WO2005106469A1 - Modulation d'une reaction immunitaire humaine - Google Patents

Modulation d'une reaction immunitaire humaine Download PDF

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WO2005106469A1
WO2005106469A1 PCT/EP2005/004740 EP2005004740W WO2005106469A1 WO 2005106469 A1 WO2005106469 A1 WO 2005106469A1 EP 2005004740 W EP2005004740 W EP 2005004740W WO 2005106469 A1 WO2005106469 A1 WO 2005106469A1
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cells
human
thl
mice
cell
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PCT/EP2005/004740
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Hendrik Schulze-Koops
Alla Skapenko
Peter Lipsky
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Friedrich-Alexander- Universitaet Erlangen- Nuernberg
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates

Definitions

  • the invention concerns an in vivo model for human Thl 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 Thl cells.
  • the present invention thus is directed to a method of screening candidate compounds for the modulation of human Thl mediated immune responses and to compounds for the modulation of human Thl 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.
  • Thl 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, Thl cell-mediated immune reaction requires certain cell populations that are involved in and carry out the different steps of the immune reaction.
  • 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 Thl cells with particular effector functions.
  • Thl cells predominantly secrete interferon-gamma (IFN-garnma) and interleukin-2 (IL-2). By secreting these cytokines, other effector cell populations get activated.
  • IFN-garnma interferon-gamma
  • IL-2 interleukin-2
  • Thl cell-mediated immune reactions examples include 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 Thl- mediated immune responses that result in destruction of tissue in these situations or diseases are the same as those activated during protective immune responses.
  • 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 Thl cells (2, 3). Therefore, the mechanisms controlling the evolution of Thl -biased immune responses play a critical role in the development of pathogenic autoimmune reactions. Delineation of the mechanisms controlling Thl -mediated immunity has largely been derived from animal models.
  • 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 (A, 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).
  • treatment with recombinant IL-4 induces a switch from a Thl -type to a Th2-type response and prevents proteoglycan-induced arthritis, a different model of human inflammatory arthritis (9).
  • IL-10 immunomodulatory cytokine
  • 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 Thl 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 Thl- mediated autoimmune responses in animals.
  • IL-4 and IL-10 clearly exhibit an anti-inflammatory effect as they induce expression of the IL-1 receptor antagonist (14, 15) and down-regulate the production of pro-inflammatory cytokines, such as IL-1 and TNF from human monocytes (16, 17).
  • IL-4 has a direct inhibitory effect on the development of human Thl cells (18) and IL-10 is able to prevent Thl effector functions by induction of long lasting T cell unresponsiveness (19).
  • the invention describes a model the permits the in vivo analysis of a human Thl 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, Thl cell mediated immune response.
  • One focus of the present invention are therefore human, Thl cell driven immune responses that evolve after injection of human cells in the body of immunocompromised mice.
  • human immune reactions 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.
  • the invention concerns an in vivo model for human Thl -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, Thl -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 is hampered by the fact that technical possibilities to delineate the development, the continuation and the resolution of a human, Thl cell-mediated immune response in an in vivo situation, are missing.
  • 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 Thl cells.
  • Thl-driven immune responses are defined as immune responses that are mediated by T helper cells that produce the proinflammatory cytokine, interferon-gamma (Thl -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:
  • the present invention provides a method of screening candidate compounds for the modulation of human Thl mediated immune responses, comprismg the following steps:
  • APCs antigen presenting cells
  • 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.
  • CD 4 + T cells as contained herein is defining a group of cells mandatorily comprising the Thl 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 ficoll gradient centrifugation.
  • PBMC peripheral blood mononuclear cells
  • 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 Thl cells.
  • 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.
  • the method of the present invention employs cells which are peripheral blood mononuclear cells or derived therefrom.
  • cells which are peripheral blood mononuclear cells or derived therefrom.
  • 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.
  • 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 APC's preferably are selected from the group consisting of monocytes, macrophages, B cells and/or dendritic cells.
  • 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.
  • a mammal in the present method 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.
  • 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-1 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.
  • the animal is a SCID mouse wherein the mouse was treated by sublethal irradiation in order to acquire a SCID phenotype.
  • the animals used have a naturally occurring SCID mutation.
  • the mouse was generated by biotechnological elemination of RAG-1 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.
  • intraperitoneal, intravenous, intramuscular, intracutanous, subcutanous and/or intraarticular administration are suitable.
  • the candidate compound is selected from the group consisting of drugs, including chemical or biological agents, or cells.
