The invention concerns an in vivo model for human ThI cell-mediated inflammatory immune responses and the application of this model for developing therapeutic approaches involving substances such as drugs, chemicals, biologicals and cells for pathologic immune responses in man, that are driven by activated ThI cells. The present invention thus is directed to a method of screening candidate compounds for the modulation of human ThI mediated immune responses and to compounds for the modulation of human ThI mediated immune responses identified by said method. The invention is further directed to the use of said compounds in the field of medicine, in particular in the prevention and treatment of autoimmune diseases and rejection of transplanted organs or tissues.

ThI cells promote inflammatory immune responses, which are physiologically involved in the elimination of pathogenic extra- or intracellular microorganisms or foreign proteins. The development and perpetuation of a specific, ThI cell-mediated immune reaction requires certain cell populations that are involved in and carry out the different steps of the immune reaction. First, cells are required that take up foreign antigens, process these antigens and present them to Th cells (so called antigen presenting cells). A subset of the Th cells recognizes the presented antigens in a highly specific manner and differentiates into ThI cells with particular effector functions. ThI cells predominantly secrete interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). By secreting these cytokines, other effector cell populations get activated. Of particular importance in this regard are macrophages and cytotoxic T cells.

These two effector populations finally eliminate the intruding microorganisms or proteins. During their effector functions, cytotoxic T cells secrete IFN-gamma themselves and thereby further enhance the evolving immune response. Specific, ThI -mediated immune reactions are a physiological occurring, complex but nevertheless very elegant mechanism of higher organisms to protect them from intruding microorganisms and foreign proteins in order to enable survival for a time that permits reproduction of the higher organisms. A disadvantage of the complicated immune system of man is however, that under certain circumstances uncontrolled or unwanted immune reactions may develop that have pathological consequences for the particular individual. Examples for such consequences of unwanted, ThI cell-mediated immune reactions are autoimmune diseases, which are caused by pathological immune responses of the immune system to antigens of the organism itself, and the majority of transplant rejection reactions after transplantation of bone marrow or solid organs for medical reasons. The mechanisms driving and involved in pathological ThI- mediated immune responses that result in destruction of tissue in these situations or diseases are the same as those activated during protective immune responses.

Currently available modern treatment approaches of these immunological disorders are complex, require long-term application and are therefore costly and economically unfavorable. Moreover, current treatment approaches are frequently associated with severe side effects. The development of novel treatment strategies, however, is hampered by the fact that possibilities to study the development, perpetuation and resolution of human ThI cell mediated immune responses in an in vivo situation, are currently missing.

Autoimmune diseases are caused by failure of self-tolerance and subsequent immune responses against autologous antigens (1). Convincing evidence exists that self-tolerance is an active dynamic state in which potentially pathogenic autoreactive cells are prevented from causing disease by regulatory mechanisms (2). The breakdown of such mechanisms might, therefore, result in the development of pathologic autoimmune reactions. It has become apparent that the destructive effector mechanisms of many systemic autoimmmune diseases are mediated by activated autoantigen specific ThI cells (2, 3). Therefore, the mechanisms controlling the evolution of ThI -biased immune responses play a critical role in the development of pathogenic autoimmune reactions. Delineation of the mechanisms controlling ThI -mediated immunity has largely been derived from animal models. For example, amelioration of autoimmune diabetes in nonobese diabetic (NOD)3 mice, a murine model of human insulin dependent diabetes mellitus, is associated with increased expression of the Th2-derived cytokines IL-4 and IL-5 (4, 5). Pancreatic expression of IL-4, moreover, completely prevents diabetes in NOD mice (6). Injection of IL-4-transduced cells reduces the incidence and severity of collagen-induced arthritis (CIA), a model of human inflammatory arthritis (7) and of experimental autoimmune encephalomyelitis (EAE), a model of human multiple sclerosis (MS) (8). Furthermore, treatment with recombinant IL-4 induces a switch from a ThI -type to a Th2-type response and prevents proteoglycan-induced arthritis, a different model of human inflammatory arthritis (9).

The anti-inflammatory role of another immunomodulatory cytokine, IL-IO, has also been shown in animal models. IL-10-deficient mice are more susceptible to EAE when compared to wild type mice (10). Diabetes induced by adoptively transferred lymphocytes into NOD mice can be prevented by IL-10-transduced islet-specific ThI lymphocytes (11). Moreover, the effect of regulatory T cells in a transfer model of colitis can be abrogated by neutralizing antibodies to transforming growth factor-beta and IL-10, resulting in the emergence of tissue pathology (12, 13). Although the pathways are complex, there is convincing evidence that anti-inflammatory cytokines play essential roles in regulating the development and perpetuation of chronic ThI- mediated autoimmune responses in animals.

Analysis of the role of cytokines in regulating T cell immunity in humans is hampered by the inability to address these questions directly in vivo. In vitro, IL-4 and IL-10 clearly exhibit an anti-inflammatory effect as they induce expression of the IL-I receptor antagonist (14, 15) and down-regulate the production of pro-inflammatory cytokines, such as IL-I and TNF from human monocytes (16, 17). Moreover, IL-4 has a direct inhibitory effect on the development of human ThI cells (18) and IL-10 is able to prevent ThI effector functions by induction of long lasting T cell unresponsiveness (19). Recent studies aiming to dissect the impact of new treatment approaches in human autoimmune diseases have revealed that clinical benefit may be associated with enhanced Th2 cell differentiation in vivo (20-22). Moreover, administration of IL-4 or IL- 10 to patients with psoriasis resulted in improvement of skin disease (23-25). On the other hand, treatment of rheumatoid arthritis (RA), a prototype human Thl-biased autoimmune disease, with IL-4 or IL-IO has largely failed to down-modulate inflammation and to provide clinical benefit (26). Therefore, the precise mechanisms of regulation of ThI -mediated immune reactions in humans remain obscure, and as a result the impact of targeted interventions remains unpredictable.

Therefore it is a problem underlying the present invention to provide an animal model and a method for screening compounds for the modulation of ThI mediated immune responses. It is a further problem to provide new compounds which are capable of modulating said immune responses in humans. Additionally, it is a problem underlying the invention to provide new therapies based on said new compounds, in particular therapies for the treatment of autoimmune diseases and transplant rejection.

These problems are solved by the subject-matter of the independent claims. Preferred embodiments are set forth in the dependent claims.

