CELL POPULATIONS HAVING IMMUNOREGULATORY ACTIVITY, METHOD FOR ISOLATION AND USES

FIELD OF THE INVENTION The present invention relates to the prevention, treatment or amelioration of one or more symptoms of disorders in which modulation of a subject's immune system is beneficial utilizing cell populations derived from adult tissues In particular, the present invention provides a population of connective tissue derived cells that respond to interferon-gamma (IFN-γ) by expressing indolamine-2,3-dioxygenase (IDO) for use in preventing, treating or ameliorating one or more symptoms associated with disorders in which modulation of a subject's immune system is beneficial, including, but not limited to, autoimmune diseases, inflammatory disorders, and immunologically mediated diseases including rejection of transplanted organs and tissues

BACKGROUND OF THE INVENTION

The immune system in higher vertebrates represents the first line of defence against vanous antigens that can enter the vertebrate body, including micro-organisms such as bacteπa, fungi and viruses that are the causative agents of a variety of diseases Moreover, the immune system is also involved in a variety of other diseases or disorders, including autoimmune or immunopathologic diseases, immunodeficiency syndromes, atherosclerosis and various neoplastic diseases Although methods are available for treating these diseases, many current therapies provide less than adequate results Among new emergent therapeutic strategies, those based on cell therapy appear to constitute a potentially useful tool for treating a great number of diseases Thus, a great effort is being currently made by researchers in order to achieve said aim

AUTOIMMUNE DISEASES

Autoimmune diseases are caused when the body's immune system, which is meant to defend the body against bacteria, viruses, and any other foreign product, malfunctions and produces a pathological response against healthy tissue, cells and organs Antibodies,

T cells and macrophages provide beneficial protection, but can also produce harmful or deadly immunological responses

Autoimmune diseases can be organ specific or systemic and are provoked by different pathogenic mechanisms Organ specific autoimmumzation is characterized by aberrant expression of major-histocompatibihty complex (MHC) antigens, antigenic mimicry and allelic variations in MHC genes Systemic autoimmune diseases involve polyclonal B cell activation and abnormalities of immunoregulatory T cells, T cell receptors and MHC genes Examples of organ specific autoimmune diseases are diabetes, hyperthyroidism, autoimmune adrenal insufficiency, pure red cell anemia, multiple sclerosis and rheumatic carditis Representative systemic autoimmune diseases are systemic lupus erythematosus, chronic inflammation, Sjogren's syndrome, polymyositis, dermatomyositis and sclerodeπna

Current treatment of autoimmune diseases involves administering immunosuppressive agents such as cortisone, aspiπn derivatives, hydroxychloroquine, methotrexate, azathiopπne and cyclophosphamide or combinations thereof The dilemma faced when administering immunosuppressive agents, however, is the more effectively the autoimmune disease is treated, the more defenseless the patient is left to attack from infections, and also the more susceptible for developing tumours Thus, there is a great need for new therapies for the treatment of autoimmune diseases

INFLAMMATORY DISORDERS Inflammation is a process by which the body's white blood cells and secreted factors piotect our bodies fiom infection by foreign substances, such as bacteria and viruses Secreted factors known as cytokines and prostaglandins control this process, and are released in an ordered and self-limiting cascade into the blood or affected tissues

Inflammatory Bowel disease (IBD)

IBD is a family of chronic, idiopathic, relapsing, and tissue-destructive diseases characteπzed by dysfunction of mucosal T cells, altered cytokine production and cellular inflammation that ultimately leads to damage of the distal small intestine and the colonic mucosa IBD is clinically subdivided into two phenotypes Crohn s disease (CD) and ulcerative colitis CD is a nowadays incurable autoimmune disease with a prevalence of 0 05% that leads to chronic inflammation resulting in a range of gastrointestinal and extraintestinal symptoms, including abdominal pain, rectal bleeding, diarrhea, weight loss, skin and eye disorders, and delayed growth and sexual maturation in children These

symptoms can greatly impact the patients' well being, quality of life, and capacity of function Because CD is chronic and typically has an onset before 30 years of age, patients generally require lifelong treatment Although its etiology remains unknown, there is circumstantial evidence to link CD to a failure of the mucosal immune system to attenuate the immune response to endogenous antigens

Therapeutic agents currently used for CD, including aminosalicylates, corticosteroids, azathiopπne, 6-mercaptopuπne, antibiotics, and methotrexate, are not entirely effective, nonspecific, and with multiple adverse side effects In most cases, surgical resection is the ultimate alternative Therefore, the present therapeutic strategy is to find drugs or agents that specifically modulate both components of the disease, i e , the inflammatory and T-cells dπven responses

Recently, the drug infliximab has been approved for the treatment of moderate to severe Crohn's disease that does not respond to standard therapies and for the treatment of open, draining fistulas Infliximab, the first treatment approved specifically for Crohn's disease, is an anti-tumour necrosis factor (TNF) antibody TNF is a protein produced by the immune system that may cause the inflammation associated with Crohn's disease Anti- TNF removes TNF from the bloodstream before it reaches the intestines, thereby preventing inflammation However, since it has a systemic effect, and TNF is a very pleiotropic factor, severe side effects are relatively common, and its long-term safety is still to be determined Also, the efficacy is also limited because many of the inflammatory processes that occur in the patients are not dependant on TNF signalling

Rheumatoid arthritis (RA)

Rheumatoid arthritis and juvenile rheumatoid arthritis are types of inflammatory arthritis Arthritis is a general term that descπbes inflammation in joints Some, but not all, types of arthritis are the result of misdirected inflammation Rheumatoid arthritis affects about 1 % of the world's population and is essentially disabling Rheumatoid arthritis is an autoimmune disorder where the body's immune system improperly identifies the synovial membranes that secrete the lubricating fluid in the joints as foreign Inflammation results, and the cartilage and tissues in and around the joints are damaged or destroyed The body replaces damaged tissue with scar tissue, causing the normal spaces within the joints to become narrow and the bones to fuse together

In rheumatoid arthritis, there is an autoimmune cycle of persistent antigen presentation, T-cell stimulation, cytokine secretion, synovial cell activation, and joint destruction

Currently available therapy for arthritis focuses on reducing inflammation of the joints with anti-inflammatory or immunosuppressive medications The first line of treatment of any arthritis is usually antiinflammatories, such as aspirin, ibuprofen and

Cox-2 inhibitors such as celecoxib and rofecoxib Anti-TNF humanized monoclonal antibodies, such as Infliximab are also used, however, it has many secondary effects or side effects and its efficacy is quite low. "Second line drugs" include gold, methotrexate and steroids. Although these are well-established treatments for arthritis, very few patients remit on these lines of treatment alone, and difficult treatment issues still remain for patients with rheumatoid arthritis.

In general, the current treatments for chronic inflammatory disorders have a very limited efficiency, and many of them have a high incidence of side effects or cannot completely prevent disease progression So far, no treatment is ideal, and there is no cure for these type of pathologies. Thus, there is a great need for new therapies for the treatment of inflammatory disorders

INHIBITION OF T-CELL RESPONSES All immune responses are controlled by T cells Self-reactive cells with the potential to elicit autoimmune responses comprise a part of the normal T cell repertoire, but in the healthy state, their activation is prevented by suppressor cells Although T suppressor cells were originally descπbed in the 1970s, significant progress in characterizing T-cell subsets has been made only recently, when they have been renamed as regulatory T cells

There are different CD4 ⁺ , CD8 ⁺ , natural killer cell, and γδ T cell subsets with regulatory (suppressor) activity. Two major types of T-reg cells have been charactenzed in the CD4 ⁺ population, i.e., the naturally-occurring, thymus-generated T-reg cells, and the peripherally-induced, IL-10 or TGF-β secreting T-reg cells (TrI cells) The CD4 ⁺ CD25 ⁺ , Foxp3 -expressing, naturally-occurring T-reg cells generated in thymus, migrate and are maintained in the periphery. The signals for their thymic generation and maintenance in the peπphery are not entirely defined, although both CD28 stimulation and IL-2 appear to be required The number of CD4 ⁺ CD25 ⁺ T-reg cells in the peπphery does not decrease with

age, although these cells are anergic and prone to apoptosis, and their site of ongin, the thymus, undergoes age-related involution This suggests that the pool of CD4 ⁺ CD25 ⁺ T-reg cells is maintained peripherally Several experimental models support the idea of peripheral generation of CD4 ⁺ CD25 ⁺ T-reg cells from CD4 ⁺ CD25 T cells The endogenous factors and mechanisms controlling the peripheral expansion of CD4 ⁺ CD25 ⁺ T-reg cells are mostly unknown

There is evidence that the cytokine transforming growth factor-beta (TGF-β) plays an important role in the expansion of thymus-denved, professional CD4+ CD25+ precursors that circulate in the blood TGF-β is also involved in the generation of peripherally induced CD4+ and CD8+ regulatory subsets

However, recent experimental data suggest that a mechanism of immunotolerance could be dependent on tryptophan metabolism, and in particular on the activity of the enzyme indoleamme 2,3-dioxygenase (IDO), which is an intracellular heme-containing enzyme that catalyzes the initial rate-limiting step in tryptophan degradation along the kynurenine pathway

There is considerable evidence that supports the hypothesis that cells expressing

IDO can suppress T cell responses and promote tolerance (Mellor and Munn, Nat Rev

Immunol 2004 Oct, 4(10) 762-74) IDO is expressed in some subsets of dendritic cells

(DCs), which are key regulators of immune response (tolerogenic DCs) These DCs are able of suppressing in vivo T-cell responses by locally depleting tryptophan (US Patent No

2002/0155104) Aside from monocyte-deπved DCs and macrophages, several tumour lines, intestinal cells, and trophoblasts express IDO The expression of IDO in trophoblasts appears to be constitutive and has been strongly correlated to tolerance of allogeneic tissue from the foetus IDO is believed to induce apoptosis in T cells, and cause spontaneous tolerance to liver allografts

The molecular mechanisms behind the immunosuppressive activity of IDO are not known However, it has been demonstrated that DCs expressing IDO are able to induce the generation of regulatory T cells IDO is induced in human cells by several inflammatory mediators, including interferons and hpopolysacchaπde (LPS), as well as by viral infection Several studies have shown that allogeneic tumour cells being rejected by the host immune system in vivo up-regulate IDO and this effect is mediated by IFN-γ

Recent experiments have indicated an in vitro immunosuppressive capacity of bone marrow derived mesenchymal stem cells (MSCs) and adipose-deπved stem cells (ASCs), as well as an in vivo immunosuppressive capacity of MSCs This in vivo activity has been studied in bone marrow transplants, in which the infusion of expanded MSCs appears to reduce acute and chronic graft versus host disease (GVHD) The in vitro effect is characterized by a suppression of lymphocyte proliferation in experiments where the lymphocytes were activated either via a mixed lymphocyte reaction (MLR) or stimulation with phytohemagglutinin (PHA) However, the molecular mechanisms responsible for the immunosuppressive effects of said cells have not been unequivocally identified

SUMMARY OF THE INVENTION

The invention is based on the discovery that certain cell populations with multilineage potential which are present in different connective tissues are capable of acting as immunoregulatory agents in vivo and in vitro Inventors have isolated a population of connective tissue deπved cells that respond to interferon-gamma (IFN-γ) by expiessing indolamine-2,3-dioxygenase (IDO) The immunoregulatory effects of said cells can be used for preventing, treating or ameliorating one or more symptoms associated with disorders in which modulation of a subject's immune system is beneficial, including, but not limited to, autoimmune diseases, inflammatory disorders, and immunologically mediated diseases including rejection of transplanted organs and tissues

Thus, in an aspect, the invention relates to an isolated cell population from connective tissue wherein the cells of said cell population (i) do not express markers specific from antigen-presenting cells (APC) in other words markers specific for antigen presenting cells, (n) do not express indolamine 2,3-dioxygenase (IDO) constitutively, (in) express IDO upon stimulation with interferon-gamma (IFN-γ), and (iv) present capacity to be differentiated into at least two cell lineages

In other aspect, the invention relates to a method for the isolation of said cell population The cell population obtainable according to said method constitutes an additional aspect of this invention In other aspect, the invention relates to said cell population for use in the prevention, treatment or amelioration of one or more symptoms of disorders in which modulation of a subject's immune system is beneficial

In other aspect, the invention relates to said cell population for use as medicament, or for inducing transplantation tolerance, or for treating autoimmune diseases, or for treating an inflammatory disease In a particular embodiment, said inflammatory disease is a chronic inflammatory disease, such as, for example, Inflammatory Bowel Disease (IBD) or Rheumatoid Arthritis (RA)