  • 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.
  • CD4 T cell subset with regulatory capacity is defined by the constitutive expression of the alpha chain of the IL-2 receptor, CD25.
  • CD25 pos 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).
  • CD25 pos 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).
  • CD25 pos CD4 T cells in contrast to their CD25 neg 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).
  • 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).
  • CTLA-4 CTLA-4
  • GITR glucocorticoid induced TNF receptor family related protein
  • Foxp3 the transcription factor Foxp3
  • the importance of Foxp3 for the regulatory function of Tregs has been demonstrated by transfection of CD25 ne ⁇ CD4 T cells with a plasmid encoding Foxp3, which conferred a regulatory capacity to the trans fected T cells (68).
  • the accumulated evidence indicates that Tregs may play an important role in maintaining peripheral tolerance and preventing the evolution of autoimmune inflammation.
  • IL-4 favors the generation of CD25 + T cells with regulatory capacity in an in vivo model of human inflammation:
  • 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.
  • the present invention in a second aspect provides a compound for the modulation of human Thl 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.
  • 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.
  • 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.
  • 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 (15 th Ed., Mack Publishing Company, Easton, Ps., 1980).
  • the compound and/or composition as identified above is used for the prevention or treatment of unwanted immune reactions in human beings.
  • the compound and/or composition of the present invention is used for the prevention or treatment of autoimmune diseases and/or transplant rejection reactions.
  • 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.
  • 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.
  • organ specific diseases such as thyroiditis, insulitis, multiple sclerosis, iridocyclitis, uveitis, orchitis, hepatitis, Addison's disease, myasthenia gravis
  • systemic illnesses such as rheumatoid arthritis, vasculitis or lupus erythematosus.
  • 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 PBMC'
  • a method of preventing or treating an autoimmune disease and/or transplant rejection reaction in a human comprising administering to a human patient in need thereof a therapeutically effective amount of a compound or composition as defined above.
  • FIGURES la and lb 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-10 (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).
  • PBS buffer phosphate buffered saline
  • 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 Thl 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.
  • FIGURE 3 Human PBMC develop a Thl-biased immune reaction after intraperitoneal injection into SCID mice. 50xl0 6 human PBMC were injected i.p. into SCID 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.
  • C Percentage of CD4 cells expressing the activation marker, HLA-DR or CD25.
  • Immunostaining of a serial section to iii identifies the majority of infiltrating cells as human CD3 positive T cells (iv).
  • F A typical epitheloid cell granuloma from perigastric fatty tissue is illustrated ( , hematoxylin and eosin staining). Immunostaining reveals a row of human CD68 positive macrophages surrounding an area of central necrosis (ii).
  • the polymorphic lymphoid cell infiltrate adjacent to the macrophages consists of human CD3 positive T cells (iii) with a large proportion of CD4 positive cells (iv) and a smaller number of CD8 positive cells (v). Adjacent to the granuloma are aggregates of human CD20 positive B cells (vi).
  • FIGURE 4 Cyclosporine prevents the Thl-biased human immune response in SCID mice.
  • 50x10 6 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-garnma after in vitro stimulation with PMA and ionomycin. For comparison, values from freshly isolated PBMC are shown ("Before").
  • 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 Thl-biased immune reaction of human cells in SCID mice.
  • Human T cells and monocytes were purified by negative selection from PBMC.
  • a - C 50xlO ⁇ T cells and 10xl0 ⁇ monocytes from the same donor were injected either separately or together into mice.
  • mice were injected with PBMC that contained 50xl0 6 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.
  • D 50x10 6 T cells were injected together with different numbers of monocytes (6.25x10 6 , 12.5xl0 6 , or 25x10 6 , 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 Thl- biased immune reaction.
  • 50x10 ⁇ freshly isolated human PBMC were injected into SCID mice.
  • mice were treated with mAbs to (A - Q human IL-4 ("anti-IL-4") or (D - F) human IL-10 ("anti-IL-10").
  • 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- 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 Thl-biased immune reaction.
  • 50x10 6 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 Thl-biased immune reaction.
  • 50x10 6 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 Thl- 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.
  • mice To induce the development of a human, Thl 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.
  • immunodeficient mice we have used animals that have a natural occurring SCID mutation on the NOD (non obese diabetic) background (NOD/SCID-mice).
  • NOD/SCID-mice non obese diabetic background
  • 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).
  • Thl 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.
  • a mouse is sacrificed.
  • Human cells can be recovered from the peritoneal cavity by irrigation.
  • tissue surrounding the peritoneal cavity is analyzed.
  • the cells harvested from the peritoneum are assessed by extra and intracellular flow cytometry.
  • FIG. 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 Thl 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.
  • histological analysis of the surrounding tissue reveals granulomatous formations, which are typical for Thl cell-mediated diseases, such as tuberculosis and Wegener's granulomatosis.