Compared to the current state-of-the art technology, the invention describes a model the permits the in vivo analysis of a human ThI cell-driven immune reaction. This model is reliable, easy to use and is suitable for the study of therapeutic approaches. The inventors have observed that injection of human cells from the peripheral blood e.g. into the peritoneal cavity of mice that lack a functional specific immune system results in the development of a human, ThI cell mediated immune response. One focus of the present invention are therefore human, ThI cell driven immune responses that evolve after injection of human cells in the body of immunocompromised mice. Of importance of its own within the framework of the invention is the use of such human immune reactions as a model to evaluate in vivo means (such as chemicals, drugs, biologicals or cells) for immunodulation or for the treatment of immunological diseases.

Thus, the invention concerns an in vivo model for human ThI -mediated inflammatory immune responses and the application of this model for the investigation of the therapeutic use of substances such as drugs, chemicals, biologicals or cells in pathological, ThI -driven situations in man. The currently available therapeutic approaches of such immunological diseases are complex, costly and require long-term application. Therefore, they are not optimal from the economic and practical point of view. Moreover, they are frequently associated with severe side effects. The development of novel treatment strategies, however, is hampered by the fact that technical possibilities to delineate the development, the continuation and the resolution of a human, ThI cell-mediated immune response in an in vivo situation, are missing.

With the present model, known and established immunological principals can be tested and verified. This is an important indication for the economical and scientific value of this model for the analysis of novel therapeutic approaches and means in Thl-driven immune diseases.

The invention is based on the observation that injection of human mononuclear cells from the peripheral blood into the peritoneal cavity of mice that lack a functional specific immune system results in an immune reaction that is driven by human ThI cells.

Specifically, Thl-driven immune responses are defined as immune responses that are mediated by T helper cells that produce the proinflammatory cytokine, interferon-gamma (ThI -cells). The invention describes a model, which permits the analysis of such human immune responses in an in vivo system and allows the evaluation of therapeutic approaches aimed to modulate these immune reactions.

The present invention is directed to the following:

1. Human Thl-driven immunreactions that develop in vivo in the body of immunocompromised animals (whether genetically or after manipulation by means, such as irradiation or application of chemical or biological substances) after injection of cells of human origin.

2. Use of a human ThI -mediated immune response generated as described in (1) for the analysis of the modulation of a human ThI -mediated immune response. 3. Use of a human Thl-mediated immune response generated as described in (1) for the evaluation of a therapeutic application for divers pathological immunological Thl-mediated processes.

4. Use of a human Thl-mediated immune response generated as described in (1) for the testing of drugs, chemicals, biologicals or cells for therapeutic treatment of the human body.

The present invention in particular is directed to the following aspects and embodiments:

According to a first aspect, the present invention provides a method of screening candidate compounds for the modulation of human ThI mediated immune responses, comprising the following steps:

a) providing human cells at least comprising CD 4+ T cells and antigen presenting cells (APCs); b) administering said cells to a non-human animal, wherein the non-human animal is lacking its own functional immune system or is immunocompromised thereby causing an immune response in said non-human animal; c) administering one or more candidate compounds to said non-human animal; and d) determining the capability of said candidate compound(s) to modulate ThI -driven immune reactions in vivo in the body of said animal.

It is important for the invention to conclude that the above indicated cell populations are necessary to mimic a Thl-mediated immune response in the present model. These cell populations at least comprise CD 4+ T cells and antigen presenting cells necessary in order to elucidate an immune response in said animal.

It is noted that the term "CD 4+ T cells" as contained herein is defining a group of cells mandatorily comprising the ThI subgroup of T cells or containing precursor cells which are capable of developing same. An example of a source of cells to be used in the present invention are mononuclear cells from the peripheral blood {peripheral blood mononuclear cells, PBMC) which constitute a fraction of cells within the blood and can be isolated from peripheral blood based on their density by fϊcoll gradient centrifugation.

This cell fraction comprises among other cells of monocytes (precursor cells of macrophages) and T cells (Th cells and cytotoxic T cells) thus fulfilling the above requirements. Inoculation of about 50 million cells of such a mixed cell population into the peritoneal cavity of mice that lack a functional immune system, results in the development of an immune reaction against murine proteins, which is driven by human ThI cells. In other words, injection of human PBMC into these mice results in a xenogenic graft versus host reaction, which is similar to a allogenic graft versus host reactions observed after transplant of bone marrow or solid organs in man.

Thus, according to a preferred embodiment, the method of the present invention employs cells which are peripheral blood mononuclear cells or derived therefrom. Thus, both essential cell populations as mentioned above may be present in a mixture with other cell populations which not necessarily contribute to the model system.

Further naturally occuring cell populations which may be used in this invention are, e.g. mononuclear cells derived from synovial fluid (SFMNC). Those are containing both, CD4+ T cells and APCs. As mentioned above, it is essential for the present method to include APCs. Those APCs preferably are selected from the group consisting of monocytes, macrophages, B cells and/or dendritic cells.

Further, it should be mentioned that CD 4+ T cells and antigen presenting cells or the mixtures of cell populations containing same may be administered to the non-human animal combined (in one step) or as separate components.

Basically, in the present method a mammal may be used as a non-human animal. It is, however, preferred to use a rodent, in particular a rat or a mouse. Mice are most preferred. o 8

The animal's immune system is lacking its own functional specific immune system or is compromised preferably due to genetical alterations, manipulation by irradiation or application of chemical or biological substances. Of particular importance for the invention is the fact that only those animals (in the above example mice) that lack their own functional immune system can be the carrier of such a human immune response. Included in this category of mice are first of all mice that carry a natural occurring genetic defect that results in the loss of T and B cells. These mice are known as SCID {severe combined immuno deficiency) mice. Knockout mice, that are characterized by the elimination of a gene by biotechnical means can also present with a SCID-phenotype. The prime example of such an approach are mice in which one of the genes encoding a critical enzyme of T and B cell development, such as RAG-I or RAG-2 {recombination activating gene), has been biotechnically eliminated. Another way to generate a SCID-phenotype is sublethal irradiation of the mice that allows the animals to survive radiation but destroys the proliferative capacity of the cells of the mice, including T and B cells and their precursor cells in the bone marrow, such that these mice cannot generate a specific immune response.

Thus, in a embodiment, the animal is a SCID mouse wherein the mouse was treated by sublethal irradiation in order to acquire a SCID phenotype. In a preferred embodiment, the animals used have a naturally occurring SCID mutation.

