cells, particularly those producing IL-10.

Furthermore, it was also surprising that by blocking the Notch signaling pathway after immunizations with antigen plus adjuvants (e.g., TLR ligands), a decrease in IL-10 production and enhancement of the inflammatory T cell response can be achieved.

The following are some terms to be explained and illustrated. Modulation of anti-inflammatory functions in T cells

By this is meant in particular an influencing of the expression of anti-inflammatory factors, in particular of IL-10, including anti-inflammatory soluble messengers and / or surface molecules. Modulation expresses that influencing in two different directions is possible:

in the case of inflammatory diseases, enhancement of these factors would be desirable and would be achieved by activating the Notch signaling pathway;

in the case of (tumor) vaccination, it is preferable to reduce anti-inflammatory factors which can be brought about by inhibition of the distress pathway.

Signal-active Notch and STAT-4 molecules

For both Notch and STAT4, they are always present in a latent form in the cell. This latent form is converted by activators into a (signal) active (signal active = active) form. This is done by proteolytic cleavage in the case of the Notch receptor or by phosphorylation in the case of STAT4. This means at the same time that the active form has a different structure than the latent form.

As a result, a signal-active form can be brought into the cell in two ways

a) By physiological activation of the receptors: Notch activators according to this definition are Notch ligands or stimulating anti-Notch antibodies. In the case of STAT4, these are (in an IFN-independent manner) the cytokines of the IL-12 family, IL-12, IL-23, IL-27 and IL-35.

b) By direct transfection of cells with the constitutively active form, that would not be the case with Notch

Notch-intracellular domain (NICD) and in STAT4, a modified STAT4 molecule that is phosphorylated in the cell independently of the receptor and thereby active.

Highly reactive inflammatory T cells

Highly reactive inflammatory T cells are inflammatory T cells in which, in particular, the production of the anti-inflammatory cytokine IL-10 is inhibited (e.g., by blocking Notch).

Notch ligand fragments or protein fragments

Notch ligand fragments or protein fragments are those as disclosed in WO 2004/024764 A1. The constructs and fragments of the Notch ligands disclosed therein can either be used for activation or inhibition of the Notch signaling pathway. Preferably, the fragments have substantially the same activity as the molecule from which they were recovered.

Substances that inhibit the natural activation or activity of STAT-4 The inhibitors of STAT4 include, in particular, blocking antibodies against the STAT4-activating cytokines (eg anti-IL-12, anti-IL-23, anti-IL-27, ano-IL-35), and in particular blocking antibodies against the corresponding cytokine receptors on the cells. In the narrower sense, STAT4 inhibitors would also be substances from the class of decoy oligonucleotides (nucleotides that contain STAT4 binding sites and thus bind the active site in the cell)

Inhibit STAT4 to the target DNA) or proteins or their fragments or derivatives from the group of STAT-interacting proteins, such as SHIP, PIAS or SOCS. The latter are STAT's natural interaction partners that can modulate activity and be used as specific inhibitors after cell introduction.

Pharmacological inhibitors of Notch activation

Pharmacological inhibitors of the Notch signaling pathway include in particular substances of the class gamma-secretase inhibitors.

In addition, other inhibitors of the Notch signaling pathway should be included, such as soluble Notch ligands, their fragments, fusion proteins or individual peptides as disclosed in WO2004 / 024764. Furthermore, in particular naturally blocking antibodies are claimed against the Notch receptors with. The inhibition of Notch activation is also possible by transfecting the cells with negative regulators of the Notch signaling pathway. Here, deltex, MINT, NRARP or dominant negative forms of mastermind are preferred agents known to those skilled in the art.

The term "modulation of the inflammatory potential" corresponds to the term "modulation of the (anti) -inflammatory function". The strengthening or weakening of the anti-inflammatory T-cell function (this is preferably the IL-10 production) are in the context of the invention alternative ways of manipulation by Notch, which can be used depending on the nature of the disease.

In a preferred embodiment of the invention, it is provided that the Th1 / Th17 cells are additionally brought into contact with signal-active STAT-4 molecules. The combined activation of the Notch and STAT-4 signaling pathway is necessary to achieve pro-inflammatory conversion

To achieve Th1 / Th17 cells in anti-inflammatory cells with suppressive properties.

In a further preferred embodiment, in the method according to the invention, the T cells are selected from the group comprising naive T cells and / or memory cells. It is particularly preferred if the T cells are memory T cells.

In a further preferred embodiment of the invention, it is provided that the T cells are selected from the group of the inflammatory Th1 and / or TM7 cells.

In a further preferred embodiment of the invention, it is provided that the Notch molecules in the context of the invention are selected from the group comprising the Notch receptors or the Notch ligands. In particular, in a preferred embodiment of the invention, the Notch receptors according to the invention are molecules which are selected from the group comprising Notch-1, -2, -3 and / or -4. The Notch ligands are in particular molecules which are selected from the group comprising molecules of the Jagged family, in particular Jagged-1 and / or -2 and / or Delta-like (DII) family, in particular DIM, 2, -3 and / or -4.