In other aspect, the invention relates to the use of said cell population in the preparation of a medicament, such as a medicament for the prevention, treatment or amelioration of one or more symptoms of disorders in which modulation of a subject's immune system is beneficial, e g , a medicament for inducing transplantation tolerance, or a medicament for treating autoimmune diseases, or a medicament for treating an inflammatory disease

In other aspect, the invention relates to the use of said cell population in the preparation or generation of regulatory T-cells (T-reg) Said T-reg cell population as well as a method for the isolation thereof constitute further aspects of the invention In other aspect, the invention relates to said T-reg cell population for use as medicament, or for inducing transplantation tolerance, or for treating autoimmune diseases, or for treating an inflammatory disease

In other aspect, the invention relates to the use of said T-reg cell population in the preparation of a medicament, such as a medicament for the prevention, treatment or amelioration of one or more symptoms of disorders in which modulation of a subject's immune system is beneficial, e g , a medicament for inducing transplantation tolerance, or a medicament for treating autoimmune diseases, or a medicament for treating an inflammatory disease, or a medicament for treating allergies, for example, but not limited to, hypersensitivity Type IV reactions In other aspect, the invention relates to a method for the isolation of an irradiated cell population which compπses irradiating said cell population with a controlled source of ionizing radiation under appropπate conditions Said irradiated cell population constitutes a further aspect of the invention

In other aspect, the invention relates to said irradiated cell population for use as medicament, or for inducing transplantation tolerance, or for treating autoimmune diseases, or for treating an inflammatory disease

In other aspect, the invention relates to the use of said irradiated cell population in the preparation of a medicament, such as a medicament for the prevention, treatment or

amelioration of one or more symptoms of disorders in which modulation of a subject's immune system is beneficial, e g , a medicament for inducing transplantation tolerance, or a medicament for treating autoimmune diseases, or a medicament for treating an inflammatory disease In other aspect, the invention relates to a method which comprises subjecting said cell population to treatment with interferon-γ (IFN-γ). Said IFN-γ-treated cell population constitutes a further aspect of the invention

In other aspect, the invention relates to said IFN-γ-treated cell population for use as medicament, or for inducing transplantation tolerance, or for treating autoimmune diseases, or for treating an inflammatory disease

In other aspect, the invention relates to the use of said IFN-γ-treated cell population in the preparation of a medicament, such as a medicament for the prevention, treatment or amelioration of one or more symptoms of disorders in which modulation of a subject's immune system is beneficial, e.g , a medicament for inducing transplantation tolerance, or a medicament for treating autoimmune diseases, or a medicament for treating an inflammatory disease

In other aspect, the invention relates to a method which composes subjecting said cell population to (i) irradiation, and (ii) stimulation with IFN-γ, wherein treatments (i) and (π) are carried out in any order Said irradiated IFN-γ-pre-stimulated cell population or IFN-γ-pre-stimulated irradiated cell population constitute a further aspect of the invention

In other aspect, the invention relates to said irradiated IFN-γ-pre-stimulated cell population or IFN-γ-pre-stimulated irradiated cell population for use as medicament, or for inducing transplantation tolerance, or for treating autoimmune diseases, or for treating an inflammatory disease In other aspect, the invention relates to the use of said irradiated IFN-γ-pre- stimulated cell population or IFN-γ-pre-stimulated irradiated cell population in the preparation of a medicament, such as a medicament for the prevention, treatment or amelioration of one or more symptoms of disorders in which modulation of a subject's immune system is beneficial, e.g., a medicament for inducing transplantation tolerance, or a medicament for treating autoimmune diseases, or a medicament for treating an inflammatory disease

In other aspect, the invention relates to the use of said cell population, or said T-reg cell population, or said irradiated cell population, or said IFN-γ-treated cell population, or

said irradiated IFN-γ-pre-stimulated cell population, or said IFN-γ-pre-stimulated irradiated cell population for preventing, treating, or ameliorating one or more symptoms associated with autoimmune diseases, inflammatory disorders, or immunologically mediated diseases including rejection of transplanted organs and tissues In other aspect, the invention relates to a method of preventing, treating, or ameliorating one or more symptoms associated with autoimmune diseases, inflammatory disorders, or immunologically mediated diseases, in a subject suffering from any of said disorders or diseases, which comprises administering to said subject in need of such treatment of a prophylactically or therapeutically effective amount of said cell population, or said T-reg cell population, or said irradiated cell population, or said IFN-γ-treated cell population, or said irradiated IFN-γ-pre-stimulated cell population, or said IFN-γ-pre- stimulated irradiated cell population The invention also relates to the use of such methods in combination therapy, in other words, a cell population of the invention is coadministered with one or more agents, either simultaneously with the second or further agent, or separately, e g , sequentially

In other aspect, the invention relates to a pharmaceutical composition comprising said cell population, or said T-reg cell population, or said irradiated cell population, or said IFN-γ-treated cell population, or said irradiated IFN-γ-pre-stimulated cell population, or said IFN-γ-pre-stimulated irradiated cell population and an acceptable pharmaceutically earner

In other aspect, the invention relates to a method for distinguishing adult multipotent cells from differentiated cells compiising the step of venfying whether the cell expresses IDO upon stimulation with IFN -γ

In other aspect, the invention relates to a kit comprising said cell population, or said T-reg cell population, or said irradiated cell population, or said IFN-γ-treated cell population, or said irradiated IFN-γ-pre-stimulated cell population, or said IFN-γ-pre- stimulated irradiated cell population

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the growth kinetics of the cells provided by the instant invention isolated from human adipose tissue and cultuied ex vivo for more than 25 cell population doublings

Figure 2 shows histograms of fluorescence immunocytometry corresponding to the profile of surface markers obtained from the cells provided by the instant invention isolated from human adipose tissue Histograms corresponding to the isotype controls (negative controls) are shown shaded in grey Figure 3 shows the analysis of IDO expression after incubating the cells provided by the instant invention isolated from human adipose tissue with different proinflammatory reagents for different time periods, detected by means of RT-PCR (Figure 3A) or western blotting (Figure 3B) IL-I , interleukin 1 , TNF-α, tumour necrosis factor- alpha, LPS, lipopolysacchaπde, IFN-γ, interferon-gamma, C-, negative control, C+, positive control, n i , cells not induced with IFN-γ GAPDH (glyceraldehyde-3 -phosphate dehydrogenase) is used as loading control of the RT-PCR

Figure 4 shows the western blotting detection of IDO expression after 48 hours of

IFN-γ treatment of the cells provided by the instant invention isolated from different human tissues (adipose, bone marrow, cartilage, and skin) Ctrl-, negative control (culture medium), Ctrl+, positive control, (-), cells not treated with IFN-γ, (+), cells treated with

IFN-γ for 48 hours

Figuie 5 shows loss of body weight in mice treated with TNBS (2,4,6- tπmtrobenzene sulfonic acid) administration The figure shows a dose-dependent impiovement of weight gained after the administration of the cells provided by the instant invention isolated from human adipose tissue After 10 days mice that received 1 x 10 ⁶ cells showed no significant weight difference compared to the control group

Figure 6 shows survival rate of TNBS treated mice after administration of the cells provided by the instant invention isolated from human adipose tissue Again, a dose dependency can be observed with 1 x10 cells showing a stronger effect than 0 3x 10 cells, although in both cases the cells significantly improved the survival rate of the TNBS treated mice

Figure 7 shows the companson of body weight in TNBS treated mice after administration of 1 x10 ⁶ cells provided by the instant invention isolated from human adipose tissue and 1 x10 ⁶ of the same cells pre-stimulated with 30 ng/ml IFN-γ during 48 hours The graph shows the severe weight loss in TNBS treated mice and a clear improvement after 3 days in mice that received cells After 8 days these mice even showed a weight gain, whereas the control mice (TNBS treated mice without cell administration) still showed a severe weight loss Furthermore, IFN-γ pre-stimulated cells showed a faster

and stronger recuperation from the TNBS treatment than non-prestimulated cells.

Figure 8 shows the data of Figure 7 as "Experiment 1" and in addition data from an additional dataset "Experiment 2" described in Example 5. The graph shows that TNBS treated mice lost weight dramatically and a clear improvement in mice that received cells. This improvement was also measurable by the severity of colitis

Figure 9 shows that all proinflammatory cytokines (TNF-a, IL-6, IL-Ib, IL- 12, and IFN-γ) and chemokines (MIP-2 and RANTES) tested, both in the colon (local response) and in the serum (systemic response), were lower in cell-treated animals compared with the non-treated mice. Figure 10 shows that neutrophil infiltration, as measured by MPO activity was lower in ASC-treated animals, and even lower when cells were pre-stimulated with IFN-γ

Figure 1 1 shows that CFSE labeled cells were localized in the draining lymph nodes of treated animals by means of cell cytometry. This is the localization expected if the administered cells were functioning as APCs. Figure 12 shows induction of APC markers in human ASCs by IFN-γ treatment.

Upper row: cytometric histograms of untreated ASCs; lower row, cytometric histograms of ASCs after treatment with IFN-γ for 4 days. Isotype controls are shown shaded in black.

Figure 13 shows the cells of the invention decrease CIA incidence and severity. A,

Severity of arthritis, assessed by clinical scoring or paw thickness measurement, in mice with established CIA injected. Numbers in parenthesis represent incidence of arthritis (% mice with arthritis score > 2 at day 50) in control, i.p. and i.a. groups. Images show representative examples of the paw swelling in mice of the different experimental groups

(control and ASC i.p.). n=8-l 1 mice per group. p<0.001 versus control after day 32.

Myeloperoxidase (MPO) activity measuring neutrophil infiltration in the joints. *p<0.001 versus control.

Figure 14 shows inhibition of inflammatory response. Systemic and local expression of inflammatory mediators in untreated (control) or ASC-treated CIA mice assayed at day 35 post-immunization. A, Cytokine/chemokine contents in joints. A paw from an unimmunized mouse was analyzed simultaneously for assessment of the basal response. B, Serum TNFα and IL-I β levels. n=6-8 mice/group. *p<0.001 versus controls.

Figure 15 shows the cells of the invention downregulate ThI -mediated response in CIA. A, Proliferative response and cytokine production of draining lymph node (DLN) cells isolated at day 30 from untreated (control) or ASC-treated CIA mice and stimulated

in vitro with different concentrations of CII Stimulation of DLN cells with anti-CD3 antibodies (T, for untreated CIA mice, V, for AM-treated CIA mice) is used for assessment of nonspecific stimulation A pool of 3 nommmunized DBA/1 DLN cells was used for assessment of the basal response n=5 mice/group B, Number of Cll-specific cytokine-producing T cells DLN cells from untreated (control) or ASC-treated CIA mice were restimulated in vitro with CII (10 μg/ml) and analyzed for CD4 and intracellular cytokine expression by flow cytometry (for IFN-γ/TNFα or IL-4/IL-10 expression in gated CD4 T cells) The number of IFN-γ-, IL-4- and IL- 10-expressing T cells relative to 10 ⁴ CD4 T cells is shown Data shown represent pooled values from two independent expeπments C, Cll-specific proliferative response in synovial membrane cells isolated from untreated (control) or ASC-treated CIA mice and stimulated in vitro with CII (10 μg/ml) for 48 h Data show the results of pooled synovial cells from 3 animals per group D, Cll-specific IgG, IgGl and IgG2a levels in serum collected at day 35 from untreated (control) or ASC-treated CIA mice (8-12 mice/group) *p<0 001 versus controls Figure 16 A shows both the DLN and the synovial membrane of CIA mice treated with the cells of the invention induce an increase in the numbers of regulatory T cells (CD4 ⁺ CD25 ⁺ Foxp3 ⁺ ), without any increase in the numbers of effectoi T cells, comparing with the untreated (control) CIA mice

Figuie 16 B shows that CIA mice treated with the cells of the invention, but not contiol (untreated) ClA mice, contain regulatory T cells that specifically inhibit the effectoi T cell response against CII

Figure 1 7 shows the co-culture of ASCs and lymphocytes results in an inhibition of lymphocyte proliferation

Figure 18 shows that ASCs plated at 5000cell/cm ² and stimulated at 3ng/ml IFN-γ for up to 120 hours produce IDO, the activity of which is measured by the metabohzation of Tryptophan and production of Kynurenine using HPLC

Figure 19 shows that ASCs plated at 5000cell/cm ² and stimulated at 3pg/ml IFN-γ for up to 120hours fail to produce IDO No Kynurenine could be detected

Figure 20 shows that ASCs plated at 500cell/cm ² and stimulated at 3ng/ml IFN-γ for up to 120 hours fail to produce significant amounts IDO

Figure 21 A shows cells which have phagocytosed dextran FITC in a bright field image Figuie 21 B shows the same population using fluorescence microscopy using Green Fluorescent Protein filters