As an alternative approach, the mouse was generated by biotechnological elemination of RAG-I and/or RAG-2. The cells in step b) are administered to said non-human animal by intraperitoneal, intravenous, intramuscular, intracutanous, subcutanous and/or intraarticular administration. Thus, basically all parenteral modes of administration are suitable.

According to a preferred embodiment, the candidate compound is selected from the group consisting of drugs, including chemical or biological agents, or cells.

Preferably the drug is selected from biological or chemical immunosuppressants. A preferred example of such a biological immunosuppressant is a preformed regulatory T cell. Those in vitro or ex vivo generated T cells turned out to have an inhibitory effect in the present method and thus are suitable candidates for an immunosuppressant.

General information on such regulatory T cells may be found in: A. Skapenko, J. Leipe, P. E. Lipsky, and H. Schulze-Koops. 2005. The role of the T cell in autoimmune inflammation. Arthritis Res Ther 7 S2:4-14 (52).

For example, one particular interesting CD4 T cell subset with regulatory capacity is defined by the constitutive expression of the alpha chain of the IL-2 receptor, CD25. CD25pos regulatory CD4 T cells (Tregs) were isolated first in mice, where it was shown that transfer of CD4 T cells that were depleted of the CD25-expressing T cell fraction into athymic syngeneic Balb/c mice resulted in the development of various organ specific autoimmune disease, such as thyroiditis, gastritis, colitis and insulin-dependent autoimmune diabetes (54).

Furthermore, cotransfer of CD4 CD25pos T cells with the pathogenic CD4 CD25neg T cells prevented the development of experimentally induced autoimmune diseases (55; 56). These data implied that CD25pos Tregs are able to actively regulate the responsiveness of autoreactive T cells that have escaped central tolerance. Subsequently, Tregs were also detected in man (57-63). Tregs are part of the physiologic peripheral T cell repertoire and constitute between 5 and 15% of the CD4 T cells in the peripheral blood of healthy individuals. Tregs are anergic, i.e. they do not proliferate in response to mitogenic stimulation (64). Of importance, CD25posCD4 T cells, in contrast to their CD25neg counterparts, are able to inhibit activation-induced proliferation of autologous responder T cells in a contact-dependent and cytokine-independent manner (58). Both, anergy and inhibition of proliferation can be prevented by the addition of exogenous IL-2 (65).

Besides their constitutive expression of CD25, Tregs are characterized phenotypically by the surface expression of CTLA-4 (66) and glucocorticoid induced TNF receptor family related protein (GITR) (67) as well as by the expression of the transcription factor Foxp3 (68). The importance of Foxp3 for the regulatory function of Tregs has been demonstrated by transfection of CD25neεCD4 T cells with a plasmid encoding Foxp3, which conferred a regulatory capacity to the trans fected T cells (68). Together, the accumulated evidence indicates that Tregs may play an important role in maintaining peripheral tolerance and preventing the evolution of autoimmune inflammation.

The experiment is outlined in the following, wherein it is shown that IL-4 favors the generation of CD25+ T cells with regulatory capacity in an in vivo model of human inflammation:

The inventors have previously shown that injection of human PBMC into NOD/SCID mice results in the development of a ThI -mediated immune response of human cells against murine tissue accompanied by an increase in the frequency of CD25+ CD4 T cells (Skapenko et al., 2004). As IL-4 had a pronounced immunoregulatory effect on the development of this reaction, we used this model to investigate the role of IL-4 in the generation of CD25+ regulatory T cells in an in vivo situation. NOD/SCID mice were injected with human PBMC and were treated either with IL-4 (Fig. 9A) or with an antibody neutralizing human IL-4 (Fig. 9B). Consistent with the previous work, treatment of the animals with IL-4 during the ongoing ThI -mediated immune reaction down-modulated systemic inflammation as assessed by a decrease of the serum levels of the human inflammatory cytokines, TNF and IFN-gamma (Fig. 9Aa), whereas neutralization of endogenous IL-4 resulted in an exaggeration of the Thl-mediated immune response (Fig. 9Ba). Concomitant analysis of the frequencies of CD25+ CD4 T cells in recovered human cells revealed that the treatment with IL-4 augmented and IL-4 neutralization diminished the increase in the frequency of CD25+ CD4 T cells (Fig. 9Ab, 9Bb).

When the immunosuppressive ability of these cells was analyzed in vitro, human CD25+ T cells purified from the mice with established inflammation on day 14 were able to inhibit the proliferative response of autologous PBMC, whereas CD25" T cells did not suppress and even enhanced their proliferation (Fig. 9Ca). When injected into animals with an ongoing Thl- mediated immune reaction driven by autologous PBMC, these purified CD25+ T cells were able to reduce serum levels of human IFN-gamma and TNF, whereas injection of CD25" T cells resulted in increased levels of both cytokines (Fig. 9Cb). Moreover, the frequency of CD25+ CD4 T cells within the human cells recovered from the peritoneal cavity at day 14 inversely correlated with the concentrations of human IFN-gamma and TNF in the serum of the animals (Fig. 9D), indicating that CD25+ CD4 T cells which accumulate during the xenogeneic Thl-mediated immune response of human cells in NOD/SCID mice possess an immunosuppressive phenotype in vivo during the ongoing immune response. Therefore, these data imply that IL-4 might have accomplished part of its immunomodulatory effect via induction of CD25+ Tregs in the in vivo model of a human Thl-mediated immune response.

According to a preferred embodiment, it is preferred in the present invention to administer human cells to the animal in the range of between 20 and 70 million cells, more preferably about 50 million cells.

As already indicated above, the present invention in a second aspect provides a compound for the modulation of human ThI mediated immune responses identified by the above explained method. That compound (or also a mixture of more than one compound) preferably is contained in a pharmaceutical composition comprising a therapeutically effective amount of that compound and a pharmaceutically acceptable carrier or excipients in doses to treat or ameliorate a disease, in particular an autoimmune disease or transplant rejection as outlined below.

Such a composition may also contain (in addition to the ingredient and the carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the activity of the active component(s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition may further contain other agents which either enhance the activity of the activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect or to minimize side-effects.