In a further preferred embodiment of the invention it is provided that the ligands used in the method are molecules of the delta-like family, in particular delta-Iike4 and / or delta-likei.

Furthermore, in a preferred embodiment it is provided that the contacting with STAT-4 molecules takes place by the activation with cytokines, in particular by the cytokines IL-12, IL-23 and / or IL-27 or by overexpression of signal-active STAT-4, in particular by transduction of T cells.

In a further preferred embodiment of the invention, the contacting with STAT-4 molecules takes place by the overexpression of molecules which increase or enhance the STAT-4 activation, in particular the cytokine receptors, preferably selected from the group comprising IL -12, IL-23 and / or IL-27 and / or fusion proteins of these cytokines with their receptor or STAT-4 or signal-active STAT-4, preferably by transduction of the T cells.

In a particularly preferred embodiment of the invention it is provided that the bringing into contact in the context of the invention by overexpression of signal-active Notch-1, -2, -3 and / or -4 takes place or by the bringing into contact of endogenous Notch molecules with Notch ligands. Methods are known to the person skilled in the art to induce the overexpression of certain FToteins as well as to produce signal-active variants of proteins. Furthermore, those skilled in the art are familiar with methods for contacting Notch endogenous molecules with Notch ligands such that the Notch signaling pathway is activated.

In a particularly preferred embodiment of the invention, the contacting with the Notch ligands is carried out by stimulation with antigen-presenting cells, wherein the contacting in particular via the activation of TolHike receptors (TLR) and / or CD40 on the Amplified and / or induced antigen-presenting cells. Methods are known to those skilled in the art to activate TLR. The ligands are preferably molecules of the Delta-Jike family, in particular Delta-Iike4 and / or Delta-likel. In particular, the TLRs are molecules selected from the group comprising TLR-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11 , -12 and / or -13.

In a further preferred embodiment of the invention, it is provided that contacting with the Notch ligands takes place by stimulation with plasmacytoid dendritic cells, the contacting in particular by stimulation of the plasmacytoid dendritic cells via TolHike receptors and / or CD40 is amplified and / or induced. In a particularly preferred embodiment of the invention, contacting with the Notch ligands takes place by stimulation with plasmacytoid dendritic cells, the contacting being intensified and / or induced in particular via TLR. Plasmacytoid dendritic cells express the Notch ligands Delta-like-1 and-4 particularly strongly.

In a further preferred embodiment of the invention, the contacting with signal-active STAT-4 molecules takes place by the addition of cytokines, in particular by the cytokines IL-12, IL-23, IL-27.

In a further preferred embodiment of the invention, the contacting with signal-active STAT-4 molecules takes place by the overexpression of signal-active STAT-4. Methods are known to those skilled in the art to induce the overexpression of certain proteins and to produce signal-active variants of proteins.

In a further embodiment of the invention, it is preferred that the generation of the protective, in particular the IL-10-producing, T cells in vitro by stimulation with Notchligand- proteins or signal-active fragments of Notch ligands, by a stimulation with Notchligand- expressing cells and / or by introducing signal-active Notch by viral and / or non-viral transduction methods in the presence of Th1 / Th17 cells.

In a further preferred embodiment of the invention, it is provided that the generation of the protective, in particular the IL-10-producing, T cells in vivo by the application of recombinant Notch ligands and / or Notchligandfusionsproteinen and / or signal-active Notchligandfrag- elements and / or by applying Notch ligand-expressing cells. In particular, plasmacytoid dendritic cells are suitable for the Notch ligand-expressing cells.

Accordingly, in a preferred embodiment, the invention relates to a method wherein the generation of IL-10 producing T cells in vitro by stimulation with Notchligand proteins, Notchligand fusion proteins and / or signal-active Notchligand fragments by stimulation with Notchligand-expressing cells and / or by introducing signal-active Notch by viral and / or non-viral transduction methods in the presence of signal-activated STAT-4 molecules.

In a further preferred embodiment, the invention relates to a method wherein the generation of IL-10 producing T cells in vivo by the application of recombinant Notch ligands and / or by the application of Notch ligand-expressing cells in the presence of signal-active STAT -4 is done.

In a further preferred embodiment, the invention relates to a method wherein the inhibition of Notch and / or STAT-4 activation by antibodies against Notch or Notch ligands and / or against STAT-4 activating cytokines, in particular IL-12, IL-23, IL-27 takes place and / or its receptor and / or by recombinant proteins or protein fragments or peptides of Notch or Notchligan- den. In a further preferred embodiment, the invention relates to a method wherein the inhibition of STAT4 activation is effected by substances which inhibit the natural activation or activity of STAT4.

In a further preferred embodiment, the invention relates to a method wherein the inhibition of the activation of the note is effected by the use of pharmacological inhibitors of Notch activation, e.g. so-called γ-secretase inhibitors.

In a further preferred embodiment, the invention relates to a method wherein the inhibition takes place in the course of an active immune reaction, in particular in the case of infections, tumor diseases, vaccinations.

In a further preferred embodiment, the invention relates to a method in which the inhibition takes place in the course of a vaccination in which antigens, in particular tumors or pathogens are used together with adjuvants, preferably ligands for TolHike receptors (TLR), antj CD40 and / or CD40 ligand.