DETAILED DESCRIPTION OF THE INVENTION

As it has been previously mentioned, inventors have found that certain cell populations with multilineage potential which are present in various, if not all, connective tissues and respond to mterferon-gamma (IFN-γ) by expressing indolamine-2,3- dioxygenase (IDO) are capable of acting as immunoregulatory agents in vivo and in vitro The immunosuppressant immunoregulatory effects of said cells can be used for preventing, treating or ameliorating one or more symptoms associated with disorders in which modulation of a subject's immune system is beneficial, including, but not limited to, autoimmune diseases, inflammatory disorders, and immunologically mediated diseases including rejection of transplanted organs and tissues

Definitions

In order to facilitate the understanding of the present description, the meaning of some terms and expressions in the context of the invention will be explained below Further definitions will be included along the description when necessary

The term "antigen presenting cells' ^" (APC) refers to a cell population that displays foreign antigen complexed with MHC (major histocompatibility complex) on its surface Although almost every cell in the body is capable of presenting antigens to T cells, the term "antigen presenting cells ^" ' (APC) is herein limited to those specialized cells, also called professional APCs, that express HLAIl in their surface, and are deπved from the monocyte-macrophage lineage (for example, dendritic cells)

The term "autoimmune disease ^"' refers to a condition in a subject charactenzed by cellular, tissue and/or organ injury caused by an immunologic reaction of the subject to its own cells, tissues and/or organs Illustrative, non-limiting examples of autoimmune diseases which can be treated with the cell population of the invention include alopecia areata, ankylosing spondylitis, antiphosphohpid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFlDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-

fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, sarcoidosis, scleroderma, progressive systemic sclerosis, Sjogren's syndrome, Good pasture's syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arteπstis/giant cell arteritis, ulcerative colitis, uveitis, vascuhtides such as dermatitis herpetiformis vasculitis, vitiligo, Wegener's granulomatosis, etc

The term "immunoregulatory agent" refers to an agent that inhibits or reduces one or more biological activities of the immune system An immunoregulatory agent is an agent that inhibits or reduces one or more biological activities (e g , the proliferation, differentiation, priming, effector function, production of cytokines or expression of antigens) of one or more immune cells (e g , T cells)

The teπn " inflammatory disease" refers to a condition in a subject characterized by inflammation, e g , chronic inflammation Illustrative, non-limiting examples of inflammatory disorders include, but are not limited to, rheumatoid arthritis (RA), Inflammatory Bowel Disease (IBD), asthma, encephalitis, chronic obstructive pulmonary disease (COPD), inflammatory osteolysis, allergic disorders, septic shock, pulmonary fibrosis (e g , idiopathic pulmonary fibrosis), inflammatory vacuhtides (e g , polyarteritis nodosa, Wegner's granulomatosis, Takayasu's arteritis, temporal arteritis, and lymphomatoid granulomatosus), post-traumatic vascular angioplasty (e g , restenosis after angioplasty), undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, chronic hepatitis, and chronic inflammation resulting from chronic viral or bacteria infections

The teπn "isolated ^" ' applied to a cell population refers to a cell population, isolated from the human oi animal body, which is substantially free of one or more cell populations that are associated with said cell population in vivo or in vitro

The term "MHC" (major histocompatibility complex) refers to a subset of genes that encodes cell-surface antigen-presenting proteins In humans, these genes are referred to as human leukocyte antigen (HLA) genes Herein, the abbreviations MHC or HLA are used interchangeably The term "subject" refers to an animal, preferably a mammal including a non- pπmate (e g , a cow, pig, horse, cat, dog, rat, or mouse) and a primate (e g , a monkey, or a human) In a preferred embodiment, the subject is a human

The term "T-cell" refers to cells of the immune system which are a subset of lymphocytes that express the T cell receptoi (TCR) The term "regulatory T-cells" (T-reg cells) refers to T cell subsets that actively suppress activation of the immune system and prevent pathological self-reactivity, i e an autoimmune disease

As used herein, the terms "treat", "treatment" and "treating" refer to the amelioration of one or more symptoms associated with a disorder including, but not limited to, an inflammatory disorder, an autoimmune disease or an immunologically mediated disease including rejection of transplanted organs and tissues, that results from the administration of the cell population of the invention, the T-reg cell population of the invention, or the IFN-γ-pre stimulated cell population of the invention, or a pharmaceutical composition comprising same, to a subject in need of said treatment The term "combination therapy' ^" refers to the use of the cell populations of the present invention with other active agents or treatment modalities, in the manner of the present invention for the amelioration of one or more symptoms associated with a disorder including, but not limited to, an inflammatory disorder, an autoimmune disease or an immunologically mediated disease including rejection of transplanted organs and tissues These other agents or treatments may include known drugs and therapies for the treatment of such disorders The cell populations of the invention may also be combined with corticosteroids, non-steroidal anti-inflammatory compounds, or other agents useful in treating inflammation The combined use of the agents of the present invention with these other therapies or treatment modalities may be concurrent, or given sequentially, that is, the two treatments may be divided up such that a cell population or a pharmaceutical composition comprising same of the present invention may be given prior to or after the other therapy or treatment modality The attending physician may decide on the

appropriate sequence of administering the cell population, or a phaπnaceutical composition comprising same, in combination with other agents, therapy or treatment modality

Cells of the invention In an aspect, the present invention relates to an isolated cell population from connective tissue, hereinafter referred to as "cell population of the invention", characteπsed in that the cells of said cell population a) do not express markers specific for antigen-presenting cells (APC), b) do not express indoleamine 2,3-dioxygenase (IDO) constitutively, wherein constitutively is understood to mean the expression of a gene without any specific induction c) express IDO upon stimulation with interferon-gamma (IFN-γ) and, d) present capacity to be differentiated into at least two cell lineages

The cells of the cell population of the invention, hereinafter referred to as the ' cells of the invention ' deπve from connective tissue The term "connective tissue" refers to tissue derived from mesenchyme and includes several tissues which are characterized in that their cells are included within the extracellular matπx Among the different types of connective tissues, adipose and cartilaginous tissues are included In a particular embodiment, the cells of the invention are from the stromal fraction of the adipose tissue In othep particular embodiment, the cells of the invention are obtained from chondrocytes, the only cells found in the hyaline cartilage In another particular embodiment, the cells of the invention are obtained from skin Also, in another particular embodiment, the cells of the invention are obtained from bone marrow

The cells of the invention can be obtained from any suitable source of connective tissue from any suitable animal, including humans In general, said cells are obtained from non-pathological post-natal mammalian connective tissues In a preferred embodiment, the cells of the invention are obtained from a source of connective tissue, such as the stromal fraction of adipose tissue, hyaline cartilage, bone marrow, skin etc Also, in a particular embodiment, the cells of the cell population of the invention are from a mammal, e g , a rodent, primate, etc , preferably, from a human

As mentioned above, the cells of the invention are characteπzed in that (i) they do not express markers specific from APCs, (π) they do not express IDO constitutively, (in) they express IDO upon stimulation with IFN-γ, and (iv) they present capacity to be differentiated into at least two cell lineages Markers

The cells of the invention are negative for at least one, two, three, four or preferably all of the following markers CDl Ib, CDl Ic, CDl 4, CD45, and HLAIl, which are specific markers for APCs lineages Thus, the cells of the invention do not constitute a previously described subpopulation of specialized APCs Moreover, the cells of the invention are negative for at least one, two of, or preferably all of the following cell surface markers CD31 , CD34 and CDl 33

As used herein, "negative" with respect to cell surface markers means that, in a cell population comprising the cells of the invention, less than 10%, preferably 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 % or none of the cells show a signal for a specific cell surface marker in flow cytometry above the background signal, using conventional methods and apparatus (for example a Beckman Coulter Epics XL FACS system used with commercially available antibodies and standard protocols known in the art) In a particular embodiment, the cells of the invention are characterised in that they express at least one, two, three, four, of or preferably all of the following cell surface markers CD9, CD44, CD54, CD90 and CD 105, i e , the cells of the invention are positive for at least one, two, three, four of and preferably all said cell surface markers (CD9, CD44, CD54, CD90 and CDl 05) Preferably, the cells of the invention are characterised in that they have significant expression levels of at least one, two, three, four, of and preferably all of said cell surface markers (CD9, CD44, CD54, CD90 and CD 105) As used herein, the expression "significant expression" means that, in a cell population compπsing the cells of the invention, more than 10%, preferably 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or all of the cells show a signal for a specific cell surface marker in flow cytometry above the background signal using conventional methods and apparatus (for example a Beckman Coulter Epics XL FACS system used with coinmeicially available antibodies and standard protocols known in the art) The background signal is defined as the signal intensity given by a non-specific antibody of the same isotype as the specific antibody used to detect each surface marker in conventional FACS analysis Thus for a markei to be considered positive

the specific signal observed is stronger than 10%, preferably 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 500%, 1000%, 5000%, 10000% or above, than the background signal intensity using conventional methods and apparatus (for example a Beckman Coulter Epics XL FACS system used with commercially available antibodies and standard protocols known in the art)

Optionally, the cells of the invention are also negative for the cell surface marker CD 106(VCAM-I ), Examples of such cells are certain populations of adipose tissue- derived stromal stem cells as described herein

Commercially available and known monoclonal antibodies against said cell-surface markers (e g , cellular receptors and transmembrane proteins) can be used to identify the cells of the invention

Expression of IDO

The cells of the invention do not express IDO constitutive! y, but they express IDO upon stimulation with IFN-γ Expeπments carried out by the inventors have shown that said cells, upon stimulation with other pro-inflammatory mediators by themselves, such us interleukin-1 (IL-I) used at a concentration of 3ng/ml, tumour necrosis factor-alpha (TNF- α) used at a concentration of 50ng/ml, or the endotoxin LPS used at a concentration of l OOng/ml, did not induce IDO expression, as measured by conventional RT-PCR and Western Blot analysis Stimulation with IFN-γ for example at 3ng/ml or higher can also induce expression of HLAII in the cells of the invention to give a positive signal as defined herein for a cell surface marker Said expression can be detected by those skilled in the art using any known technique that allows the detection of the expression of specific proteins Preferably, said techniques are cell cytometry techniques

Differentiation The cells of the invention present the capacity to proliferate and be differentiated into at least two, more preferably three, four, five, six, seven or more cell lineages Illustrative, non-limiting examples of cell lineages in which the cells of the invention can be differentiated include osteocytes, adipocytes, chondrocytes, tenocytes, myocytes, cardiomyocytes, hematopoietic-supporting stromal cells, endothelial cells, neurons, astrocytes, and hepatocytes

Cells of the invention can proliferate and differentiate into cells of other lineages by conventional methods Methods of identifying and subsequently isolating differentiated cells from their undifferentiated counterparts can be also earned out by methods well known in the art The cells of the invention are also capable of being expanded ex vivo That is, after isolation, the cells of the invention can be maintained and allowed to proliferate ex vivo in culture medium Such medium is composed of, for example, Dulbecco's Modified Eagle's Medium (DMEM), with antibiotics (for example, 100units/ml Penicillin and lOOμg/ml Streptomycin) or without antibiotics, and 2 rnM glutamine, and supplemented with 2-20% fetal bovine serum (FBS) It is within the skill of one in the art to modify or modulate concentrations of media and/or media supplements as necessary for the cells used Sera often contain cellular and non-cellular factors and components that are necessary for viability and expansion Examples of sera include FBS, bovine serum (BS), calf serum (CS), fetal calf serum (FCS), newborn calf serum (NCS), goat serum (GS), horse serum (HS), porcine serum, sheep serum, rabbit serum, rat serum (RS), etc Also contemplated is, if the cells of the invention are of human oπgin, supplementation of cell culture medium with a human serum, preferably of autologous origin It is understood that sera can be heat- lnactivated at 55-65°C if deemed necessary to inactivate components of the complement cascade Modulation of serum concentrations, withdrawal of serum from the culture medium can also be used to promote survival of one or more desired cell types Preferably, cells of the invention will benefit from FBS concentrations of about 2% to about 25% In another embodiment, the cells of the invention can be expanded in a culture medium of definite composition, in which the serum is replaced by a combination of serum albumin, serum transferrin, selenium, and recombinant proteins including but not limited to insulin, platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF) as known in the art

Many cell culture media already contain amino acids, however some require supplementation prior to cultunng cells Such amino acids include, but are not limited to, L-alanine, L- arginine, L-aspartic acid, L-asparagine, L cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine, and the like