Techniques for formulation and administration of the compounds of the instant application may be found in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, latest edition. Whenever the compositions of the invention are to be used for medical purposes, they will contain a therapeutically effective dose of the respective ingredient. A therapeutically effective dose refers to that amount of the compound/ingredient sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of such conditions. In the context of the present invention, a therapeutically effective dose is to be understood as an amount of the compound/ingredient, which results in a statistically significant reduction of the degree of disadvantageous autoimmune reactions involved in an autoimmune disease or a transplant rejection.

Suitable routes of administration may, for example, include those indicated above.

A typical composition for intravenous infusion can be made up to contain 250 ml of sterile Ringer's solution, and 10 mg of active compound. See Remington's Pharmaceutical Science (15th Ed., Mack Publishing Company, Easton, Ps., 1980).

According to a third aspect the compound and/or composition as identified above is used for the prevention or treatment of unwanted immune reactions in human beings. According to an embodiment, the compound and/or composition of the present invention is used for the prevention or treatment of autoimmune diseases and/or transplant rejection reactions.

In particular, their use for preventing and/or treating is for rheumatoid arthritis, multiple sclerosis, Morbus Crohn, Psoriasis, diabetes mellitus or systemic lupus erythematosus and related autoimmune diseases as autoimmune vasculitis or autoimmune connective tissue diseases, ulcerative colitis, Hashimoto's disease or Morbus Basedow.

Basically, the spectrum of autoimmune disorders ranges from organ specific diseases (such as thyroiditis, insulitis, multiple sclerosis, iridocyclitis, uveitis, orchitis, hepatitis, Addison's disease, myasthenia gravis) to systemic illnesses such as rheumatoid arthritis, vasculitis or lupus erythematosus. According to a fourth aspect, the invention provides a non-human animal lacking its own functional immune system or being immunocompromised, in which an immunologically effective amount of human human cells at least comprising CD 4+ T cells and antigen presenting cells, preferably PBMCs was introduced.

In a fifth aspect, there is a method of preventing or treating an autoimmune disease and/or transplant rejection reaction in a human provided comprising administering to a human patient in need thereof a therapeutically effective amount of a compound or composition as defined above.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The invention is now further illustrated by the accompanying figures, in which:

FIGURES Ia and Ib: Mononuclear cells from the peripheral blood of healthy volunteers are injected into the peritoneal cavity of NOD/SCID mice. After 14 days, IL-4 (0.1 mg) or IL-IO (0.01 mg) are injected intraperitoneally for 5 days daily. These two cytokines are so called immunomodulatory cytokines that down modulate inflammatory immune reactions. Control animals are treated with daily injections of PBS buffer {phosphate buffered saline). At day 19, the animals are sacrificed and the extent of the ThI -reaction is assessed by determination of the human inflammatory cytokines IFN-gamma and TNF (tumor necrosis factor).

FIGURE 2: Mononuclear cells from the peripheral blood of healthy human volunteers were injected into the peritoneal cavity of 7 mice that lack an own specific immune system. This results in the development of a specific ThI cell-driven immune response of human cells against murine antigens. As described in example 1, the development of this immune reaction is associated with an increase of CD25 positive Th cells. After 14 days, 4 mice are sacrificed. Human cells are recovered from the peritoneal cavity of the animals, and CD25 positive and CD25 negative cells are isolated from the recovered cells. On the same day, 150.000 of the CD25 χ

positive or negative T cells are injected into the peritoneal cavity of 2 of the remaining 3 mice. The third mouse serves as the untreated control and receives an injection of PBS buffer only. At day 19, the extent of inflammation is determined by analysis of the production of human inflammatory cytokines.

FIGURE 3: Human PBMC develop a ThI -biased immune reaction after intraperitoneal injection into SCID mice. 5OxIO6 human PBMC were injected i.p. into SCED mice. At the indicated time points human cells were recovered from the peritoneal cavity, and analyzed by flow cytometry. (A) Before injection PBMC were labeled with CFSE. Frequencies of cells with reduced CFSE fluorescence indicative of proliferative cycles were assessed after counter staining with mAbs to CD4 or CD8. (B) The CD4/CD8 ratio in recovered cell populations was calculated after surface staining with mAbs to CD4 and CD8. (Q Percentage of CD4 cells expressing the activation marker, HLA-DR or CD25. (D) The frequency of cells capable of IFN-gamma production was assessed by intracellular flow cytometry after a 5 hour in vitro stimulation with PMA and ionomycin. One representative of eight independent experiments with different donors is shown. (E) Liver sections from day 14 are illustrated after staining with hematoxylin and eosin (i-iii), or with a mAb to human CD3 (zv). Polymorphic lymphoid cell infiltrates are observed in a large (i) and in small portal tracts (H, Ui) around small bile ducts. Note, that bile duct epithelial cells occasionally show degenerative changes (U). Immunostaining of a serial section to Hi identifies the majority of infiltrating cells as human CD3 positive T cells (zv). Original magnification: i, Hi, iv x200; H x400. (F) A typical epitheloid cell granuloma from perigastric fatty tissue is illustrated (i, hematoxylin and eosin staining). Immunostaining reveals a row of human CD68 positive macrophages surrounding an area of central necrosis (H). The polymorphic lymphoid cell infiltrate adjacent to the macrophages consists of human CD3 positive T cells (Ui) with a large proportion of CD4 positive cells (zv) and a smaller number of CD8 positive cells (v). Adjacent to the granuloma are aggregates of human CD20 positive B cells (vz). Original magnification: i, ii, Ui, vi x200; zv, v x400.

FIGURE 4. Cyclosporine prevents the Thl-biased human immune response in SCID mice. 5OxIO6 freshly isolated human PBMC were injected into SCID mice. Mice were treated daily by i.p. injection with cyclosporine or PBS as control. Analysis was performed on day 14. Human cells were recovered from the peritoneal cavity and analyzed by flow cytometry (A) for extracellular expression of CD4 and HLA-DR, and (B) for cytoplasmic IFN-gamrna after in vitro stimulation with PMA and ionomycin. For comparison, values from freshly isolated PBMC are shown ("Before"). (Q Serum levels of the human inflammatory cytokines, IFN-gamma and TNF, were determined by ELISA. One representative of six independent experiments with different donors is shown.