In a further preferred embodiment, the invention relates to a process wherein the adjuvants from the group of ligands for TLR9, preferably oligonucleotides comprising methylated CpG motifs.

In a further preferred embodiment, the invention relates to a method wherein the adjuvants from the group of antibodies and / or ligands for CD40 originate.

In a further preferred embodiment, the invention relates to a method wherein the inhibition takes place in the course of a vaccination with dendritic cells or other antigen-presenting cells. In particular, by the use of dendritic cells or other antigen-presenting cells for immunization in which the expression of the Notch ligands is inhibited (for example by transduction with siRMA or antisense oligo-nucleotides against the Notch ligands).

In a further preferred embodiment, the invention relates to a method in which the expression of the Notch ligands of the delta-like family is inhibited in the dendritic cells or other antigen-presenting cells, in particular the ligands Delta-likel and Delta-Jike 4.

In a further preferred aspect, the invention relates to the use of the method according to the invention for the inhibition of inflammation.

In a further preferred aspect, the invention relates to the use of the method according to the invention for immunosuppression.

In a further preferred aspect, the invention relates to the use of the method according to the invention for the induction of interleukin-10 (IL-10).

In a further preferred aspect, the invention relates to the use of the method according to the invention for activating the immune response. In a further preferred aspect, the invention relates to the use of the method according to the invention for enhancing vaccinations.

In a further preferred aspect, the invention relates to the use of the method according to the invention for the inhibition of interleukin-10 (IL-10) expression.

In another preferred aspect, the invention relates to the use of the method of the invention for the manufacture of a medicament for inducing interleukin-10 (IL-10) for immunosuppression in organ transplantation.

In a further preferred embodiment of the invention, it is envisaged to inhibit joint activation of Notch and STAT4 in T cells in order to achieve an enhancement of the immune response by reducing IL-10 production. This can be achieved by pharmacological inhibition of the Notch signal (the person skilled in methods are known to pharmacologically inhibit the Notch activation, eg by the drug class of the γ-secretase inhibitors) or by administering antibodies to Notch ligands (especially the delta-like Family and in particular against delta-like 4) or by parts of the ligands or peptides or other molecules that prevent the signal-activating interaction between Notch and Notch ligands. Furthermore, the effect can be achieved by inhibiting STAT4 activation. This can be achieved by antibodies to STAT4 activating cytokines (IL-12, IL-23, IL-27, type I interferons) or their receptors, or by pharmacological agents that block STAT4 activation or function.

In a further aspect, the invention relates to the use of the methods according to the invention for the inhibition of inflammation, for immunosuppression and / or for the induction of IL-10.

In a further aspect, the invention relates to the use of the methods according to the invention for the activation of pro-inflammatory T-cell functions, for vaccinations in infections or in tumor diseases and / or for the inhibition of IL-10 production.

The invention also relates to the use of the method according to the invention for the production of a medicament for the inhibition of inflammation, in particular for the treatment of diseases selected from the group comprising HASHIMOTO thyroiditis, primary myxedema, thyrotoxicosis (BASEDOW disease), pernicious anemia, ADDISON disease, myasthenia gravis, juvenile Diabetes mellitus, GOODPASTURE syndrome, autoimmune hemolytic anemia, autoimmune leukopenia, pemphigus vulgaris, sympathetic ophthalmia, primary biliary cirrhosis, especially primary biliary cirrhosis, autoimmune hepatitis, especially chronic aggressive autoimmune hepatitis,

SJÖGREN syndrome, rheumatoid arthritis, rheumatic fever, systemic lupus erythematosus, dermatomyositis / polymyositis, progressive systemic sclerosis, WEGENER granulomatosis, panoritic nodosa, hypersensitivity angiitis, thyrotoxicosis, thyroid-related myxedema, generalized endocrinopathy, chronic type A gastritis, autoimmune diseases of some or all corpuscular elements of the blood, especially idiopath. Thrombocytopenia or pathophysiology; idiopath. Leukopenia, agranulocytosis, pemphigoid, uveitis, diabetes mellitus type I, CROHN disease, ulcerative colitis, ADDISON disease, lupus erythematosus disseminatus and as a discoid form of this Disease, as dermatomyositis and scleroderma, rheumatoid arthritis (= primary chronic polyarthritis), antiglomerulus basal membrane nephritis, an aggressive immune response due to collapse of immune tolerance to self-termerminants and a decrease in the activity of T-suppressor cells favored with lymphocyte marker T 8 or overweight T helper cells preferentially with T 4 lymphocyte marker via the suppressor cells; Formation of autoantigens, in particular by linking host proteins with haptens, preferably pharmaceuticals, immune reactions by ontogenetic tissue, which develops only after development of self-tolerance and for protein components unmasked by changes in the conformation of the proteins, preferably in connection with infection by viruses or bacteria and / or for new proteins associated with neoplasia.