Antimicrobial agents are also typically used in cell culture to mitigate bacteπal, mycoplasmal, and fungal contamination Typically, antibiotics or anti-mycotic compounds

used are mixtures of penicillin/streptomycin, but can also include, but are not limited to amphotericin (Fungizone®), ampicilhn, gentamicin, bleomycin, hygromacin, kanamycin, mitomycin, etc

Hormones can also be advantageously used in cell culture and include, but are not limited to, D-aldosterone, diethylstilbestrol (DES), dexamethasone, b-estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin/human growth hormone (HGH), etc

The maintenance conditions of the cells of the invention can also contain cellular factors that allow cells to remain in an undifferentiated form It is apparent to those skilled in the art that pπor to differentiation, supplements that inhibit cell differentiation must be removed from the culture medium It is also apparent that not all cells will require these factors In fact, these factors may elicit unwanted effects, depending on the cell type

Advantageously, the cells of the invention lack in vivo tumoπgenic activity Thus, said cells are characterized in that they do not present tumoπgenic activity, i e , they do not present an altered behaviour or proliferative phenotype which gives rise to a tumour cell

In an embodiment, the cells of the invention can be administered to a subject suffering from autoimmune diseases, inflammatory diseases or immunologically mediated diseases, such as rejection of transplanted organs and tissues, for suppressing the immune response Thus, it is necessary that the cells of the invention do not present tumoiigenic activity

The tumongenic activity of the cells of the invention can be tested by performing animal studies using immunodeficient mice strains In these expenments, several million cells are implanted subcutaneously in the recipient animals, which are maintained for several weeks and analyzed for tumour formation A particular assay is disclosed in Example 3

The cells of the invention can be transfected or genetically engineered to express, at least, one antigenic polypeptide In an embodiment, the antigen comprises a purified or a synthetic or recombinant polypeptide representing a specific antigen to which it is desired that tolerance is to be induced, or a short synthetic polypeptide fragment derived from the amino acid sequence of such an antigen Preferably, the source of antigen comprises antigens expressed by a donor tissue graft Also preferably, the source of antigen comprises a protein to which a patient has an autoimmune disorder

Method for isolating IDO-expressing cells

In an aspect, the present invention relates to a method for isolating a cell population from connective tissue, wherein the cells of said cell population present a phenotype characterized in that (i) they do not express markers specific from APCs; (n) they do not express IDO constitutively, (in) they express IDO upon stimulation with IFN-γ, and (iv) they present capacity to be differentiated into at least two cell lineages, said method comprising the steps of:

(i) prepaπng a cell suspension from a sample of a connective tissue, (n) recovering the cells from said cell suspension,

(in) incubating said cells in a suitable cell culture medium on a solid surface under conditions which allow cells to adhere to the solid surface and proliferate;

(iv) washing said solid surface after incubation to remove non-adhered cells,

(v) selecting the cells which after being passaged at least twice in such medium remain adhered to said solid surface, and

(vi) confirming that the selected cell population presents the phenotype of interest

As used herein, the term ' solid surface ^" ' refeis to any material that allows the cells of the invention to adhere In a particular embodiment said material is a plastic material treated to promote the adhesion of mammalian cells to its surface, for example commercially available polystyrene plates optionally coated with poly-D-Lysine or other reagents.

Steps (ι)-(vi) can be earned out by conventional techniques known by those skilled in the art. Briefly, the cells of the invention can be obtained by conventional means from any suitable source of connective tissue from any suitable animal, including humans, e g , from human adipose tissue or cartilaginous tissue The animal can be alive or dead, so long as connective tissue cells within the animal are viable Typically, human adipose cells are obtained from living donors, using well-recognized protocols such as surgical or suction hpectomy Indeed, as liposuction procedures are so common, liposuction effluent is a particularly preferred source from which the cells of the invention can be derived Thus, in a particular embodiment, the cells of the invention are from the stromal fraction of human

adipose tissue obtained by liposuction In another particular embodiment, the cells of the invention are from human hyaline articular cartilage obtained by arthroscopic techniques In another particular embodiment, the cells of the invention are from human skin obtained by biopsy techniques. Also in another particular embodiment, the cells of the invention are from human bone marrow obtained by aspiration

The sample of connective tissue is, preferably, washed before being processed to separate the cells of the invention from the remainder of the material. In a protocol, the sample of connective tissue is washed with physiologically-compatible saline solution (e.g , phosphate buffered saline (PBS)) and then vigorously agitated and left to settle, a step that removes loose matter (e.g., damaged tissue, blood, erythrocytes, etc) from the tissue Thus, the washing and settling steps generally are repeated until the supernatant is relatively clear of debπs. The remaining cells generally will be present in clumps of various sizes, and the protocol proceeds using steps gauged to degrade the gross structure while minimizing damage to the cells themselves. One method of achieving this end is to treat the washed lumps of cells with an enzyme that weakens or destroys bonds between cells (e g., collagenase, dispase, trypsin, etc ). The amount and duration of such enzymatic treatment will vary, depending on the conditions employed, but the use of such enzymes is generally known in the art Alternatively or in conjunction with such enzymatic treatment, the lumps of cells can be degraded using other treatments, such as mechanical agitation, sonic energy, thermal energy, etc If degradation is accomplished by enzymatic methods, it is desirable to neutralize the enzyme following a suitable period, to minimize deleteπous effects on the cells

The degradation step typically produces a slurry or suspension of aggregated cells and a fluid fraction containing generally free stromal cells (e.g., red blood cells, smooth muscle cells, endothelial cells, fibroblast cells, and stem cells). The next stage in the separation process is to separate the aggregated cells from the cells of the invention This can be accomplished by centπfugation, which forces the cells into a pellet covered by a supernatant. The supernatant then can be discarded and the pellet suspended in a physiologically-compatible fluid. Moreover, the suspended cells typically include erythrocytes, and in most protocols it is desirable to lyse them Methods for selectively lysing erythrocytes are known in the art, and any suitable protocol can be employed (e g , incubation in a hyper -or hypotonic medium, by lysis using ammonium chloride, etc ) Of

course, if the erythrocytes are lysed, the remaining cells should then be separated from the lysate, for example by filtration, sedimentation, or density fractionation

Regardless of whether the erythrocytes are lysed, the suspended cells can be washed, re-centπfuged, and resuspended one or more successive times to achieve greater purity Alternatively, the cells can be separated on the basis of cell surface marker profile or on the basis of cell size and granularity

Following the final isolation and resuspension, the cells can be cultured and, if desired, assayed for number and viability to assess the yield Desirably, the cells will be cultured without differentiation, on a solid surface, using a suitable cell culture media, at the appropnate cell densities and culture conditions Thus, in a particular embodiment, cells are cultured without differentiation on a solid surface, usually made of a plastic mateπal, such as Petπ dishes or cell culture flasks, in the presence of a suitable cell culture medium [e g , DMEM, typically supplemented with 5-15% (e g , 10%) of a suitable serum, such as fetal bovine serum or human serum], and incubated under conditions which allow cells to adhere to the solid surface and proliferate After incubation, cells are washed in order to remove non-adhered cells and cell fiagments The cells are maintained in culture in the same medium and under the same conditions until they reach the adequate confluence, typically, about 80% cell confluence, with replacement of the cell culture medium when necessary After reaching the desired cell confluence, the cells can be expanded by means of consecutive passages using a detachment agent such as trypsin and seeding onto a bigger cell culture surface at the appropnate cell density (usually 2,000- 10,000 cells/cm ² ) Thus, cells are then passaged at least two times in such medium without differentiating, while still retaining their developmental phenotype, and more preferably, the cells can be passaged at least 10 times (e g , at least 15 times or even at least 20 times) without losing developmental phenotype Typically, the cells are plated at a desired density such as between about 100 cells/cm ² to about 100,000 cells/cm ² (such as about 500 cells/cm ² to about 50,000 cells/cm ² , or, more particularly, between about 1,000 cells/cm ² to about 20,000 cells/cm ) If plated at lower densities (e g , about 300 cells/cm ), the cells can be more easily clonally isolated For example, after a few days, cells plated at such densities will proliferate into an homogeneous population In a particular embodiment, the cell density is between 2,000-10,000 cells/cm ²

Cells which remain adhered to the solid surface after such treatment comprising at least two passages are selected and the phenotype of interest is analyzed by conventional methods in order to confirm the identity of the cells of the invention as will be mentioned below Cells which remain adhered to the solid surface after the first passage are from heterogeneous oπgin, therefore, said cells must be subjected to at least another passage As a result of the above method, a homogeneous cell population having the phenotype of interest is obtained Example 1 descπbes in a detailed manner the isolation of the cells of the invention from human adipose tissue and from human cartilaginous tissue

Usually, cells which remain adhered to the solid surface after the second passage show the phenotype of interest, although it has to be confirmed so that the cells can be used according to the invention Therefore, the adhesion of cells to the solid surface after at least two passages constitutes a preferred embodiment of the invention for selecting the cells of the invention Confirmation of the phenotype of interest can be carried out by using conventional means Cell-surface markers can be identified by any suitable conventional technique, usually based on a positive/negative selection, for example, monoclonal antibodies against cell-surface markers, whose presence/absence in the cells has to be confirmed, can be used, although other techniques can also be used Thus, in a particular embodiment, monoclonal antibodies against one, two, three, four, five, six, seven of or preferably all of CDl Ib, CDl I c, CD14, CD45, HLAII, CD31, CD34 and CD133 are used in order to confirm the absence of said markers in the selected cells, and monoclonal antibodies against one, two, three, four, of or preferably all of CD9, CD44, CD54, CD90 and CD 105 are used in order to confirm the presence thereof or detectable expression levels of, at least one of and preferably all of, said markers Said monoclonal antibodies are known, commercially available or can be obtained by a skilled person in the art by conventional methods

IFN-γ-inducible IDO activity in the selected cells can be determined by any suitable conventional assay For example, the selected cells can be stimulated with IFN-γ and assayed for IDO expression, then conventional Western-blot analysis for IDO protein expression can be performed and IDO enzyme activity following IFN-γ stimulation of the selected cells can be measured by tryptophan-to-kynuremne conversion with for example via High Performance Liquid Chromatography (HPLC) analysis and photometric determination of kynuremne concentration in the supernatant as the readout Since the cells of the invention express IDO under certain conditions, any suitable technique which allows

the detection of IDO activity following IFN-γ stimulation may be used for selecting the cells of the invention A suitable assay for determining IFN-γ-inducible IDO activity in the selected cells is disclosed in Example 2 The amount of IDO produced depends on the number of cells per square centimetre, which is preferably at a level of 5O00cells/cm ² or more, but not limited to this concentration and the concentration of IFN-γ, which ideally is 3ng/ml or more, but not limited to this concentration The activity of IDO produced under the described conditions will result in a detectable production of kynurenine in the μM range after 24hours or more

The capacity of the selected cells to differentiate into at least two cell lineages can be assayed by conventional methods as known in the art

The cells and cell populations provided by the instant invention can be clonally expanded, if desired, using a suitable method for cloning cell populations For example, a proliferated population of cells can be physically picked and seeded into a separate plate (or the well of a multi-well plate) Alternatively, the cells can be subcloned onto a multi- well plate at a statistical ratio for facilitating placing a single cell into each well (e g , from about O 1 to about 1 cell/well or even about 0 25 to about 0 5 cells/well, such as 0 5 cells/well) Of course, the cells can be cloned by plating them at low density (e g , in a Petπ dish or other suitable substrate) and isolating them from othei cells using devices such as a cloning rings The production of a clonal population can be expanded in any suitable culture medium In any event, the isolated cells can be cultured to a suitable point when their developmental phenotype can be assessed

Further investigations carried out by the inventors have shown that ex vivo expansion of the cells of the invention without inducing differentiation can be accomplished for extended time periods for example by using specially screened lots of suitable serum (such as fetal bovine serum or human serum) Methods for measuπng viability and yield are known in the art (e g , trypan blue exclusion)

Any of the steps and procedures for isolating the cells of the cell population of the invention can be performed manually, if desired Alternatively, the process of isolating such cells can be facilitated and/or automated through one or more suitable devices, examples of which are known in the art

Uses of the cells of the invention

The cells of the invention can be used for preventing, treating or ameliorating one or more symptoms associated with disorders in which modulation of a subject's immune system is beneficial, including, but not limited to, autoimmune diseases, inflammatory disorders, and immunologically mediated diseases including rejection of transplanted organs and tissues

Thus, in another aspect, the cells of the invention are used as a medicament In a particular embodiment, medicaments containing the cells of the invention may be used for inducing transplantation tolerance, or for treating, and thereby alleviating, symptoms of autoimmune or inflammatory disorders, or immunologically mediated diseases including rejection of transplanted organs and tissues, in a subject suffering from any of said disorders or diseases Thus, the cells of the invention can be used to therapeutically or prophylactically treat and thereby alleviate symptoms of autoimmune or inflammatory disorders in a subject suffering from any of said disorders or to alleviate symptoms of immunologically mediated diseases in a subject suffeπng from said diseases