  • the inventional human Thl cell-mediated immune response can be used as a model to assess the impact of different immuno-suppressants in vivo.
  • Figures la and lb illustrate examples of such an approach.
  • mononuclear cells from the peripheral blood of healthy volunteers are injected into the peritoneal cavity of NOD/SCID mice.
  • IL-4 0.1 mg
  • IL-10 0.01 mg
  • 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).
  • FIG. 1 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 Thl cell-driven immune response of human cells against murine antigens.
  • the development of this immune reaction is associated with an increase of CD25 positive Th cells.
  • 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.
  • 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.
  • the extent of inflammation is determined by analysis of the production of human inflammatory cytokines.
  • injection of CD25 positive T cells results in amelioration of the Thl -immune response, whereas the injection of CD25 negative T cells enhances the immune response.
  • 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-10 and the neutralizing mAb to IL-10 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 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 (Er Weg, Germany). Mice were used at 6-12 weeks of age.
  • PBMC Human cell preparation.
  • PBMC were obtained by ficoll-hypaque (Sigma) gradient- centrifugation of heparinized venous blood from young healthy volunteers not taking any medications.
  • 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.
  • T cells were negative for the activation markers CD25, CD30, CD69, and HLA-DR.
  • Cytokine determination Human cells were recovered from the peritoneal cavity of the mice. To assess the acquired capacity of T cells for cytokine production, 2x10 s 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.
  • mice 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 ⁇ m paraffin sections were stained with hematoxylin and eosin for morphological assessment by light microscopy.
  • 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.
  • Fig. 3C 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 frequency of IFN- gamma producing T cells increased with time of the xenogeneic immune reaction.
  • IL-4 producing T cells could not be detected by intracytoplasmic stain after ex vivo stimulation in the recovered T cells (data not shown).
  • Fig. 3E,F histopathological analysis revealed infiltration of activated human lymphocytes into the portal tracts of the liver, and into the perigastrointestinal and perirenal fatty tissues.
  • the lymphocytic infiltrates were frequently organized in granuloma-like structures with pallisading human macrophages and central necrosis (Fig. 3F), further indicating the Thl-biased nature of the inflammatory immunity (29-31).
  • eosinophils indicative of Th2 activation, could not be detected in the granulomas (data not shown).
  • Cylosporine prevents the human Thl immune reaction in SCID mice.
  • 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. 4R).
  • 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 Thl-biased immune response requires APC.
  • the development of a specific immune response requires APC (32).
  • Monocytes represent potential APC in the pool of PBMC.
  • 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.
  • IL-4 and IL-10 are regulators of human Thl immunity.
  • cytokines such as IL-4 and IL-10 (26).
  • IL-4 and IL-10 26
  • conclusive evidence from in vivo human immune responses has not been provided to date.
  • human endogenously produced IL-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-10 (Fig. 6).
  • Fig. 6A-C The blockade of endogenous human IL-4 lead to a significant enhancement of the Thl-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. 6-4), 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).
  • 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 modulator in humans. Therefore, we sought to delineate the effect of IL-4 as a means to down- modulate an established Thl-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. 7-4).
  • 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. 7B).
  • 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. 7Q.
  • the data indicate that administration of exogenous DL-4 is able to down- modulate an established Thl-biased immune response.
  • IL-10 targets Thl 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.
  • Fig. 8 we investigated the effect of exogenous human IL-10 on an established Thl-mediated immune reaction of human PBMC (Fig. 8).
  • IL-10 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. SA,B).
  • 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 ⁇ dp ⁇ 0.03; Fig. 5C).
  • IL-10 might inhibit effector functions of the human Thl-mediated immune response by preventing the production of these inflammatory cytokines.
  • Proteoglycan (aggrecan)-induced arthritis in BALB/c mice is a Thl -type disease regulated by Th2 cytokines. J. Immunol. 163:5383.
  • IL-10 is critical in the regulation of autoimmune encephalomyelitis as demonstrated by studies of EL-10- and IL-4-deficient and transgenic mice. J. Immunol. 161:3299.
  • Interleukin 10 upregulates EL-1 receptor antagonist production from lipopolysaccharide-stimulated human polymorphonuclear leukocytes by delaying mR ⁇ A degradation. J Exp. Med. 179:1695.
  • Interleukin (IL) 4 differentially regulates monocyte IL-1 family gene expression and synthesis in vitro and in vivo. J. Exp. Med. 177:775.
  • Interleukin 10 inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J. Exp. Med. 174:1209.
  • Interleukin- 10 induces a long-term antigen-specific anergic state in human CD4+ T cells. J. Exp. Med. 184:19.
  • IL-10 is a key cytokine in psoriasis. Proof of principle by EL- 10 therapy: a new therapeutic approach. J. Gin. Invest. 101:783.
  • IL-10 improves skin disease and modulates endothelial activation and leukocyte effector function in patients with psoriatic arthritis.
  • IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells. J. Immunol. 149:2378.
  • Sakaguchi S, Sakaguchi ⁇ , Asano M, Itoh M, Toda M Immunologic self-tolerance maintained by activated T Cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 1995, 155: 1151-1164.