In a further preferred embodiment, the invention also relates to the use of the method according to the invention for the production of a medicament for immunosuppression for the treatment of diseases from the group comprising AIDS, acne, albuminuria (proteinuria), alcohol withdrawal syndrome, allergies, alopecia (hair loss), ALS ( Amyotrophic lateral sclerosis), Alzheimer's disease, AMD (age-related macular degeneration), anemia, anxiety disorders, anthrax (anthrax), aortic sclerosis, arterial occlusive arteriosclerosis, arterial occlusion, temporal arteritis, atherosclerosis, arteriovenous fistulas, arthritis, arthrosis, asthma, respiratory insufficiency, Autoimmune disease, AV block, acidosis, herniated disc, peritonitis, pancreatic cancer, Becker muscular dystrophy, benign prostatic hyperplasia (BPH), bladder carcinoma, haemophilia, bronchial carcinoma, breast cancer, BSE, Budd-Chiari syndrome, bulimia nervosa, bursitis

(Bursitis), Byler syndrome, Bypass, Chlamydia infection, Chronic pain, Cirrhosis, Commotio cerebri (concussion), Creutzfeld ^ Jakob disease, colon carcinoma, colon cancer, intestinal tuberculosis, depression, diabetes insipidus, diabetes mellitus, juvenile diabetes mellitus, diabetic Retinopathy, Duchenne muscular dystrophy, duodenal carcinoma, dystrophia musculorum progressiva, dystrophy, Ebola, eczema, erectile dysfunction, obesity fibrosis, cervix cancer, uterine cancer, cerebral hemorrhage, encephalitis, alopecia, hemiplegia, hemolytic anemia, hemophilia, pet allergy (animal hair allergy), skin cancer, Herpes zoster, myocardial infarction, heart failure, heart valve inflammation, brain metastasis, stroke, brain tumor, testicular cancer, ischemia, Kahler's disease (plasmocytoma), polio (poliomyelitis), bone loss, contact eczema, paralysis, liver cirrhosis, leukemia, pulmonary fibrosis, lung cancer, pulmonary edema, lymph node cancer (Hodgk's disease in), lymphogranulomatosis, lymphoma, lyssa, gastric carcinoma, breast carcinoma, meningitis, anthrax, cystic fibrosis, multiple sclerosis (MS), myocardial infarction, atopic dermatitis, neurofibromatosis, neural tumors, kidney cancer (renal cell carcinoma), osteoporosis, pancreatic carcinoma, pneumonia, polyneuropathies , Potency Disorders, Progressive Systemic Sclerosis (PSS), prostate cancer, urticaria, cross-sectional syndrome, traumatic, rectal, pleurisy, craniocerebral trauma, vaginal carcinoma, sinusitis, esophageal cancer, tremor, tuberculosis, tumor pain, vaginal carcinoma, burn - Scalding, intoxication, viral meningitis, menopause, soft tissue sarcoma, soft tissue tumor, cerebral circulatory disorders and / or CNS tumors. In a further preferred embodiment, the invention also relates to the use of the method according to the invention for the production of a medicament for immunosuppression in organ transplantations.

In a further preferred embodiment, the invention relates to the use of the method according to the invention for the induction of interleukin-10 in vivo or in vitro, in particular in a patient, for the treatment of the above-mentioned. Diseases.

In addition, the invention will be explained in more detail by means of examples, without being limited to this example.

example 1

Naive, i. antigen-unexercised, CD4 + CD25-CD62L + T helper cells were isolated by flow cytometry. The cells were then cultured in a ratio of 1: 4 with irradiated (30 Gy) MHCII + antagonist cells in the presence of interleukin-12 (10 ng / ml). The stimulation was carried out in a polyclonal manner with 0.5 μg / ml antπCD3 and 1 μg / ml anti-CD28. After 24 h, the cells were retrovirally transduced with active Notch-3 (Notch-3IC). For this purpose, the cells were centrifuged with virus supernatant for 75 min at 75Ox g and 32 ° C. After 5 days, the cells were restimulated with PMA / ionomycin and their cytokine expression analyzed by intracellular staining.

As shown in Figure 1, control cells produce IFN-gamma and IL-2 under these conditions, but not immunosuppressive IL-10. Notch transduced helper T cells, on the other hand, produce less IL-2, but high levels of IL-10 and IFN-gamma. No IL-4 expression is observed.

The Notch-modified Th1 cells simultaneously express the cytokines IFN-gamma and IL-10, as can be seen in FIG

Example 2

Naive CD4 + CD25-CD62L + Th cells were isolated by flow cytometry and incubated for a week under Th1 conditions (10 ng / ml IL-12, 10 μg / ml WL4, 0.5 μg / ml anti-CD3, 1 μg / ml antithrombin). CD28). Subsequently, IFN-gamma producing cells were isolated by flow cytometry using the Miltenyi IFN-gamma secretion assay. The cells were cultured again with irradiated MHCII + antjg presenting cells under Th1 conditions. After 24 h, cells with active Notch were retrovirally transduced. After a further 5 days, the cells were restimulated with PMA / ionomycin and their cytokine expression analyzed. As shown in Figure 3, control cells produce IFN-gamma under these conditions, but no IL-10. Notch-transduced T helper cells, on the other hand, produce high levels of IL-10 and IFN-gamma. The differences in the IL-10 production are particularly evident when measuring the cytokine in the culture supernatant (by ELISA). Example 3