FIGURE 5. Monocytes are required for the development of the ThI -biased immune reaction of human cells in SCID mice. Human T cells and monocytes were purified by negative selection from PBMC. (A - C) 5OxIO6 T cells and lOxlO5 monocytes from the same donor were injected either separately or together into mice. As a control, mice were injected with PBMC that contained 5OxIO6 T cells. Analysis was performed on day 14. Human cells were recovered from the peritoneal cavity and analyzed by flow cytometry for (A) CD4 and HLA-DR, and (B) cytoplasmic IFN-gamma. (C) Serum levels of the human inflammatory cytokines, IFN-gamma and TNF, were determined by ELISA. One representative of three independent experiments with different donors is shown, n.d. - non-detectable. (D) 5OxIO6 T cells were injected together with different numbers of monocytes (6.25x106, 12.5x106, or 25x106, respectively) from the same donor into mice. Serum levels of the human inflammatory cytokines IFN-gamma and TNF were determined at day 14 by ELISA. (E) Serum concentrations of human IFN-gamma (day 14) were plotted as a function of the monocyte to T cell ratio within the injected PBMC from 19 independent experiments, and the linear regression was calculated. The serum IFN-gamma concentration significantly correlated with the monocyte to T cell ratio of the inoculum.

FIGURE 6. Endogenously produced IL-4 and IL-10 control the development of the human ThI- biased immune reaction. 5OxIO6 freshly isolated human PBMC were injected into SCID mice. At days 0, 5 and 10, mice were treated with mAbs to (A - C) human IL-4 ("anti-IL-4") or (D - F) human IL-10 ("anti-IL-10"). As a control, animals were treated with isotype-matched control mAbs ("IgG"). Analysis was performed on day 14. Human cells were recovered from the peritoneal cavity and analyzed by flow cytometry for (A, D) CD4 and HLA-DR, and (B, E) cytoplasmic IFN-gamma. (C, F) Serum levels of the human inflammatory cytokines, IFN- 1

gamma and TNF, were determined by ELISA. One representative of 13 independent experiments for anti-IL-4 and one of 13 independent experiments for anti-IL-10 with different donors are shown.

FIGURE 7. Exogenous IL-4 diminishes the human ThI -biased immune reaction. 5OxIO6 freshly isolated human PBMC were injected into SCID mice. Starting at day 14, mice were treated daily with human IL-4 for 5 days. As a control, animals were treated with PBS. Analysis was performed on day 19. Human cells were recovered from the peritoneal cavity and analyzed by flow cytometry for (A) CD4 and HLA-DR, and (B) cytoplasmic IFN-gamma after in vitro stimulation with PMA and ionomycin. (C) Serum levels of the human inflammatory cytokines, IFN-gamma and TNF, were determined by ELISA. One representative of six independent experiments with PBMC from different donors is shown.

FIGURE 8. Exogenous IL-10 diminishes the human ThI -biased immune reaction. 5OxIO6 freshly isolated human PBMC were injected into SCID mice. Starting at day 14, mice were treated daily with human IL-10 for 5 days. As a control, animals were treated with PBS. Analysis was performed on day 19. Human cells were recovered from the peritoneal cavity and analyzed by flow cytometry for (A) CD4 and HLA-DR, and (B) cytoplasmic IFN-gamma. (C) Serum levels of the human inflammatory cytokines IFN-gamma and TNF were determined by ELISA. One representative of six independent experiments with PBMC from different donors is shown.

FIGURE 9. Effect of IL-4 on CD25+ regulatory T cells in an in vivo model of a human ThI- mediated immune response. Human PBMC were injected into NOD/SCID mice and allowed to develop a human Thl-mediated immune response against murine tissue. The animals were left untreated or treated where indicated. (A) Animals were treated with human IL-4 ("IL-4") or with PBS ("PBS") as described in Materials and Methods. On day 19, levels of the human inflammatory cytokines IFNγ and TNF were determined in the serum of the animals (a) and recovered human cells were analyzed for frequencies of CD25+ CD4 T cells (b). "pre": freshly isolated PBMC. Results of six independent experiments using cells from different donors are shown as mean ± SD. (B) Animals were treated with an antibody neutralizing human IL-4 ("αEL- 4") or with an isotype-matched antibody ("IgGl") as described in Materials and Methods. On day 14, levels of the human inflammatory cytokines IFNγ and TNP were determined in the serum of the animals (a) and recovered human cells were analyzed for frequencies of CD25+ CD4 T cells (b). "pre": freshly isolated PBMC. Results of six independent experiments using cells from different donors are shown as mean ± SD. (C) Human CD25+ and CD25- CD4 T cells were isolated from human cells recovered from the peritoneal cavity of the mice on day 14 and the ability of the sorted cells to inhibit in vitro proliferation of autologous PBMC in response to anti-CD3 (a) or the ongoing autologous ThI -mediated immune reaction in NOD/SCID mice in vivo as described in Materials and Methods (b) were analyzed. Results of a representative experiment out of seven independent experiments using cells from different donors for in vitro and of three independent experiments for the in vivo inhibitory capacity are shown. (D) Concentrations of human IFNγ and TNP in the serum of PBMC-injected mice at day 14 were plotted as a function of the frequency of CD25+ CD4 T cells within recovered human cells, and the linear regression was calculated. Results of 46 independent experiments using cells from different donors (each shown as a dot) are summarized.

In the following examples are provided that illustrate the invention in more detail. In particular, the analysis according to the invention of therapeutic approaches will become clear from these examples.

Example 1.

To induce the development of a human, ThI cell-mediated immune response in vivo, mononuclear cells are isolated from the peripheral blood of a healthy human donor. 50 million of this mixture of cells is injected into the peritoneal cavity of immundeficient mice. As immunodeficient mice we have used animals that have a natural occurring SCID mutation on the NOD (non obese diabetic) background (NOD/SCID-mice). The use of SCID mice on the NOD background has the advantage that these mice are characterized by a reduced activity and number of other players of the specific immune system, in particular of natural killer cells (NK cells). This reduces the natural immune response of the mice against foreign cells (in our case human cells) even further and permits better engraftment of the human cells and therefore a stronger immune response. The development of a ThI cell-mediated immune response can be followed by assessment of parameters indicative of activation and/or inflammation which are typical for such immune responses, on the PBMC in the days following the injection. On days 2, 5, 7, 10 and 14, a mouse is sacrificed. Human cells can be recovered from the peritoneal cavity by irrigation. In addition, tissue surrounding the peritoneal cavity is analyzed. The cells harvested from the peritoneum are assessed by extra and intracellular flow cytometry. First, by staining of the recovered cells for specific molecules that are expressed on the cell surface we have analyzed the behavior of the cell populations that are of particular importance for ThI cell-mediated immune responses.