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Abstract

L'invention concerne un modèle in vivo pour des réactions immunitaires inflammatoires humaines médiées par les cellules Th1 et l'application de ce modèle pour développer des approches thérapeutiques faisant appel à des substances, telles que des médicaments, des produits chimiques, des substances biologiques et des cellules pour des réactions immunitaires pathologiques chez l'homme, induites par des cellules Th1 activées. La présente invention se rapporte donc à une méthode de sélection de composés candidats pour la modulation de réactions immunitaires humaines médiées par les Th1 et à des composés pour la modulation de réactions immunitaires humaines médiées par les Th1, identifiés par ladite méthode. Cette invention concerne en outre l'utilisation desdits composés dans le domaine de la médecine, en particulier dans la prévention et le traitement de maladies auto-immunes et du rejet de greffons.
PCT/EP2005/004740 2004-05-03 2005-05-02 Modulation d'une reaction immunitaire humaine WO2005106469A1 (fr)

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US20030135875A1 (en) * 2000-05-12 2003-07-17 Rolf Ehrhardt Models of chronic and acute inflammatory diseases

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US20030135875A1 (en) * 2000-05-12 2003-07-17 Rolf Ehrhardt Models of chronic and acute inflammatory diseases

Non-Patent Citations (4)

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Title
ALFONZO M ET AL: "Temporary restoration of immune response against Toxoplasma gondii in HIV-infected individuals after HAART, as studied in the hu-PBMC-SCID mouse model", CLINICAL AND EXPERIMENTAL IMMUNOLOGY, vol. 129, no. 3, September 2002 (2002-09-01), pages 411 - 419, XP002341512, ISSN: 0009-9104 *
FUJIKI YUTAKA ET AL: "Dominant expansion of human T cells in non-obese diabetic/severe combined immunodeficiency mice implanted with human bone fragments", EXPERIMENTAL HEMATOLOGY (CHARLOTTESVILLE), vol. 28, no. 7, July 2000 (2000-07-01), pages 792 - 801, XP002341511, ISSN: 0301-472X *
MOSIER D E ET AL: "TRANSFER OF A FUNCTIONAL HUMAN IMMUNE SYSTEM TO MICE WITH SEVERE COMBINED IMMUNODEFICIENCY", NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 335, 15 September 1988 (1988-09-15), pages 256 - 259, XP000650830, ISSN: 0028-0836 *
SKAPENKO ALLA ET AL: "Regulation of human Th1 inflammation in vivo.", FASEB JOURNAL, vol. 17, no. 7, 14 April 2003 (2003-04-14), & 90TH ANNIVERSARY ANNUAL MEETING OF THE AMERICAN ASSOCIATION OF IMMUNOLOGISTS; DENVER, CO, USA; MAY 06-10, 2003, pages C131, XP009052647, ISSN: 0892-6638 *

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