Naive CD4 + CD25-CD62L + T helper cells from wild type (C57BL / 6) or STAT4 deficient mice were isolated by flow cytometry. Cells were then cultured 1: 4 with irradiated (30 Gy) MHCII + antigen-presenting cells in the presence of interleukin-12 (10 ng / ml). Stimulation was polyclonal with 0.5 μg / ml anti-CD3 and 1 μg / ml anti-CD28. After 24 h, the cells were retrovirally transduced with active Notch-3 (Notch-3IC). For this purpose, the cells were centrifuged with virus supernatant for 75 min at 75Ox g and 32 ° C. After 5 days, the cells were restimulated with PMA / ionomycin and their cytokine production analyzed by ELISA.

As shown in Figure 4, Notch-transduced Th cells from wild-type mice produce high levels of IL-10. In the case of STAT4 deficient cells, no IL-10 production is observed. It is thus advantageous to activate the Notch and STAT4 signaling pathways to induce IL-10.

Example 4

Naive CD4 + CD25-CD62L + Th cells were isolated by flow cytometry and analyzed under Th1 conditions (10 ng / ml IL-12, 10 μg / ml anti-IL4, 0.5 μg / ml anti-CD3, 1 μg / ml anti-CD28 ). After 24 h, cells were transduced retrovirally with active forms of the various Notch isotypes (Notch-1 to -4). For this purpose, the cells were centrifuged with virus supernatant for 75 min at 75Ox g and 32 ° C. After 5 days, the cells were restimulated with PMA / ionomycin and their cytokine expression analyzed by intracellular staining.

As Figure 5 shows, in each case IL-10 induction is observed. Thus, all four Notch isoforms can equally lead to IL-10 induction.

Example 5

Naive CD4 + CD25-CD62L + T helper cells from wild type (C57BL / 6) or STAT4 deficient mice were isolated by flow cytometry. Cells were then cultured 1: 4 with irradiated (30 Gy) MHCII + antigen-presenting cells in the presence of interleukin-27 (10 ng / ml). Stimulation was polyclonal with 0.5 μg / ml anti-CD3 and 1 μg / ml anti-CD28. After 24 h, the cells were retrovirally transduced with active Notch-3 (Notch-3IC). For this purpose, the cells were centrifuged with virus supernatant for 75 min at 75Ox g and 32 ° C. After 5 days, the cells were restimulated with PMA / ionomycin and their cytokine production analyzed by ELISA.

As shown in Figure 6, Notch-transduced Th cells from wild-type mice produce high levels of IL-10. In the case of STAT4 deficient cells, no IL-10 production is observed. Thus, the administration of IL-27, as well as IL-12 STAGE-dependent with Notch, induce induction of IL-10.

Example 6

Naive CD4 + CD25-CD62L + Th cells were isolated by flow cytometry and cultured in the absence of antigen-presenting cells under Th1 conditions (10 ng / ml IL-12, 10 μg / ml anti-IL4). The Stimulation was carried out with immobilized anti-CD3 (3 μg / ml) and 1 μg / ml anti-CD28. After 24 h, the cells were retrovirally transduced with active Notch-3 (Notch-3IC). For this purpose, the cells were centrifuged with virus supernatant for 75 min at 75Ox g and 32 ° C. After 5 days, the cells were restimulated with PMA / ionomycin and their cytokine expression analyzed by intracellular staining.

As shown in Figure 7, even in the absence of antigen presenting cells under Th1 conditions, score induces the immunosuppressive cytokine IL-10. It can therefore be concluded that no further costimulatory signals are necessary for this purpose.

Example 7

Naive CD4 + CD25-CD62L + T helper cells were isolated by flow cytometry. Cells were then cultured 1: 4 with irradiated (30 Gy) MHCII + antigen-presenting cells in the presence of interleukin-12 (10 ng / ml). Stimulation was polyclonal with 0.5 μg / ml anti-CD3 and 1 μg / ml anti-CD28. After 24 h, the cells were retrovirally transduced with active Notch-3 (Notch-3IC). For this purpose, the cells were centrifuged with virus supernatant for 75 min at 75Ox g and 32 ° C. After 5 days, GFP +, i. Notch or control transduced cells isolated by flow cytometry. Subsequently, naive CD4 + CD25-CD62L + Thy1.1 + congenic Th cells and CD11 c + dendritic cells from spleen and lymph nodes were isolated from naive mice. The naive T cells were labeled with the proliferation marker CFDA-SE and cultured in a ratio of 2: 1 with the Notch or control-transduced cells together with dendritic cells (total T cell to dendritic cell ratio 35: 1) for 3 days , Subsequently, the proliferation of the naive T cells was analyzed by flow cytometry.

As can be seen in Figure 8, Notch-transduced cells suppress the proliferation of naive Th cells. This suppression mediated by the Notch-modified Th1 cells is particularly pronounced with the addition of IL-12 (10 ng / ml).