Figure 3B demonstrates that during the 14 days following injection enrichment of CD4 positive Th cells occurs first which is later superseded by expansion of CD8 positive cytotoxic T cells. Figure 3C illustrates that a growing part of the recovered Th cells expresses classical activation markers, such as HLA-DR and CD25, on their surface. The ultimate proof for a ThI cell-driven immune response derives from the fact that, as shown in figure 3D, the frequency of IFN-gamma producing cells increases with time, whereas their counterparts (Th2 cells that produce IL-4) are absent. Moreover, histological analysis of the surrounding tissue reveals granulomatous formations, which are typical for ThI cell-mediated diseases, such as tuberculosis and Wegener's granulomatosis.

Example 2.

The inventional human ThI cell-mediated immune response can be used as a model to assess the impact of different immuno-suppressants in vivo. Figures Ia and Ib illustrate examples of such an approach. As described above, mononuclear cells from the peripheral blood of healthy volunteers are injected into the peritoneal cavity of NOD/SCID mice. After 14 days, EL-4 (0.1 mg) or IL-IO (0.01 mg) are injected intraperitoneally for 5 days daily. These two cytokines are so called immunomodulatory cytokines that down modulate inflammatory immune reactions. Control animals are treated with daily injections of PBS buffer (phosphate buffered saline). At day 19, the animals are sacrificed and the extent of the Thl-reaction is assessed by determination of the human inflammatory cytokines IFN-gamma and TNF (tumor necrosis factor). Figures Ia and Ib demonstrate that application of either IL-4 or IL-IO results in reduction of the concentration of secreted IFN-gamma and TNF. This observation should be regarded as the fulfillment of an important prerequisite for the application of the model for the analysis of therapeutic approaches, as in these two examples well established immunological principles have been tested and verified.

Example 3

A further example for the application of this model for the delineation of the therapeutic use of different biological or chemical agents is the application of CD25 positive regulatory T cells to ameliorate an inflammatory immune response. Th cells, which constitutively express CD25 on their surface have been shown to express regulatory capacities for immune responses. In other words, such cells can regulate the immune response and can control the extent of an immune reaction and might even completely resolve the immunological activity, if necessary. Figure 2 demonstrates a representative experiment. Mononuclear cells from the peripheral blood of healthy human volunteers were injected into the peritoneal cavity of 7 mice that lack an own specific immune system. This results in the development of a specific ThI cell-driven immune response of human cells against murine antigens. As described in example 1, the development of this immune reaction is associated with an increase of CD25 positive Th cells. After 14 days, 4 mice are sacrificed. Human cells are recovered from the peritoneal cavity of the animals, and CD25 positive and CD25 negative cells are isolated from the recovered cells. On the same day, 150.000 of the CD25 positive or negative T cells are injected into the peritoneal cavity of 2 of the remaining 3 mice. The third mouse serves as the untreated control and receives an injection of PBS buffer only. At day 19, the extent of inflammation is determined by analysis of the production of human inflammatory cytokines. As clearly shown in figure 2, injection of CD25 positive T cells results in amelioration of the ThI -immune response, whereas the injection of CD25 negative T cells enhances the immune response. This experiments shows that the inventional ThI cell-mediated human immune reaction can be modulated by application of CD25 regulatory T cells. In other words, the inventional human Thl-driven immune response can be used as a model for the analysis of therapeutic applications of different immunomodulatory principles in man.

Materials and Methods

Reagents and antibodies. The following mAbs were used for purification and staining of human cells: anti-CD 16, anti-CD19; FITC-conjugated anti-CD3, PE-labeled anti-CD4, FITC-labeled anti-CD4 (Sigma, Taufkirchen, Germany); FITC-labeled anti-CD14, PE-labeled anti-CD25 (Cymbus Biotechnology, Hants, UK); FITC-labeled anti-HLA-DR (Dako Diagnostika, Hamburg, Germany); PE-labeled anti-CD8, PE-labeled anti-IL-4 (MP4-25D2), FITC-labeled anti-IFN- gamma (4S.B3) (Pharmingen, Heidelberg, Germany). CFSE was obtained from Molecular Probes (Leiden, The Nertherlands). Cyclosporine was purchased from Sigma. Human recombinant IL-4 and the neutralizing mAb to IL-4 were from Perbio Science (Bonn, Germany). Human recombinant IL-IO and the neutralizing mAb to IL-IO were from R&D Systems (Wiesbaden, Germany). The neutralizing mAbs were highly specific for human cytokines with no measurable cross reactivity to murine cytokines. Isotype antibodies (mouse IgG2b and rat IgGl) were purchased from Pharmingen.

Mice. Mice congenic for the scid mutation on the NOD genetic background were purchased from M&B (Ry, Denmark). The animals were maintained under pathogen-free conditions in the animal facility of Nikolaus Fiebiger Center (Erlangen, Germany). Mice were used at 6-12 weeks of age.

Human cell preparation. PBMC were obtained by ficoll-hypaque (Sigma) gradient- centrifugation of heparinized venous blood from young healthy volunteers not taking any medications. For further T cell or monocyte preparation, PBMC were incubated with sheep erythrocytes and T cells were isolated from the rosette-positive cells by negative-selection panning using anti-CD 16 and anti-CD 19 as previously described (20). Monocytes were purified from the fraction of rosette-negative cells using the monocyte isolation kit from Miltenyi Biotec (Bergisch Gladbach, Germany), according to the manufacturer's instructions. The frequencies of cell populations within PBMC and homogeneity and purity of the isolated T cells and monocytes were routinely assessed by flow cytometry. Typically, = 95 % of the T cells were positive for CD3 and CD4, = 90 % of the monocytes stained brightly with a mAb to CDl 4, and more than 98 % of the cells were viable after the purification procedure. T cells were negative for the activation markers CD25, CD30, CD69, and HLA-DR.

Injection with human cells and treatment of mice. Isolated cell populations were resuspended in PBS and injected i.p. into mice in a total volume of 0.2 ml. For CFSE labeling, 10x106 PBMC were resuspended in 1 ml PBS and labeled for 8 minutes with 10 μM CFSE at room temperature. Cyclosporine treatment was performed daily with 0.02-0.04 mg from day 0 through day 14 by i.p. injection. Treatment with IL-4 (0.008-0.2 mg) or IL-10 (0.0016-0.04 mg) was performed daily by i.p. injection of recombinant cytokines from day 14 to day 19. Treatment with blocking antibodies for human IL-4 or IL-10 (100 μg) was performed by 3 i.p. injections during the 14 day experiments, at days 0, 5 and 10.