As used herein, the terms 'disorder ^" ' and "'disease" are used interchangeably to refer to a condition in a subject In particular, the term "'autoimmune disease' ^" is used interchangeably with the term "autoimmune disorder" to refer to a condition in a subject characterized by cellular, tissue and/or organ injury caused by an immunologic reaction of the subject to its own cells, tissues and/or organs The term "inflammatory disease ' is used interchangeably with the term "inflammatory disorder ^" ' to refer to a condition in a subject characterized by inflammation, preferably chronic inflammation Autoimmune disorders may or may not be associated with inflammation Moreover, inflammation may or may not be caused by an autoimmune disorder Thus, certain disorders may be characterized as both autoimmune and inflammatory disorders

The mechanisms by which certain conditions may result in autoimmunity in some subjects are generally not well understood, but may involve both genetic and extrinsic factors For example, bacteria, viruses or drugs may play a role in tπggeπng an autoimmune response in a subject who already has a genetic predisposition to the autoimmune disorder It has been proposed, for example, that subjects with certain common allergies are more susceptible to autoimmune disorders

Practically any autoimmune disease, inflammatory disorder or immunological mediated disease can be treated with the cells of the invention Illustrative, non-limiting examples of said diseases and disorders which can be treated are those previously listed under heading "Definitions" In a particular embodiment, said inflammatory disease is a chronic inflammatory disease, such as, e g , IBD or RA

In other aspect, the present invention relates to the use of the cells of the invention for the preparation of a medicament for preventing, treating or ameliorating one or more symptoms associated with disorders in which modulation of a subject's immune system is beneficial, including, but not limited to, autoimmune diseases, inflammatory disorders, and immunologically mediated diseases including rejection of transplanted organs and tissues Thus, the invention further refers to the use of the cells of the invention for the preparation of a medicament for suppressing the immune response, or for inducing transplantation tolerance, or for treating autoimmune diseases, or for treating inflammatory disorders Examples of said autoimmune diseases and inflammatory diseases have been previously mentioned In a particular embodiment, disease is an inflammatory disease, such as a chronic inflammatory disease, e g , IBD or RA

In another aspect, the present invention relates to the use of the cells of the invention for the preparation or generation of regulatory T-cells (T-reg), i e , cells that actively suppress activation of the immune system and prevent pathological self-reactivity, i e an autoimmune disease

T-reg cells of the invention

The invention further refers, in other aspect, to regulatory T-cells (T-reg), i e , cells

(including Foxp3+CD4+CD25+ T-reg and IL-10/TGFb-producing TrI cells) that actively suppress activation of the immune system and prevent pathological self-reactivity, i e an autoimmune disease, obtainable from the cells of the invention, hereinafter referred to T- reg cells of the invention

Thus, in other aspect, the present invention relates to a method for the isolation of a T-reg cell population of the invention, which comprises (a) contacting a cell population of the invention with peripheral blood leukocytes, and

(b) selecting the T-reg cell population of the invention

Consequently, the cells of the invention can be used to produce a subset of T-cells, the T-reg cells of the invention, which constitutes an additional aspect of the present invention. The T-reg cells of the invention can be isolated by conventional means known by a skilled person in the art. The T-reg cells of the invention can be used for preventing, treating or ameliorating one or more symptoms associated with disorders in which modulation of a subject's immune system is beneficial, including, but not limited to, autoimmune diseases, inflammatory disorders, and immunologically mediated diseases including rejection of transplanted organs and tissues. Said use constitutes an additional aspect of the present invention.

Thus, in another aspect, the T-reg cells of the invention are used as a medicament. In a particular embodiment, medicaments containing the T-reg cells of the invention may be used for inducing transplantation tolerance, or for treating, and thereby alleviating, symptoms of autoimmune or inflammatory disorders, or immunologically mediated diseases including rejection of transplanted organs and tissues, in a subject suffering from any of said disorders or diseases. Thus, the T-reg cells of the invention can be used to therapeutically or prophylactically treat and thereby alleviating symptoms of autoimmune or inflammatory disorders in a subject suffering from any of said disorders or to alleviate symptoms of immunologically mediated diseases in a subject suffering from said diseases. Practically any autoimmune disease, inflammatory disorder or immunological mediated disease can be treated with the T-reg cells of the invention. Illustrative, non- limiting examples of said diseases and disorders which can be treated are those previously listed under heading "Definitions". In a particular embodiment, said inflammatory disease is a chronic inflammatory disease, such as, e.g., IBD or RA. In other aspect, the present invention relates to the use of the T-reg cells of the invention for the preparation of a medicament for preventing, treating or ameliorating one or more symptoms associated with disorders in which modulation of a subject's immune system is beneficial, including, but not limited to, autoimmune diseases, inflammatory disorders, and immunologically mediated diseases including rejection of transplanted organs and tissues. Thus, the invention further refers to the use of the T-reg cells of the invention for the preparation of a medicament for suppressing the immune response, or for inducing transplantation tolerance, or for treating autoimmune diseases, or for treating

inflammatory disorders Examples of said autoimmune diseases and inflammatory diseases have been previously mentioned In a particular embodiment, disease is an inflammatory disease, such as a chronic inflammatory disease, e g , IBD or RA

The invention also provides the use of cell populations of the invention in the production of Treg cells specific for a chosen antigen or group of antigens and the use of these in the treatment of disease or disorders relating to that antigen or group of antigens Examples of such antigens are those that play a role in autoimmune diseases, such as, for example, rheumatoid arthritis, Crohn's disease, hypersensitivity reaction Type IV, lupus, psoiiasis and other autoimmune disoiders known in the art and described elsewheie herein Briefly, cell populations of the invention are cultured in vitro in the presence of a chosen antigen, group of antigens or cell types expressing and/or presenting this antigen or antigens The cells of the invention can optionally be prestimulated with IFN-γ, LPS or other activating agents known in the art After a culture period of about 2, 4, 6, 12, 24, 48 or more hours, preferably between about 12 to about 24 hours, the cell population of the invention is further co-cultured, optionally after the removal of the antigen, group of antigens or cells carrying said antigen, with peπpheral blood leukocytes obtained from a subject This co-cultuπng will result in the production of Treg cells specific for the chosen antigen, which can be used for treatment of the subject Optionally these Treg cells can be expanded in numbei e\ vivo using culture techniques known in the art before being administered to the patient Without wishing to be bound by theory, the Inventors believe that the cell populations of the invention are capable of presenting the chosen antigen via HLA Class Il on the cell suiface (seeming induced by IFN-γ) to the peπpheral blood leukocytes such that Treg cells are augmented and or activated within the population of penpheral blood leukocytes As shown in Example 1 1, the Inventors have demonstrated that cell populations of the invention are able to phagocytose small molecular weight molecules and thus are capable of presenting such molecules after IFN-γ stimulation via HLA Class II molecules The presentation of chosen antigen via this mechanism with the interaction with the peπpheral blood leukocytes is believed to result in the above descπbed Treg cell production As an alternative treatment methodology, as descπbed in Example 7 a cell population of invention is administered directly in vivo without any co-cultuiing and can generate specific Treg cells, which in turn can treat a disorder

Thus the invention provides an in vitro method of obtaining Treg cells specific for a chosen antigen or group of antigens, which comprises

(a) contacting a cell population of the invention with said chosen antigen or group of antigens, (b) bringing said cell population into contact with peπpheral blood leukocytes,

(c) selecting a T-reg cell population specific for said chosen antigen or group of antigens

The invention also provides the use of the specific Treg cells of step (c) in the treatment of diseases and disorders related to said chosen antigen or groups of antigens by administration of said Treg cells to the subject from which the peπpheral blood leukocytes were obtained The cell population of the invention as used in this method may be from the subject (autologous) or from a donor (allogeneic)

Irradiated cells of the invention If desired, the cells of the invention can be irradiated using a suitable controlled source of ionizing radiation, such a gamma irradiator device The irradiation conditions must be experimentally adjusted by a person skilled in the art to determine the required exposure time to impart a radiation dose that cause the long term growth arrest of the cells of the invention Said radiation dose can be for example 1-100, 5 85, 10-70, 12-60 Gy or more pi eferably 15-45 Gy

Since the cells of the invention can be used for therapeutic uses, irradiation of the cells of the invention before administration to the subject may result beneficial since said irradiation treatment makes cells incapable to proliferate or survive for long time periods in the subject Said irradiated cells constitute a further aspect of the instant invention The irradiated cells of the invention can be used for preventing, treating or ameliorating one or more symptoms associated with disorders in which modulation of a subject's immune system is beneficial, including, but not limited to, autoimmune diseases, inflammatory disorders, and immunologically mediated diseases including rejection of transplanted organs and tissues Said use constitutes an additional aspect of the present invention

Thus, in another aspect, the irradiated cells of the invention are used as a medicament In a particular embodiment, medicaments containing the irradiated cells of the invention may be used for inducing transplantation tolerance, or for treating, and thereby alleviating, symptoms of autoimmune or inflammatory disorders, 01 immunologically mediated diseases including rejection of transplanted organs and tissues, in a subject suffering from any of said disorders or diseases Thus, the irradiated cells of the invention can be used to therapeutically or prophylactically treat and thereby alleviating symptoms of autoimmune or inflammatory disorders in a subject suffeπng from any of said disorders or to alleviate symptoms of immunologically mediated diseases in a subject suffeπng from said diseases

Practically any autoimmune disease, inflammatory disorder or immunological mediated disease can be treated with the irradiated cells of the invention Illustrative, non- hmiting examples of said diseases and disorders which can be treated are those previously listed under heading "Definitions" In a particular embodiment, said inflammatory disease is a chronic inflammatory disease, such as, e g , IBD or RA

In other aspect, the present invention relates to the use of the irradiated cells of the invention for the preparation of a medicament for preventing, treating or ameliorating one or more symptoms associated with disorders in which modulation of a subject's immune system is beneficial, including, but not limited to, autoimmune diseases, inflammatory disorders, and immunologically mediated diseases including rejection of transplanted organs and tissues Thus, the invention further refers to the use of the irradiated cells of the invention for the preparation of a medicament for suppressing the immune response, or for inducing transplantation tolerance, or for treating autoimmune diseases, or for treating inflammatory disorders Examples of said autoimmune diseases and inflammatory diseases have been previously mentioned In a particular embodiment, disease is an inflammatory disease, such as a chronic inflammatory disease, e g , IBD or RA

IFN-γ-pre-stimulated cells of the invention

Also, if desired, the cells of the invention can be pre-stimulated with IFN-γ The methods for pre-stimulation with IFN-γ are evident to those skilled in the art, and a procedure is given in Example 2 Preferably, the cells are pre-stimulated using a concentration of IFN-γ between 0 1 and 100, 0 5 and 85, 1 and 70, 1 5 and 50, 2 5 and 40

ng/ml or more preferably 3 and 30 ng/ml, and a stimulation time preferably longer than 12 hours, for example, 13, 18, 24, 48, 72 hours or more

Since the cells of the invention can be used for therapeutic uses, pre-stimulation of the cells of the invention with IFN-γ before administration to the subject may result beneficial since the time period between IFN-γ-pre-stimulated cell administration and IDO expression in the subject can be reduced

Thus, in another aspect, the present invention refers to a method which comprises the treatment of the cells of the invention with IFN-γ in order to pre-stimulate said cells The cells obtainable according to said method, hereinafter referred to "IFN-γ-pre- stimulated cells of the invention", constitutes an additional aspect of the present invention The IFN-γ-pre-stimulated cells of the invention can be isolated by conventional means known by a skilled person in the art

The IFN-γ-pre-stimulated cells of the invention can be used for preventing, treating or ameliorating one or more symptoms associated with disorders in which modulation of a subject's immune system is beneficial, including, but not limited to, autoimmune diseases, inflammatory disorders, and immunologically mediated diseases including rejection of transplanted organs and tissues Said use constitutes an additional aspect of the present invention

Thus, in another aspect, the IFN-γ-pre-stimulated cells of the invention are used as a medicament In a particular embodiment, medicaments containing the IFN-γ-pre- stimulated cells of the invention may be used for inducing transplantation toleiance, or for treating, and thereby alleviating, symptoms of autoimmune or inflammatory disorders, or immunologically mediated diseases including rejection of transplanted organs and tissues, in a subject suffering from any of said disorders or diseases Thus, the IFN-γ-pre- stimulated cells of the invention can be used to therapeutically or prophylactically treat and thereby alleviating symptoms of autoimmune or inflammatory disorders in a subject suffering from any of said disorders or to alleviate symptoms of immunologically mediated diseases in a subject suffering from said diseases