Example 8

Antigen-specific (OVA-TZR transgenic) naive CD4 + CD25-CD62L + T helper cells were isolated by flow cytometry. Cells were then cultured 1: 4 with irradiated (30 Gy) MHCII + antigen-presenting cells in the presence of interleukin-12 (10 ng / ml). Stimulation was with 0.2 μg / ml OVA peptide. After 24 h, the cells were retrovirally transduced with active Notch-3 (Notch-3IC). For this purpose, the cells were centrifuged with virus supernatant for 75 min at 75Ox g and 32 ° C. After 5 days, GFP +, i. Notch or control transduced cells, isolated by flow cytometry.

If antigen-specific Notch-transduced Th1 cells are transferred into recipient animals and the animals subsequently immunized with the antigen (OVA peptide in IFA), an inflammatory reaction does not occur, as in the case of transferred control Th1 cells (DTHX figure 9A) , Noteh-modified Th1 cells invert their pro-inflammatory potential. Moreover, when transferred together with control Th1 cells, they are capable of suppressing the inflammatory response caused by the control cells, ie, of acting protective (Figure 9B). If in the recipient animals IL- 10, the suppression mediated by Notch-transduced Th1 cells is abolished (Figure 9C).

Example 9

Antigen-specific (OVA-TZR transgenic) naive CD4 + CD25-CD62L + T helper cells were isolated by flow cytometry and incubated with A20 B cells expressing the Notch ligands, DII-1 or DII-4 (in the ratio 2: 1), cultured in the presence of IL-12 (10 ng / ml) and stimulated with OVA peptide (0.2 μg / ml). After 5 days, the cells were restimulated with PMA / ionomycin and their cytokine expression analyzed by intracellular staining.

As shown in Figure 10, after coculture with delta-like ligands, IL-10 production is induced in the T cells. This induction is mediated by an inhibitor of the Notch signaling pathway (gamma-

Secretase inhibitor, 125 nM Insolution gamma-secretase inhibitorX, Calbiochem) specifically blockable (Figure 10A). Further cocultivation in the presence of the ligands DIM and DIW dramatically enhances IL-10 production (FIG. 10B).

Example 10

CD11c positive dendritic cells were isolated by flow cytometry from spleen and lymph nodes from naive mice and incubated for 15 h with 50 μg / ml pIC (TLR3), 1 μg / ml LPS (TLR4), 1 μg / ml flagellin (TLR5) or 1 μM CpG (TLR9). As shown in FIG. 11, this leads to increased expression of the Notch ligand Delta-like 4. The expression of Delta-like 1 and Jagged-1 remains largely unchanged.

Example 11

Antigen specific (OVA-TZR transgenic) naive CD4 + CD25-CD62L + T helper cells were isolated by flow cytometry and incubated with spleen and lymph node CD11c + dendritic cells (20: 1 ratio) in the presence of IL-12 (10 ng / ml). and / or 1 μM CpG and stimulated with OVA peptide (0.2 μg / ml). After 5 days, the cells were restimulated with PMA / ionomycin and their cytokine expression analyzed by intracellular staining.

As shown in Figure 12, IL-10 is induced in the T cells under these conditions. IL-10 expression is blocked by a specific Notch signaling inhibitor (gamma-secretase inhibitor Calbiochem, 125 nM).

Example 12

1 x 1 OV antigen-specific (OVA-TZR transgenic) naive TH cells were transferred into recipient mice and subsequently immunized with 50 μg OVA / 20 μg CpG sc. After 5 days, lymph node cells were restimulated with PMA / ionomycin and analyzed by intracellular cytokine staining. Under these conditions, IFN-gamma / IL-10 double-positive cells were induced. The expression of IL-10 was blocked in mice receiving 10 μM / kg of a Notch inhibitor (gamma-secretase inhibitor, DBZ, Syncom) (Figure 13).

Example 13

Different subpopulations of dendritic cells were isolated by flow cytometry from the spleen and lymph nodes and compared to the expression of Notch ligands. As shown in Figure 14, only plasmacytoid dendritic cells express the delta-like 1 and delta-like 4 ligands. The Jagged ligands are not present here, whereas all other DC types studied express Jagged but not delta-like.

Example 14

Antigen-specific (OVA-TZR transgenic) naive CD4 + CD25-CD62L + T helper cells were isolated by flow cytometry and probed with various, also flow cytometrically isolated subtypes of dendritic cells from spleen and lymph nodes in the ratio 20: 1, in the presence of IL-12 (10 ng / ml) and stimulated with OVA peptide (0.2 μg / ml). After 5 days restimulation with PMA / ionomycin and analysis of IL-10 production by ELISA were performed.

As shown in Figure 15, primarily plasmacytoid dendritic cells induce greater levels of IL-10 under these conditions.