Cytokine determination. Human cells were recovered from the peritoneal cavity of the mice. To assess the acquired capacity of T cells for cytokine production, 2x10s recovered cells were restimulated with ionomycin (1 mM, Calbiochem, Schwalbach, Germany) and PMA (20 ng/ml, Sigma) for 5 h in the presence of 2 microM monensin (Sigma). Cells were fixed with 4 % paraformaldehyde (Sigma), and cytoplasmic human IFN-gamma and IL-4 were detected by flow cytometry after intracellular staining with FITC-labeled anti-IFN-gamma and PE-labeled anti-IL- 4. The numbers of cytokine producing T cells were determined from the total population of gated lymphocytes. Analysis of extracellular markers revealed that recovered lymphocytes contained less than 1 % CD 19 positive B cells.

To analyze the serum levels of the human inflammatory cytokines, IFN-gamma and TNF and of human IL-4, blood was taken from the tail vein of the mice, sera were collected, and the cytokine levels were measured using commercially available high sensitivity ELISA kits that were highly specific for the human cytokines (R&D Systems, sensitivity thresholds 0.12 pg/ml, 8 pg/ml and 0.13 pg/ml for TNF, IFN-gamma and IL-4, respectively). Histopathologic analysis. Mouse tissues were sampled immediately after sacrifice, fixed in 5% neutral buffered formalin, and embedded in paraffin wax using standard histological procedures. 3 μm paraffin sections were stained with hematoxylin and eosin for morphological assessment by light microscopy.

For immunohistochemistry, a polyclonal CD3 antiserum, monoclonal CD4, CD8, CD20, and CD68 antibodies (all specific for human antigens), and a monoclonal eosinophilic peroxidase antibody (all from Dako) were employed. Paraffin sections were dewaxed and subjected to antigen retrieval in 0.1 M citrate buffer (pH 6.0) using a pressure cooker. Following incubation with appropriately diluted primary antibodies, sections were incubated with a biotin-labeled goat anti-rabbit serum (for CD3, Dako) or with biotinylated rabbit anti-mouse immunoglobulins (for all others, Dako). Bound antibodies were detected using a streptavidin-biotinylated alkaline phosphatase complex (Dako) and Fast Red as a chromogen (Sigma). Stained sections were counterstained with hematoxylin and examined by light microscopy.

Statistical and mathematical analysis. Linear regression was calculated using the InStat computer program (GraphPad, San Diego, CA). Based on CFSE fluorescence intensity, the frequencies (F) of CD4 and CD8 cells that had undergone divisions (from d-\ for the first cycle to n cell divisions) was calculated as:

F=∑(2-d)Fd d=l

Results Development of a human ThI immune reaction in SCID mice. Previous studies have shown that human PBMC remain functionally active after injection into SCID mice (27). Moreover, transfer of human cells into SCID mice results in the development of a xenogeneic graft vs. host (GvH) disease (28). For characterization of the developing immune reaction of human cells against mouse tissue, human PBMC were labeled with CFSE and injected into SCID mice (Fig. AA). Human CD4 and CD8 T cells spontaneously proliferated after introduction into SCID mice. The degree of CD8 T cell expansion exceeded that of CD4 cells. As CFSE pattern analysis indicated a similar proliferative rate of CD4 and CD8 cells (data not shown), this was likely to be a consequence of increased precursor frequencies of CD8 cells capable of undergoing proliferation in response to murine antigens (Table I). Of note, the percentage of T cells initially reacting to mouse tissue was different among donors. Further experiments employing non-labeled PBMC indicated that greater expansion of CD8 compared to CD4 cells lead to an inverted CD4/CD8 ratio (Fig. 3B).

Analysis of activation markers on the T cell surface revealed that the frequency of CD4 T cells expressing HLA-DR and CD25 increased with time (Fig. 3C). The percentage of T cells capable of producing IFN-gamma and IL-4 as indicators of a ThI or a Th2 response, respectively, was determined by intracellular flow cytometry. As demonstrated in Fig. 3D, the frequency of IFN- gamma producing T cells increased with time of the xenogeneic immune reaction. In contrast, IL-4 producing T cells could not be detected by intracytoplasmic stain after ex vivo stimulation in the recovered T cells (data not shown). Importantly, histopathological analysis revealed infiltration of activated human lymphocytes into the portal tracts of the liver, and into the perigastrointestinal and perirenal fatty tissues (Fig. 3E,F). The lymphocytic infiltrates were frequently organized in granuloma-like structures with pallisading human macrophages and central necrosis (Fig. 3F), further indicating the ThI -biased nature of the inflammatory immunity (29-31). By contrast, eosinophils, indicative of Th2 activation, could not be detected in the granulomas (data not shown).

Cylosporine prevents the human ThI immune reaction in SCID mice. To define the role of T cells in the initiation of the xenogeneic GvH reaction in the SCID mouse more precisely, experiments were carried out in which PBMC-injected mice were treated with cyclosporine, an inhibitor of T cell activation (Fig. 4). Cyclosporine treatment had a marked inhibitoiy effect on the activation of CD4 T cells (Fig. AA). The effect of cyclosporine was even more pronounced with regard to the expansion of IFN-gamma producers, which was completely prevented by cyclosporine (Fig. AB). As a reflection of the activity of effector T cells in vivo, the inflammatory human cytokines, IFN-gamma and TNF and human IL-4 were measured in the serum of the animals. The serum level of human IFN-gamma was markedly diminished in the cyclosporine-treated mice compared to PBS-treated mice, whereas the serum level of human TNF was only slightly decreased (Fig. AC). Human IL-4 was not detectable in any of the mice (data not shown). Together, the data strongly indicate that the immune reaction of human PBMC to mouse tissue generates a T cell- mediated, Thl-biased immune response.

The xenogeneic ThI -biased immune response requires APC. The development of a specific immune response requires APC (32). Monocytes represent potential APC in the pool of PBMC. To evaluate the impact of monocytes on the development of the human Thl-biased xenogeneic GvH reaction, T cells and monocytes were purified from the peripheral blood of the same donor and injected into SCID mice separately or at a purposeful ratio of five T cells to one monocyte (Fig. 5A-C). Activation of CD4 T cells, their differentiation into effectors capable of IFN-gamma production, and in vivo secretion of IFN-gamma and TNF were all observed only in the mice that had received T cells together with monocytes.