Practically any autoimmune disease, inflammatory disorder or immunological mediated disease can be treated with the IFN-γ-pre-stimulated cells of the invention Illustrative, non-limiting examples of said diseases and disorders which can be treated are

those previously listed under heading "Definitions" In a particular embodiment, said inflammatory disease is a chronic inflammatory disease, such as, e g , IBD or RA

In other aspect, the present invention relates to the use of the IFN-γ-pre-stimulated cells of the invention for the preparation of a medicament for preventing, treating or ameliorating one or more symptoms associated with disorders in which modulation of a subject's immune system is beneficial, including, but not limited to, autoimmune diseases, inflammatory disorders, and immunologically mediated diseases including rejection of transplanted organs and tissues Thus, the invention further refers to the use of the IFN-γ- pre-stimulated cells of the invention foi the preparation of a medicament for suppressing the immune response, or for inducing transplantation tolerance, or for treating autoimmune diseases, or for treating inflammatory disorders Examples of said autoimmune diseases and inflammatory diseases have been previously mentioned In a particular embodiment, disease is an inflammatory disease, such as a chronic inflammatory disease, e g , IBD or RA

Irradiated IFN-γ-pre-stimulated cells of the invention and IFN-γ-pre-stimulated irradiated cells of the invention

Furthermore, if desired, the cells of the invention can be subjected to the treatments of irradiation and IFN-γ-stimulation, in any order, i e , cells of the invention can be subjected firstly to irradiation and the resulting cells can be subsequently subjected to IFN- γ-stimulation, or vice versa, cells of the invention can be subjected firstly to IFN-γ stimulation and subsequently the resulting cells can be subjected to irradiation

Thus, in an aspect, the cells of the invention can be pre-stimulated with IFN-γ and the resulting cells (IFN-γ-pre-stimulated cells of the invention) can be irradiated to render irradiated cells hereinafter referred to as "irradiated IFN-γ-pre-stimulated cells of the invention"

In another aspect, the cells of the invention can be irradiated and the resulting cells (irradiated cells of the invention) can be pre-stimulated with IFN-γ to render IFN-γ- prestimulated cells hereinafter referred to as "'IFN-γ-pre-stimulated irradiated cells of the invention"

Methods for pre-stimulation cells with IFN-γ as well as methods for irradiating cells are well-known for those skilled in the art and some of them have been previously mentioned above. Any of said methods can be used

Thus, in another aspect, the present invention refers to a method which comprises subjecting the cells of the invention to (i) irradiation, and (n) stimulation with IFN-γ, wherein treatments (i) and (ii) can be carried out in any order, in order to irradiate IFN-γ- pre-stimulated cells or to INF-γ-pre-stimulate irradiated cells The cells obtainable according to said method, herein referred to as "irradiated IFN-γ-pre-stimulated cells of the invention" or "IFN-γ-pre-stimulated irradiated cells of the invention", respectively, constitutes additional aspects of the present invention. Said irradiated IFN-γ-pre-stimulated cells of the invention as well as said IFN-γ-pre-stimulated irradiated cells of the invention can be isolated by conventional means known by a skilled person in the art.

Since the cells of the invention can be used for therapeutic uses, administration to a subject of the cells of the invention previously subjected to irradiation and IFN-γ- stimulation, in any order, may result beneficial for the reasons previously mentioned (e g , subjecting cells to an irradiation treatment to make the cells incapable of proliferating or surviving for long time periods in the subject, whereas pre-stimulation of cells with IFN-γ before administration to the subject may involve a reduction in the time period between IFN-γ-pie-stimulated cell administration and IDO expression in the subject The irradiated IFN-γ-pre-stimulated cells of the invention as well as the IFN-γ-pre- stimulated irradiated cells of the invention can be used for preventing, treating or amehoiating one or more symptoms associated with disorders in which modulation of a subject's immune system is beneficial, including, but not limited to, autoimmune diseases, inflammatory disorders, and immunologically mediated diseases including rejection of transplanted organs and tissues Said use constitutes an additional aspect of the present invention

Thus, in another aspect, the irradiated IFN-γ-pre-stimulated cells of the invention as well as the IFN-γ-pre-stimulated irradiated cells of the invention are used as a medicament In a particular embodiment, medicaments containing the irradiated IFN-γ-pre-stimulated cells of the invention or the IFN-γ-pre-stimulated irradiated cells of the invention may be used for inducing transplantation tolerance, or for treating, and thereby alleviating, symptoms of autoimmune or inflammatory disorders, or immunologically mediated

diseases including rejection of transplanted organs and tissues, in a subject suffering from any of said disorders or diseases Thus, the irradiated IFN-γ-pre-stimulated cells of the invention as well as the IFN-γ-pre-stimulated irradiated cells of the invention can be used to therapeutically or prophylactically treat and thereby alleviating symptoms of autoimmune or inflammatory disorders in a subject suffering from any of said disorders or to alleviate symptoms of immunologically mediated diseases in a subject suffeπng from said diseases

Practically any autoimmune disease, inflammatory disorder or immunological mediated disease can be treated with the irradiated IFN-γ-pre-stimulated cells of the invention or with the IFN-γ-pre-stimulated irradiated cells of the invention Illustrative, non-limiting examples of said diseases and disorders which can be treated are those previously listed under heading "Definitions" In a particular embodiment, said inflammatory disease is a chronic inflammatory disease, such as, e g , IBD or RA

In other aspect, the present invention relates to the use of the irradiated IFN-γ-pre- stimulated cells of the invention or the IFN-γ-pre-stimulated irradiated cells of the invention for the preparation of a medicament for preventing, treating or ameliorating one or more symptoms associated with disorders in which modulation of a subject's immune system is beneficial, including, but not limited to, autoimmune diseases, inflammatory disorders, and immunologically mediated diseases including rejection of transplanted organs and tissues Thus, the invention further refers to the use of the irradiated IFN-γ-pre- stimulated cells of the invention or the lFN-γ-pre-stimulated irradiated cells of the invention for the preparation of a medicament for suppressing the immune response, or for inducing transplantation tolerance, or for treating autoimmune diseases, or for treating inflammatory disorders Examples of said autoimmune diseases and inflammatory diseases have been previously mentioned In a particular embodiment, disease is an inflammatory disease, such as a chronic inflammatory disease, e g , IBD or RA

Pharmaceutical compositions

The present invention provides pharmaceutical compositions for the treatment, prophylaxis, and amelioration of one or more symptoms associated with a disorder in which modulation of a subject's immune system is beneficial such as autoimmune diseases,

inflammatory disorders, and immunologically mediated diseases including rejection of transplanted organs and tissues

Thus, in another aspect, the invention relates to a pharmaceutical composition, hereinafter referred to as the pharmaceutical composition of the invention, comprising a cell of the invention, or a T-reg cell of the invention, or an irradiated cell of the invention, or an IFN-γ-pre-stimulated cell of the invention, or an irradiated IFN-γ-pre-stimulated cell of the invention, or an IFN-γ-pre-stimulated irradiated cell of the invention, and an acceptable pharmaceutically carrier Combinations of two or more of said type of cells are included within the scope of the pharmaceutical compositions provided by the instant invention

The pharmaceutical composition of the invention compπses a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic agents (i e , cell of the invention, or a T-reg cell of the invention, or an irradiated cell of the invention, or an IFN-γ-pre-stimulated cell of the invention, or an irradiated IFN-γ-pre-stimulated cell of the invention, or an IFN-γ-pre-stimulated irradiated cell of the invention, or a combination thereof), and a pharmaceutically acceptable earner In a specific embodiment, the term ''pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U S Pharmacopeia, or European Pharmacopeia, or other generally recognized pharmacopeia for use in animals, and more particularly in humans The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered The composition, if desired, can also contain minor amounts of pH buffering agents Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E W Martin Such compositions will contain a prophylactically or therapeutically effective amount of a prophylactic or therapeutic agent preferably in puπfied form, together with a suitable amount of earner so as to provide the form for proper administration to the subject The formulation should suit the mode of administration In a preferred embodiment, the pharmaceutical compositions are stenle and in suitable form for administration to a subject, preferably an animal subject, more preferably a mammalian subject, and most preferably a human subject

The pharmaceutical composition of the invention may be in a vanety of forms These include, for example, solid, semi-solid, and liquid dosage forms, such as lyophihzed

preparations, liquids solutions or suspensions, injectable and infusible solutions, etc The preferred form depends on the intended mode of administration and therapeutic application

The administration of the cell population of the invention, or the pharmaceutical composition comprising same, to the subject in need thereof can be earned out by conventional means In a particular embodiment, said cell population is administered to the subject by a method which involves transferring the cells to the desired tissue, either in vitro (e g , as a graft prior to implantation or engrafting) or in vivo, to the animal tissue directly The cells can be transferred to the desired tissue by any appropriate method, which generally will vary according to the tissue type For example, cells can be transferred to graft by bathing the graft (or infusing it) with culture medium containing the cells Alternatively, the cells can be seeded onto the desired site within the tissue to establish a population Cells can be transferred to sites in vivo using devices such as catheters, trocars, cannulae, stents (which can be seeded with the cells), etc The cells of the invention can be irradiated before administration to the subject

This treatment makes cells incapable to proliferate or survive for long time peπods in the subject Thus, in a particular embodiment, the pharmaceutical composition of the invention comprises irradiated cells of the invention

Also, the cells of the invention can be pre stimulated with IFN-γ, pπor to administration to the subject in order to reduce the time period between cell administration and IDO expression in the subject Thus, in a particular embodiment, the pharmaceutical composition of the invention comprises IFN γ pre-stimulated cells of the invention

Further, the cells of the invention can be both irradiated and pre-stimulated with IFN-γ, in any order, pnor to administration to the subject Thus, in a particular embodiment, the pharmaceutical composition of the invention comprises irradiated IFN-γ - pre-stimulated cells of the invention or IFN-γ-pre-stimulated irradiated cells of the invention

The cell populations and pharmaceutical compositions of the invention can be used in a combination therapy In a specific embodiment, the combination therapy is administered to a subject with an inflammatory disorder that is iefractory to one or more anti-inflammatory agents In another embodiment, the combination therapy is used in conjunction with other types of anti-inflammatory agents including, but not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs, beta-

agonists, anticholingeπc agents, and methyl xanthines Examples of NSAIDs include, but are not limited to, lbuprofen, celecoxib, diclofenac, etodolac, fenoprofen, lndomethacin, ketoralac, oxaprozin, nabumentone, suhndac, tolmentin, rofecoxib, naproxen, ketoprofen, nabumetone, etc Such NSAIDs function by inhibiting a cyclooxgenase enzyme (e g , COX-I and/or COX-2) Examples of steroidal anti-inflammatory drugs include, but are not limited to, glucocorticoids, dexamethasone, cortisone, hydrocortisone, prednisone, prednisolone, triamcinolone, azulfϊdine, and eicosanoids such as thromboxanes, and leukotπenes Monoclonal antibodies, such as Infliximab, can also be used

In accordance with the above embodiment, the combination therapies of the invention can be used prior to, concurrently or subsequent to the administration of such anti-inflammatory agents Further, such anti-inflammatory agents do not encompass agents characteπzed herein as lymphoid tissue inducers and/or immunomodulatory agents

Method for distinguishing adult multipotent cells from differentiated cells The expression of IDO upon stimulation with IFN-γ can be used for distinguishing cells which express said enzyme from cells which do not express IDO

Thus, in another aspect, the invention relates to a method for distinguishing adult multipotent cells from differentiated cells comprising the step of veπfying whether the multipotent cell expresses IDO upon stimulation with IFN-γ The determination of IDO upon stimulation with IFN-γ can be carried by any conventional technique, in an embodiment, the determination of IDO upon stimulation with IFN-γ can be earned out as disclosed in Example 2

As previously mentioned, the cells of the cell population of the invention are characteπzed in that they do not express IDO constitutively, but only upon stimulation with IFN-γ Moreover, aside from IFN-γ no other pro-inflammatory molecule such us IL-I , TNF-α, or endotoxin is able to induce by itself the expression of IDO in the cells of the cell population of the invention This feature can be used for distinguishing the cells of the cell population of the invention from other cells

Kits

In another aspect, the invention refers to a kit comprising a cell population containing (i) cells of the invention and/or (n) T-reg cells of the invention and/or (in) irradiated cells of the invention and/or (iv) IFN-γ-pre-stimulated cells of the invention,

and/or (v) irradiated IFN-γ-pre-stimulated cells of the invention, and/or (vi) IFN-γ-pre- stimulated irradiated cells of the invention Kits of the invention may comprise one, two, three, four, five or all of such cell types