Example 15

From whole blood, human CD4 + CD45RO-CD45RA + naive and CD4 + CD45RO + CD45RA memory T cells as well as plasmacytoid (BDCA-4 +) and myeloid (CD19-CD1c +) dendritic cells were isolated by flow cytometry. The T cells were co-cultivated with the dendritic cells in a ratio of 15: 1 and stimulated with 1 μg / ml SEB. In some preparations, 3 μg / ml CpG, 10 ng / ml IL-12 or 85 nM gamma-secretase inhibitor (insolution gamma-secretase inhibitorX, calbiochem) were added. After 6 days, the cells were restimulated with PMA / ionomycin and their cytokine expression analyzed by intracellular staining. After co-culture with plasmacytoid, but not with myeloid dendritic cells, IL-10 production is observed. The IL-10 expression is through

CpG amplifies and is blocked by the inhibition of the Notch signaling pathway (Figure 16A). Memory T cells show greater IL-10 production after co-culture with plasmacytoid dendritic cells than with myeloid dendritic cells. In both cases, this is blocked by Notch inhibitors (Figure 16B).

Example 16

Human CD4 + T cells were stimulated without antigen-presenting cells by immobilized anti-CD3 (3 μg / ml), anti-CD28 (1 μg / ml) and 10 ng / ml IL-12 and retrovirally transduced after 36 h with active Notch. For this purpose, the cells were centrifuged with virus supernatant for 75 min at 75Ox g and 32 ° C. After 6 days, the cells were restimulated with PMA / ionomycin and cytokine production analyzed by intracellular staining. As shown in Figure 17, after Notch transduction induction of IL-10 occurs.

Example 17

Isolated naive CD4 + CD45RB + TH cells or grade or control-induced TH1 cells T lymphocytes were transferred to SCID mice. To study the suppressive potential of Notch-modified cells, isolated naïve CD4 + CD45RB + TH cells were co-transferred with grade- or control-transduced TH1 cells. The state of health of the animals was regularly checked by weighing. After about 3 weeks, the first signs of colitis could be detected: weight loss, shaggy coat and soft stool. To analyze the course of the disease, the average weight change from the baseline was plotted. In addition, the number of sick animals per experimental group was determined. For this purpose, a weight loss of more than 5% from baseline was considered a sign of illness.

As shown in Figure 18A, animals that had not received cell transfer (PBS) recorded a consistent weight gain throughout the analysis period. Between day 15 and day 30, animals that had received naive T cells only lost weight. In animals receiving control TH1 cells, the weight remained unchanged until day 40. Subsequently, a weight loss occurred here as well. In contrast, animals that had received Notch Th1 cells continued to increase until day 40. This corresponded exactly to the control group without cell transfer. If the number of sick animals per experimental group was used for the evaluation, the following picture emerged. In the PBS control group, as expected, 0/6 animals were affected, in the control group with transfer of naive cells, 4/6 animals became ill after about 30 days. At the same

At the time point there were 2/5 animals in the group receiving control Th1 cells. This number rose to 5/5 by day 60. In the case of the animals which had received Notch-modified Th1 cells, only 51 1/6 animals became ill after day 52 (FIG. 18B).

Animals that had been co-transferred from control-induced T cells showed a similar weight loss to the control group with naive T cell transfer alone. The number of sick animals per group was comparable. It was an average of 4/6 for the control group from day 25 onwards. In the group with co-transfer of control Th1 cells, it varied between 3/6 and even 5/6. After the co-transfer of Notch-modified Th1 cells, a weakened disease course was observed. The average weight in this group was 100% until about day 35. Only later did it fall to an average of 95%. Thus, the outbreak was delayed by 10-15 days and weakened in the further course compared to the control group. The observation of the incidence shows a similar picture with some fluctuations. It was 1/6 to 2/6 by day 35, compared to 3/6 to 4/6 after control co-transfer. In the further course, the incidence in the Notch co-transfer group varied greatly between 2/6 and 4/6, which was either significantly below that of the control group or at different times (Figure 18C / D).