To characterize the role of monocytes in the development of the human xenogeneic Thl-biased reaction in greater detail, we carried out experiments with different ratios of purified T cells to monocytes keeping the absolute number of injected T cells constant (Fig. 5D). Levels of human cytokines indicative of an in vivo Thl-biased effector cell activation (IFN-gamma and TNF) were measured in the serum of the animals. A marked increase in the concentration of -IFN- gammaγ and TNF was detected in the serum of the animals that had been injected with mixtures that contained higher ratios of monocytes to T cells. Notably, when the in vivo production of IFN-gamma-γ from PBMC of various donors was analyzed as a function of the monocyte to T cell ratio within the injected PBMC, the IFN-gamma concentration in the serum of the animals correlated directly with this ratio (Fig. 5E). Thus, the ratio of human monocytes to T cells regulates the strength of the ThI -mediated xenogeneic reaction.

IL-4 and IL-IO are regulators of human ThI immunity. A large body of in vitro data suggests that the development of a human Thl-mediated immune reaction is controlled by cytokines, such as IL-4 and EL-IO (26). However, conclusive evidence from in vivo human immune responses has not been provided to date. To address this question, human endogenously produced EL-4 and IL- 10 were neutralized during the development of the xenogeneic Thl-biased reaction resulting from the injection of PBMC into SCED mice by mAb that were specific for the human cytokines with no cross reactivity to murine IL-4 and IL-IO (Fig. 6). The blockade of endogenous human IL-4 lead to a significant enhancement of the ThI -biased immune response (Fig. 6A-C). Whereas the frequency of activated CD4 T cells within the cells recovered from anti-IL-4-treated mice was comparable to control mice (Fig. 6A), neutralization of endogenous IL-4 resulted in a significantly increased frequency of T cells capable of IFN-gamma production (1.2 ± 0.2 fold increase, p < 0.004; Fig. 6B). When the in vivo activity of effector T cells was assessed, the effect of IL-4 neutralization became even more pronounced, as neutralization of IL-4 resulted in a more than twofold increase in the serum concentrations of human IFN-gamma (3.0 ± 2.4 fold increase, p < 0.05) and human TNF (2.2 ± 1.5 fold increase, p = 0.06) in the serum of the mice (Fig. 6C). As IFN-gamma production is not affected directly by IL-4 (33) this is likely to be a consequence of markedly increased numbers of activated cytokine producing T cells rather than of enhanced secretion of human IFN-gamma and TNF by the activated T cells.

Neutralization of endogenously produced IL-10 during the xenogeneic human Thl-biased immune response did not interfere with the activation of CD4 T cells as the frequencies of HLA- DR positive CD4 T cells recovered from control mice and from anti-IL-10-treated mice were comparable (Fig. 6D). Inhibition of IL-10, however, resulted in a significant increase of the frequencies of effector T cells capable of IFN-gamma production (1.2 ± 0.3 fold increase, p < 0.02; Fig. 6E). Consequently, blockade of IL-10 resulted in a tendency for increased ThI- mediated effector functions in vivo as documented by increased serum levels of IFN-gamma (1.9 ± 1.0 fold increase, p = 0.08) and TNF (1.8 ± 0.8 fold increase, p < 0.05; Fig. 6F). Thus, the development of a Thl-biased human immune reaction is tightly controlled by the endogenously produced anti-inflammatory cytokines, IL-4 and IL-10. It should be noted, that no IL-10 mRNA could be detected in recovered cells even when using highly sensitive real-time PCR, and IL-4 mRNA was only found at a very low level (data not shown), suggesting that the mechanisms of action of IL-4 and IL-10 on the development of a human Thl-biased immune response, although different, are very precise and efficient processes.

IL-4 down-modulates established Thl-biased immunity. Neutralization of endogenous IL-4 lead to an unbalanced and exaggerated development of the xenogeneic human Thl-biased immune response (Fig. 6A-C), suggesting the potential of this cytokine to function as an immune ,Λ 26

modulator in humans. Therefore, we sought to delineate the effect of EL-4 as a means to down- modulate an established ThI -mediated immune response. SCID mice were injected with PBMC, and starting at day 14 they were treated daily for 5 days with recombinant human IL-4 (Fig. 7). A five-day-treatment with EL-4 did not alter the frequency of HLA-DR expressing T cells (Fig. IA). However, the Thl-biased differentiation of T cells into IFN-gamma producing effectors was significantly inhibited in response to IL-4 treatment (reduction to 92 ± 7 % of control, p < 0.03; Fig. IB). Moreover, EL-4 caused a significant suppression of the Thl-biased effector functions in vivo, as the concentrations of human IFN-gamma and TNF were substantially decreased in the serum of the treated animals (reduction to 50 ± 43 % and 63 ± 32 % of control, respectively, p < 0.04; Fig. 1C). Together, the data indicate that administration of exogenous EL-4 is able to down- modulate an established Thl-biased immune response.

IL-10 targets ThI effector functions. IL-10 has been shown to be a very powerful inhibitor of IFN- gamma-mediated effector functions in the mouse by preventing the production of IFN- gamma (34). The data from the anti-IL-10 experiments (Fig. 6D-F) suggest a similar mechanism of IL-10 activity in humans. To test this hypothesis, we investigated the effect of exogenous human IL-10 on an established ThI -mediated immune reaction of human PBMC (Fig. 8). EL-IO treatment reduced the frequency of HLA-DR postive CD4 T cells, but, in contrast to IL-4, had no effect on the differentiation of cells capable of producing IFN-gamma (Fig. 8A,B). However, IL- 10 treatment significantly inhibited the in vivo production of IFN-gamma and TNF as assessed by measurement of serum levels of these cytokines (reduction to 51 ± 19 % and 60 ± 32 %, respectively; p < 0.002 andp < 0.03; Fig. 5C). Thus, IL-10 might inhibit effector functions of the human ThI -mediated immune response by preventing the production of these inflammatory cytokines.

TaWe 1. FnψtL'Α'ia qf Traih imitating .ψomationu proliferation itfh'f άitmήiction into SClO niks CD4 Ce1Ib i'%j CDS CCII1- ») Donor 1 0.46 1.D7 Donor 2 1 ,09 2.43 References

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