Methods of treatment

In other aspect, the present invention refers to the use of a cell population containing cells of the invention, T-reg cells population of the invention, irradiated cells of the invention, IFN-γ-pre-stimulated cells of the invention, irradiated IFN-γ-pre-stimulated cells of the invention, or IFN-γ-pre-stimulated irradiated cells of the invention for preventing, treating, or ameliorating one or more symptoms associated with autoimmune diseases, inflammatory disorders, or immunologically mediated diseases including rejection of transplanted organs and tissues In a particular embodiment, said cell populations may be used for inducing transplantation tolerance, or for treating, and thereby alleviating, symptoms of autoimmune or inflammatory disorders, or immunologically mediated diseases in a subject suffering from said disorders or diseases Examples of said autoimmune diseases and inflammatory diseases have been previously mentioned In a particular embodiment, disease is an inflammatory disease, such as a chronic inflammatory disease, e g , IBD or RA

In other aspect, the present invention provides methods of preventing, treating, or ameliorating one or more symptoms associated with autoimmune diseases, inflammatory disorders, or immunologically mediated diseases, in a subject suffering from said disorders or diseases, which comprises administering to said subject in need of such treatment of a prophylactically or therapeutically effective amount of a cell population containing cells of the invention, T-reg cells of the invention, irradiated cells of the invention, IFN-γ-pre- stimulated cells of the invention, irradiated IFN γ-pre-stimulated cells of the invention, or IFN-γ-pre-stimulated irradiated cells of the invention In a particular embodiment, said cell populations may be used for inducing transplantation tolerance, or for treating, and thereby alleviating, symptoms of autoimmune or inflammatory disorders, or immunologically mediated diseases in a subject suffering from said disorders or diseases Examples of said autoimmune diseases and inflammatory diseases have been previously mentioned In a particular embodiment, disease is an inflammatory disease, such as a chronic inflammatory disease, e g , IBD or RA

EXAMPLES

The invention will now be described in more detail, by way of examples which in no way are meant to limit the scope of the invention, but, rather, these examples will serve to illustrate the invention with reference to the accompanying figures

EXAMPLE 1 Isolation and expansion of cells of the invention

I. Material and Methods

Isolation of cells of the invention from adipose tissue

Human adipose tissue was obtained by liposuction, under local anaesthesia and general sedation A hollow blunt-tipped cannula was introduced into the subcutaneous space through a small incision (less than 0 5 cm in diameter) With gentle suction, the cannula was moved through the adipose tissue abdominal-wall compartment for mechanical disruption of the fatty tissue A saline solution and the vasoconstrictor epinephrine were injected into the adipose tissue compartment to minimize blood loss In this way, 80 to 100 ml of raw lipoaspirate were obtained from each patient to be treated

The raw lipoaspirate was washed extensively with steπle phosphate-buffered saline (PBS, Gibco BRL, Paisley, Scotland, UK) to remove blood cells, saline and local anaesthetic The extracellular matrix was digested with a solution of type II collagenase (0 075%, Gibco BRL) in balanced salt solution (5 mg/ml, Sigma, St Louis, USA) for 30 minutes at 37°C to release the cellular fraction Then the collagenase was inactivated by addition of an equal volume of cell culture medium (Dulbecco's modified Eagle ^' s medium (DMEM, Gibco BRL) that contained 10% fetal bovine serum (FBS, Gibco BRL) The suspension of cells was centπfuged at 250 x g for 10 minutes Cells were resuspended in 0 16 M NH ₄ Cl and allowed to stand for 5 minutes at room temperature (RT) for lysis of erythrocytes The mixture was centπfuged at 250 x g, and cells were resuspended in DMEM plus 10% FBS and 1% ampicillin/streptomycin mixture (Gibco BRL) and then they were filtered through a 40 μm mesh and were plated in tissue culture flasks at a concentration of 10-30 x 10 cells/cm

Isolation of cells of the invention from articular cartilage

Human hyaline articular cartilage was obtained from the knee joint of a donor by means of arthroscopic techniques About 4 cm ² of cartilage were taken from the external margin of the phemoral condile, but the size of the biopsy may vary depending on the donor's age, the structure of the articulation and the surgeon's consideration The biopsy was suspended in a steπle saline solution and stored at 3-8°C until its use Live cartilage samples should not be stored for more than 48 hours

The cartilage biopsy was transferred to 1 ml of stenle cell culture medium containing 1 % FBS, and minced to obtain tissue fragments as small as possible The resulting cartilage fragments were suspended in a similar medium containing 0 1 % (w/v) collagenase, and incubated at 37 ⁰ C with continuous and gentle agitation After the digestion, the cell suspension obtained was filtered through a 40 μm mesh and the cells were plated onto tissue culture flasks at a concentration of 10-30 x 10 cells/cm

Ex vivo expansion of cells Cells both from adipose tissue and articular cartilage were separately cultured for

24 hours at 37°C in an atmosphere of 5% CO ₂ in air Then, the culture flasks were washed with PBS to remove non-adhenng cells and cell fragments The cells were maintained in culture in the same medium and under the same conditions until they reached approximately 80% confluence, with replacement of the culture medium every 3 to 4 days Cells were then passaged with trypsin-EDTA (Gibco BRL) at a dilution of 1 3 which corresponds to a cell density of approximately about 5-6 x 10 ³ cells/cm ² The cellular growth kinetics of the cells isolated from human adipose tissue and cultured ex vivo for more than 25 cell population doublings is shown in Figure 1

Cell charactenzation

Cell characterization was performed using cells at culture passages 1 to 25 Cells both from adipose tissue and articular cartilage were analyzed by means of flow cytometry by using antibodies labeled with a fluorescent marker (1 e , by fluorescence lmmunocytometry) for the presence/absence of a series of surface markers, which included

- Markers of antigen presenting cells (APCs) CD 1 1 b, CD 1 1 c, CD 14, CD45, and HLAII

- Markers of endothelial cells CD31

- Other markers: CD9, CD34, CD90, CD44, CD54, CD105 and CD133. The antibodies used in the flow cytometry assay were the following:

- CD9: clone MM2/57 Mouse IgG2b - FITC labeled antibody (Serotec);

- CDl Ib: clone ICRF44 Mouse IgGl - FITC labeled antibody (Serotec); - CDl Ic: clone BUl 5 Mouse IgGl - FITC labeled antibody (Serotec);

- CD 14: clone UCHM 1 Mouse IgG2a - FITC labeled antibody (Serotec);

- CD31 : clone WM59 Mouse IgGl - FITC labeled antibody (Serotec);

- CD34: clone QBEND 10 Mouse IgGl - FlTC labeled antibody (Serotec);

- CD44: clone Fl 0-44-2 Mouse IgG2a - FITC labeled antibody (Serotec); - CD45: clone Fl 0-89-4 Mouse IgG2a- FITC labeled antibody (Serotec);

- CD54: clone 15.2 Mouse IgGl - FITC labeled antibody (Serotec);

- CD90: clone Fl 5-42-1 Mouse IgGl -FITC labeled antibody (Serotec);

- CD 105: clone SN6 Mouse IgGl - FITC labeled antibody (Serotec); and

- Anti Human HLA class II DP, DQ, DR: clone WRl 8 Mouse IgG2a - FITC labeled antibody (Serotec);

- CD 133: clone 293C3 Mouse IgG2b- PE labeled antibody (Miltenyi Biotec).

II. Results

The results are collected in Figure 2 which shows that the cells analyzed were positive for CD9. CD44, CD54, CD90 and CD 105, and negative for CD l Ib, CDl I c,

CD14, CD31 , CD34, CD45, CD 133 and HLAII The cells were negative for all of the tested markers which are specific for the endothelial or APC lineages (CDl Ib, CD l I c,

CD14, CD45, and HLAII).

EXAMPLE 2

Induction of indolamine 2.3-dioxygenase (IDO) by interferon-gamma (IFN-γ)

I. Material and Methods

The cells of the invention isolated from human adipose tissue (Example 1), were seeded onto tissue culture plates at a density of 10,000 cells/cm ^" , and incubated for 48 hours in the conditions previously described for cell expansion. Then, different proinflammatory stimuli were added to the culture medium, including:

• Interleukin-1 (IL-I ) 3 ng/ml

• Interferon-gamma (IFN-γ) 3 ng/ml

• Tumor necrosis factor-alpha (TNF-α) 5 ng/ml

• Lipopolysacchaπde (LPS) 100 ng/ml The cells were incubated in the presence of the corresponding stimulus for periods ranging form 30 minutes to 48 hours, and then they were collected by trypsin digestion, and lysed in RIPA buffer (50 raM Tπs-HCl pH 7 4, 150 mM NaCl, 1 mM PMSF (phenyl- methylsulphonylfiuoπde), 1 mM EDTA (ethylenediaminetetraacetic acid), 5 μg/ml Aprotinin, 5 μg/ml Leupeptm, 1% Tπton x-100, 1 % Sodium deoxycholate, 0 1% SDS) containing protease inhibitors Cell lysates were then used in a western blot experiment using an IDO-specific monoclonal antibody (mouse monoclonal IgG, clone 10 1, from Upstate cell signaling solutions) Also, RNA was isolated from the treated cells, and tested by reverse transcnption - polymerase chain reaction (RT-PCR) experiments using primers specific for the IDO cDNA (GenBank Accession No M34455 (GI 185790)) forward 5" GGATTCTTCCTGGTCTCTCTATTGG 3', backward 5' CGGACTGAGGGATTTGACTCTAATG 3')

II. Results

The results of this experiment [Figure 3A (RT-PCR) and 3B (western blotting)] show that the cells provided by the instant invention do not express IDO constitutively The IDO mRNA is induced after 2 hours of IFN-γ stimulation, but the expression of the protein can only be detected between 8-24 hours of induction

Similar results were obtained when the cells of the invention were isolated from other human tissues, including bone marrow, articular cartilage, and skin (Figure 4)

EXAMPLE 3 Tumorigenic behaviour

I. Material and Methods

This experiment was performed with cells of the invention isolated from human adipose tissue as descπbed in Example 1 The cell samples were cultivated for 2-7 weeks prior to the subcutaneous implantation in immunodeficient mice (5 x 10 cells/mouse) The

mice were nu/nu strain obtained from Charles River Laboratories Mice lacked thymus and were T-cell deficient The implanted mice were followed-up for 4 months prior to sacrifice and pathological study

Pathological study. A necropsy was performed on all animals The animals were examined for gross abnormalities in the brain, lungs, heart, liver, kidneys, spleen, abdominal lymph nodes and injection site. Tissues were collected for a histological examination (parafin section and hematoxihn-eosin (H&E) staining), including injection site, lungs and lymph nodes

The teratome cellular line (N-TERA) was used as a positive control, which was implanted under identical conditions.

II. Results

The results show that, whereas all mice implanted with teratoma cells developed tumours after a few weeks, none of the animals implanted with the cells of the invention developed tumours within the first 4 months following implantation [data not shown]

EXAMPLE 4 Treatment of experimentally-induced IBD in mice

I. Materials and Methods

Colitis was induced in Balb/c mice (6-8 weeks old, Jackson Laboratories, Bar Harbor, ME) as previously descπbed (Neurath, M F , et al 1995 Antibodies to IL- 12 abrogate established experimental colitis in mice J Exp Med 182, 1281-1290) In brief, mice were lightly anesthetized with halothane, and a 3 5 F catheter was inserted intrarectally 4 cm from the anus. To induce colitis, 100 μl of 50 or 30 mg/ml of TNBS (2,4,6-tπnitrobenzene sulfonic acid) (Sigma Chemical Co, St Louis, MO) in 50% ethanol (to break the intestinal epithelial barrier) was slowly administered into the lumen via the catheter filled to a 1 ml syπnge Control mice received 50% ethanol alone (100 μl) Animals were treated intrarectally with different numbers of the cells of the invention obtained from human adipose tissue as described in Example 1 (0.3 x 10 ⁶ and 1 x 10 ⁶ cells, suspended in phosphate-buffered saline, PBS) 12 hours after TNBS instillation In some experiments, said cells were pretreated with 200 U/ml IFN-γ for 24 hours before injection

Animals were monitored daily for survival, appearance of diarrhea, and loss of body weight (Figures 5, 6 and 7)

II. Results As shown in Figure 5 there was a dose-dependent improvement of weight gained after the administration of the cells of the invention Indeed, a dose dependency can be observed in Figure 6 with 1x10 ⁶ cells showing a stronger effect than 0 3x10 ⁶ cells In both cases the cells improved the survival rate of the TNBS treated mice significantly

Furthermore, IFN-γ-pre-stimulated cells showed a faster and stronger recuperation from the TNBS treatment than non-prestimulated cells (Figure 7) The graph shows that TNBS treated mice lost weight dramatically and a clear improvement in mice that received cells

EXAMPLE 5 Treatment of experimentally-induced inflammatory bowel disease (IBD) in mice - additional experiments:.