Claims

claims 1. A method for the modulation of anti-inflammatory functions in T cells and generation of protective, anti-inflammatory, in particular of IL-10-producing T cells, characterized in that note and STATΦ signals are modulated, wherein by inhibiting the signals the IL 10 production is reduced and the inflammatory response amplified and activation of the signals enhances IL-10 production and reduces the inflammatory response 2. The method according to claim 1 for the modulation of anti-inflammatory functions in T cells and generation of protective, anti-inflammatory, in particular of IL-10-producing T cells, wherein T cells with signal-active Notch molecules and with signal-active STAT 4 Molecules are brought into contact and so the T cells are obtained. 3. The method according to claim 1 or 2 for the modulation of anti-inflammatory functions in T cells, in particular for the shutdown of IL-10 in these T cells, and generation of highly reactive, inflammatory T cells, wherein the contacting of the T Cells with signal-active Notch or STAT-4 are prevented and thus the T-cells are obtained. 4. The method according to any one of the preceding claims, characterized in that the T cells are selected from the group comprising naive T cells and / or memory T cells. 5. The method according to any one of the preceding claims, characterized in that the T cells are selected from the group of memory T cells. 6. The method according to any one of the preceding claims, characterized in that the T cells are selected from the group of pro-inflammatory Th1 and / or Th17- Cells. 7. The method according to any one of the preceding claims, characterized in that the Notch molecules are selected from the group comprising the Notch receptors and / or the Notch ligands. 8. Method according to the preceding claim, characterized in that the Notch receptors are selected from the group comprising Notch-1, -2, -3 and / or -4 and the Notch ligands are selected from the group comprising the Jagged- Family, particular Jagged-1 and / or -2 and / or Delta-like (DII) family, especially DII-1, -2, and / or -A. 9. The method according to any one of the preceding claims, characterized in that the ligands are molecules of the delta-like family, in particular Delta-Iike4 and / or delta-likel 10. The method according to any one of the preceding claims, characterized in that the bringing into contact with STAT4 molecules by activation with cytokines takes place, in particular by the cytokines IL-12, IL-23 and / or IL-27, or by the overexpression of signal-active STAT-4, in particular by transduction of the T cells. 11. The method according to any one of the preceding claims, characterized in that the bringing into contact with STAT4 molecules takes place by the overexpression of molecules that increase or enhance the STAT-4 activation, in particular the cytokine receptors, preferably IL-12, IL -23, IL-27 and / or fusion proteins of these cytokines with their receptor or STAT4 or signal-active STAT4, preferably by transduction of the T cells. 12. The method according to any one of the preceding claims, characterized in that the bringing into contact takes place by overexpression of signal-active Notch-1, -2, -3 and / or -4, by contacting with endogenous notch Molecules and / or by contact with Notch ligands. 13. The method according to any one of the preceding claims, characterized in that the bringing into contact with the Notch ligands by stimulation with antigen-presenting Cells, in particular via the stimulation of the antigen-presenting cells with TolMike receptors and / or CD40 amplified and / or induced. 14. The method according to any one of the preceding claims, characterized in that the bringing into contact with the Notch ligands by stimulation with plasmacytoid dendritic cells takes place, wherein the bringing into contact in particular by stimulation of plasmacytoid dendritic cells via TolHike receptors and / or CD40 is amplified and / or induced. 15. The method according to any one of the preceding claims, characterized in that the generation of IL-10 producing T cells in vitro by stimulation with Notchgland protein, Notchligand fusion proteins and / or signal-active Notchligand fragments, by stimulation with Notchligand-expressing cells and / or by introducing signal-active Notch by viral and / or non-viral transduction methods in the presence of signal-activated STAT-4 molecules. 16. The method according to any one of the preceding claims, characterized in that the generation of IL-10 producing T cells in vivo by the application of recombinant Notch ligands and / or by the application of Notch ligand-expressing cells in the presence signal-active STAT-4. 17. The method according to any one of the preceding claims, characterized in that the inhibition of Notch and / or STAT-4 activation by antibodies against Notch or Notch ligands and / or against STAT-4 activating cytokines, in particular IL-12, IL-23 , IL-27 and / or its receptor and / or by recombinant proteins or protein fragments or peptides of Notch or Notchliganden. 18. The method according to any one of the preceding claims, characterized in that the inhibition of STAT4 activation is carried out by substances that inhibit the natural activation or activity of STAT4. 19. Method according to one of the preceding claims, characterized in that the inhibition of Notch activation is effected by the use of pharmacological inhibitors of Notch activation, e.g. so-called γ-secretase inhibitors. 20. The method according to any one of the preceding claims, characterized in that the inhibition takes place in the course of an active immune response, in particular in infections, tumor diseases, vaccinations. 21. The method according to any one of the preceding claims, characterized in that the inhibition takes place in the course of a vaccination in the antigens are used in particular of tumors or pathogens together with adjuvants, preferably ligands for TolWike Receptors (TLR), anti CD40 and / or CD40 ligand. 22. The method according to the preceding claim, characterized in that the adjuvants from the group of ligands for TLR9, preferably oligonucleotides comprising methylated CpG motifs. 23. The method according to any one of the preceding claims, characterized in that the adjuvants from the group of antibodies and / or ligands for CD40 originate. 24. The method according to any one of the preceding claims, characterized in that the inhibition takes place during the course of a vaccination with dendritic cells or other antigen-presenting cells. In particular also by the use of dendritic cells or other antigen-presenting cells for immunization in which the expression of the Notch ligands is inhibited (for example by transduction with siRNA or antisense oligonucleotides against the Notch ligands). 25. Method according to the preceding claim, characterized in that in the dendritic cells or other antigen-presenting cells, the expression of the Notch ligands of the delta-like family is inhibited, in particular the ligands delta-likel and delta-like 4. 26. Use of the method according to one of the preceding claims, preferably 1 and 4 to 16 for the inhibition of inflammation. 27. Use of the method according to one of the preceding claims, preferably 1 and 4 to 16 for immunosuppression. 28. Use of the method according to one of the preceding claims, preferably 1 and 4 to 16 for the induction of interleukin-10 (IL-10). 29. Use of the method according to any one of the preceding claims, preferably 3 to 11 and 17 to 25 for activating the immune response. 30. Use of the method according to any one of the preceding claims, preferably 3 to 11 and 17 to 25 for enhancing vaccinations. 31. Use of the method according to one of the preceding claims, preferably 3 to 11 and 17 to 25 for the inhibition of interleukin-10 (IL-10) expression. 32. Use of the method according to one of the preceding claims, in particular 1 and 4 to 16 for the manufacture of a medicament for the induction of interleukin-10 (IL-10) for immunosuppression in organ transplantations.