I. Materials and Methods

In an extension of the same experiments of Example 4, Colitis was induced in Balb/c mice (6-8 weeks old, Jackson Laboratories, Bar Harboi, ME) as previously descπbed (Neurath, M F , et al 1995 Antibodies to IL- 12 abrogate established expeπmental colitis in mice J Exp Med 182, 1281-1290) In brief, mice were lightly anesthetized with halothane, and a 3 5 F catheter was inserted intrarectally 4 cm from the anus To induce colitis, 100 μl of 50 or 30 mg/ml of TNBS (2,4,6-tπnitrobenzene sulfonic acid) (Sigma Chemical Co, St Louis, MO) in 50% ethanol (to break the intestinal epithelial barrier) was slowly administered into the lumen via the catheter filled to a 1 ml syringe Control mice received 50% ethanol alone (100 μl) Animals were treated intrarectally or intrapeπtoneally (i p ) with different numbers of the cells of the invention obtained from human adipose tissue (ASC) as descπbed in Example 1 (0 3 x 10 ⁶ and 1 x 10 ⁶ cells, suspended in phosphate-buffered saline, PBS) 12 hours after TNBS instillation

In some experiments, said cells were pretreated with 200 U/ml IFN-γ for 24 hours before injection Also, in some expeπments, cells were labeled with CFSE (a fluorescent probe) before administration to the mice Animals were monitored daily for survival, appearance

and seventy of diarrhea, and loss of body weight Serum was collected and protein extracts were obtained from colons at the acute phase of the disease (day 3) Cytokine/chemokine contents in protein extracts and in serum were determined by ELISA The presence of CSFE-labeled cells in the mesenteric lymph nodes were analyzed by flow cytometry

H. Results

In all cases, mice treated with the cells of the invention (ASCs) showed a clear improvement in their inflammatory symptoms compared with non-treated animals The improvement was dose-dependent and statistically significant in all parameters tested, when cells were administered locally (intra-rectally) or systemically (i p ), although this last route seems to be more effective As previously shown in Figure 5 there was a dose- dependent improvement of weight gained after the administration of the cells of the invention Indeed, a dose dependency can be observed in Figures 6, 7 and 8 with 1x10 ⁶ cells showing a stronger effect than 0 3x10 cells In both cases the cells improved the survival rate of the TNBS treated mice significantly

Furthermore, IFN-γ-pre-stimulated cells showed a faster and stronger recuperation from the TNBS treatment than non-prestimulated cells (Figure 8) The graph shows that TNBS treated mice lost weight dramatically and a clear improvement in mice that received cells This improvement was also measurable by the seventy of colitis The inflammatory immune response is clearly diminished in animals treated with the cells of the invention As shown in Figure 9, all proinflammatory cytokines (TNF-a, IL-6, IL-Ib, IL-12, and IFN-γ) and chemokines (MIP-2 and RANTES) tested, both in the colon (local response) and in the serum (systemic response), were lower in cell-treated animals compared with the non-treated mice This inhibitory response was enhanced in animals treated with cells pre-stimulated with IFN-γ On the other hand, the immunoregulatory cytokine IL-I O was increased in the colon of ASC-treated mice, comparing with both non-treated TNBS-injured and control animals Also, neutrophil infiltration, as measured by MPO activity was lower in ASC-treated animals, and even lower when cells were pre-stimulated with IFN-γ (Figure 10) The labeled cells were localized in the draining lymph nodes of treated animals by means of cell cytometry (Figure 1 1 ) This is the localization expected if the administered cells were functioning as APCs

EXAMPLE 6 Induction of APC markers in the cells of the invention after IFN-γ stimulation

I. Materials and Methods

The cells of the invention were obtained from human subcutaneous adipose tissue (ASCs) as described in Example 1 After a minimum of 3 culture passages, the cells were incubated in standard culture medium or in culture medium containing 3 ng/ml IFN-γ for 4 days After that, the cells were stained for some surface markers related with the immune response (specifically related with the activity of antigen presenting cells (APCs)) These markers included the following'

• HLA-II (DP, DQ, DR). This receptor presents fragments of foreign antigens to T cells, initiating the adaptive immune response (it is the first signal for T cell activation). The cells of the invention do not express HLA-II constitutively. The antibody used was obtained from Serotec

• CD40 This protein binds to CD40L, which is expressed in the surface of activated T cells The cells of the invention express undetectable or very low levels of CD40 constitutively. The antibody used was obtained from Serotec

• ICAM-I (CD54) Is the major protein involved in the binding between T cells and APCs Its expression is needed for other interactions between APCs and T cells to be earned out properly The cells of the invention express low-medium levels of ICAM-I constitutively The antibody used was obtained from Serotec

• Members of the B7 family of co-stimulatory proteins (they deliver the second signal for T cell activation): o CD80 (B7-l ) Antibody obtained from Serotec o CD86 (B7-2) Antibody obtained from Serotec o ICOSL (B7-H2) Antibody obtained from e-Bioscience o B7-H4 Antibody obtained from e-Bioscience o PD-Ll (B7-H1 ) Antibody obtained from e-Bioscience o PD-L2 (B7-DC) Antibody obtained from e-Bioscience

The first four deliver mainly a stimulatory signal (promoting induction of T cell effector clones), while PD-Ll and PD-L2 are mainly tolerogenic (promoting

induction of T cell anergy-inactivation) None of them are expressed by the cells of the invention constitutively

II. Results After IFN-γ treatment, the cells of the invention induce the expression of HLA-II,

PD-Ll and PD-L2, and a strong upregulation of CD40 and ICAM-I The results of this experiment are shown in Figure 12

These results are very relevant because, together with the induction of IDO activity, they demonstrate that the cells of the invention, upon IFN-γ treatment, display a phenotype characteristic of tolerogenic APCs

EXAMPLE 7 Treatment of collagen-induced arthritis (CIA) with ASCs

I. Materials and Methods

Experimental arthritis was induced in DBAl/Jlac male mice (6-8 weeks of age) by injecting subcutaneously (s c ) an emulsion containing 200 μg of chicken type II collagen (CII) in complete Freund's adjuvant (CFA) and 200 μg of Mycobacterium tuberculosis H37RA The evolution of CIA was followed daily by two different technicians, by measuring the lnflammation-redness-ankylosis of the joints of upper and lower limbs, according to a pre-established scoring system

When clinical symptoms showed the establishment of CIA (day 23 post- lmmunization, p i ), animals were injected i p daily for 5 days with 2x10 ³ cells of the invention obtained from human adipose tissue as descπbed in Example 1 (ASCs), or with PBS as control Alternatively, CIA mice were injected intrarticularly (i a ) once in one of the affected joints The evolution of the treated animals were followed as previously descπbed, and at day 50 p i they were euthanized Several parameters were measured in the blood and the joints, including joint cytokines, serum cytokines, immunoglobin isotypes, as well as phenotype and cytokine production of lymphocytes

II. Results

As shown in the Figures 13, 14, and 15, the cells of the invention clearly decrease CIA incidence and seventy in the mouse model In particular, the effect on the immune

response is consistent with a strong inhibition of the ThI response (IFN-γ, TNFα, IL-2, IL- l β, IL-6, IL- 12, MIP2, RANTES, and lgG2a) without any increase in the Th2 response (IL-4, IgGl ), and with the induction of high levels of immunmoregulatory cytokines (IL- lO and TGF-β).

EXAMPLE 8 In vivo induction of regulatory T cells with the cells of the invention

I. Materials and Methods

In a study similar to the one described in Example 7, effector (CD4 ⁺ CD25 ^~ Foxp3 ^~ ) and regulatory T cells (CD4 ⁺ CD25 ⁺ Foxp3 ⁺ ) were isolated from the drain lymph node (DLN) and the synovial membrane of untreated and ASC-treated CIA mice, by means of cell cytometry, and the number of cells in each population was evaluated In order to evaluate the capacity of regulatory T cells present in the ASC-treated

CIA mice to inhibit the Cll-specific effector cells, a proliferative assay was performed in which autoreactive T cells isolated from ClA mice weie co-cultured with increasing numbers of DLN T cells (regulatory T cells) from untreated (control) or ASC-treated CIA mice (ratios from 1/64 to 1/1), and stimulated with CII ( 10 μg/ml) and splenic APCs.

II. Results

As shown in Figure 16. A., both the DLN and the synovial membrane of CIA mice treated with the cells of the invention induce an increase in the numbers of regulatory T cells (CD4 ⁺ CD25 ⁺ Foxp3 ⁺ ), without any increase in the numbers of effector T cells, comparing with the untreated (control) CIA mice.

The data shown in Figure 16.B. demonstrates that CIA mice treated with the cells of the invention, but not control (untreated) CIA mice, contain regulatory T cells that specifically inhibit the effector T cell response against ClI.

In conclusion, the treatment of an animal model of an experimental autoimmune disease (CIA) with the cells of the invention induces the emergence of antigen-specific regulatory T cells able to suppress the autoreactive T cell effector response.

EXAMPLE 9

Lymphocyte proliferation assay

Adipose derived ASCs of the invention, obtained via the methods of Example 1 , were plated at 5000 cells/cm ² with and without 200 000 lymphocytes (activated w/ lOμgPHA/ml) and co-cultured for 3 days Proliferation of the lymphocytes was measured by H3 incorporation As shown in Figure 17, the co-culture of ASCs and lymphocytes resulted in an 86% inhibition of lymphocytes proliferation The addition different concentrations ot I M ethyl -trytophan (1-MT) reverted this suppression 1-MT is a non- metabohzable tryptophan analog The assay demonstrates the necessity of tryptophan catabolism via IDO to induce the immunosuppressive activity of the cells of the invention

EXAMPLE 10 ASCs potency (IDO production) in relation to cell number and IFN-γ concentration

Materials and Methods

HPLC

Conventional HPLC was carried out using a Waters 1515 Isocratic HPLC pump, aWaters 717 Autosampler and a Wateis 2487 Dual Absorbance Detector

HPLC - protocol

Fresh solutions in the range of l OOμM to 10OmM of trytophan and kynuienine were prepared in 10% acetonitπle in potassium-phosphate buffer (5OmM pH 6 0) From these stock solutions 50μl tryptophan and l Oμl kynuremne and 940μl BSA (70g/l) or 10%FCS were combined to make up the control sample and stored at -80 ⁰ C

Sample preparation 200μl or more of supernatant from samples (cell cultures) were collected in Eppendorf tubes and stored at 80 ⁰ C Samples and control samples were thawed and 200μl 5OmM potassium-phosphate buffer pH6 0 was added to each 200μl sample in an Eppendorf tube 50μl of 2M TCA (trichloroacetic acid) was added to the Eppendorf tube The tube was vortexed and centπfuged for l Omin at 13 00Og at 4°C From the Eppendorf tube 150μl was removed for measuring

Column preparation for HPLC measurement

The HPLC column was prepaied as known in the art and equilibrated with mobile phase, which consisted of 4OmM sodium-citrate pH5 - in 5% acteonitπle 50μl of above described sample of 150 μl sample was injected into the column (Cl 8 reverse phase) Separation occurs by an isocratic flow rate of 700μl/min The photometric detection of L- kynurenine occurs at 365nm, for L-tryptophan at 280nm

Results

As shown in Figure 18, ASCs plated at 5000cell/cm ² and stimulated at 3ng/ml IFN-γ for up to 120 hours produce IDO, the activity of which is measured by the metabolisation of tryptophan and production of kynurenine using HPLC ASCs plated at 5OOOcell/cm ² and stimulated at 3pg/ml IFN-γ for up tol20hours did not produce IDO No kynurenine could be detected (Figure 19) Similarly, ASCs plated at 500cell/cm ² and stimulated at 3ng/ml IFN-γ for up to 120 hours did not produce significant amounts of IDO (Figure 20)

EXAMPLE 10 Ability of ASCs to Phagocvtose small molecules:

Materials and Methods 4kda- Dextran-FITC (Sigma) was added to the cells of Example 1 for 24 hours in culture The cell were washed and analyzed for the incorporation of the fluorescent FITC

Results

Figure 21 A shows the cells bright field image of the washed cell population Figure 21 B shows the same population using fluorescence microscopy using Green Fluorescent Protein filters known in the art The uptake of the fluorescent marker visible in Figure 21 B shows that the cells are able to phagocytose small weight molecules and this indicates that these cells are capable of antigen-presentation via HLA class II induced by additional treatment of the cells with IFN-γ