Regulatory macrophages for treatment of vascular disorders

Abstract

To provide the use of immunoregulatory macrophages for treating microvascular or macrovascular disorders of the lower extremities, such as diabetic foot ulcers or venous leg ulcers.SOLUTION: An immunoregulatory macrophage expressing the markers CD258, DHRS9 and IDO for use in a method of treating a microvascular or macrovascular disorder of the lower extremities in a subject is provided. Also provided is a pharmaceutical composition for use in a method of treating a microvascular or macrovascular disorder of the lower limb in a subject comprising said macrophages.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the use of immunoregulatory macrophages for treating diseases associated with pathological changes in blood vessels. In particular, the present invention relates to the use of immunoregulatory macrophages for treating microvascular and macrovascular disorders of the lower extremities. The present invention further relates to the use of immunoregulatory macrophages for promoting tissue remodeling and facilitating wound healing. Pharmaceutical compositions comprising immunoregulatory macrophages for use in the listed treatments are also disclosed. [Background technology]

[0002] Vascular ulcers of the lower extremities pose serious problems for patients and the healthcare system. Foot ulcers can be acute or chronic. Acute foot ulcers are defined as those that occasionally follow a normal phase of healing. These ulcers are expected to show signs of healing in less than four weeks and include traumatic and post-operative wounds. Chronic foot ulcers persist for more than four weeks and are often of complex and poorly understood etiology. Chronic foot ulcers typically occur in patients with diabetes.

[0003] Minimally invasive methods for managing superficial saphenous venous trunk insufficiency, including endovenous laser ablation, radiofrequency ablation, and foam sclerotherapy, have been used to treat patients with leg ulcers. Additionally, for patients with leg ulcers, perforator ligation is usually combined with superficial venous surgery. However, the effectiveness of this method has so far been insufficient. A few drugs have been shown to be beneficial in promoting leg ulcer healing. However, leg ulcer healing remains complex, and recurrence is frequently observed.

[0004] Despite the progress made in the field of foot ulcer treatment, there remains a continuing need to provide additional compounds and methods for treating acute or chronic foot ulcers, particularly chronic foot ulcers. Surprisingly, it has now been discovered that certain immunomodulatory cells can be used to treat chronic wounds, such as diabetic foot ulcers. As shown herein, administering these cells directly to or near a chronic wound results in accelerated healing.

[0005] The use of immunomodulatory cells for the treatment of specific diseases, particularly to establish immunological tolerance in recipients of these cells [1], has attracted considerable attention in recent years. Currently, several immunomodulatory cell types, including regulatory T cells [2], tolerogenic dendritic cells [3], and regulatory macrophages [4], are reaching the point of preclinical development, which will allow them to be investigated in early-stage clinical trials as immunosuppressive agents.

[0006] The use of immunomodulatory cells to treat numerous immunological conditions, including T-cell and B-cell mediated autoimmune diseases, chronic inflammatory diseases, graft-versus-host disease (GVHD), and transplant rejection, is contemplated. In these conditions, cell-based immunomodulatory therapies are intended to reduce or even eliminate the need for systemic immunosuppressive or anti-inflammatory therapy, thereby sparing patients from their associated complications. Because the type of immunological tolerance supported by regulatory cells is dominant and self-sustaining, cell-based immunomodulatory therapies may potentially offer a treatment option for diseases that would otherwise require long-term systemic immunosuppressive or anti-inflammatory therapy.

[0007] One particularly promising candidate cell type for use as an adjuvant immunosuppressant in transplantation and other clinical indications is immunoregulatory macrophages (referred to herein and in the literature as "Mregs"). Mreg cells reflect a unique state of macrophage differentiation and are distinguished from other activated macrophages by their robust phenotype and potent T cell suppressor function. [5] Human Mregs potently suppress mitogen-stimulated T cell proliferation in vitro, which may be due to interferon (IFN)-γ-induced indoleamine 2,3-dioxygenase activity and contact-dependent elimination of activated T cells. In addition, Mregs promote the development of activation-induced regulatory T cells, which in turn suppress effector T cell proliferation and dendritic cell maturation. Therefore, it is hypothesized that administration of Mregs to recipients initiates a feed-forward immunoregulatory loop, resulting in long-term antigen-specific immunological hyporesponsiveness of the foreign transplant.

[0008] In a series of case studies and two early clinical trials, Mreg-containing cell preparations have already been used in clinical trials and administered to a total of 19 kidney transplant recipients as a form of adjuvant immunosuppressive treatment.[5][9] These pilot studies clearly demonstrate the relevance of this technology for solid organ transplantation. Another two living donor kidney transplant recipients are currently receiving approximately 8.0 × 10 6 Currently, patients are treated with purer donor-derived Mregs at a dose of 2.5 × 10 cells / kg [5]. Further trials of Mreg therapy in living donor kidney transplantation have now received regulatory approval within the framework of the ONE Study (Clinicaltrials.gov: NCT02085629). This trial involves administering 2.5 × 10 Mregs at a dose of 2.5 × 10 Mregs supported by 500 mg / day of mycophenolate mofetil 7 days before surgery. 6 ~7.5×10 6 The goal is to treat 16 patients with donor-derived Mreg cells at a dose of 100 / kg body weight. Ulcer treatment with Mregs has not been attempted in prior art to date, and will make a significant contribution to the management of foot ulcers. Summary of the Invention

[0009] The present invention is based on the insight that Mreg cells prepared according to established protocols accelerate the healing of foot ulcers, particularly chronic foot ulcers. Accordingly, in a first aspect, the present invention relates to immunomodulatory macrophages expressing the markers CD258, DHRS9, and IDO for use in methods of treating micro- and macrovascular disorders of the lower limbs in a subject.

[0010] As used herein, microangiopathy is a vascular disease that adversely affects small blood vessels and capillaries in the body, such as small blood vessels in the brain, coronary arteries, or feet. During microangiopathy, the basement membrane of capillaries thickens and stiffens, causing blockage or rupture of capillaries or arterioles. This results in tissue necrosis and loss of function. In contrast, macroangiopathy is a vascular disease that adversely affects large blood vessels, such as arteries. Blockage of larger arteries leads to a high incidence of heart attacks, strokes, and peripheral vascular disease in diabetics. Blockage of arterial vessels in the feet often results in slow-healing ulcers throughout the legs. Peripheral vascular disease also causes intermittent claudication, which is pain during walking and severely impairs mobility. In many cases, particularly in diabetic patients, macroangiopathy has necessitated amputation of one or both legs.

[0011] One cause of microvascular and macrovascular damage is long-term diabetes. In diabetic patients, high blood sugar levels cause vascular endothelial cells to absorb more glucose than normal. These endothelial cells then produce more glycoproteins than normal on their surface. These cells also cause the basement membrane of the blood vessel walls to become abnormally thicker and weaker than before. As a result, the blood vessel walls become leaky, slowing blood flow through the body. As a result, some damaged tissues may not receive enough oxygen for an hour.

[0012] According to the present invention, the microangiopathy to be treated is preferably selected from the group of diseases consisting of vasculitis, arteritis, vascular dysplasia, atrophy alba, scleroderma, Determann syndrome, diabetic vasculopathy, endarteritis obliterans, erythromelalgia, fibromuscular dysplasia, perforation of the foot, Mönckeberg's calcific sclerosis, Osler's disease, compartment syndrome, Paget-von-Schroetter syndrome, Raynaud's disease, and foot ulcers. In a particularly preferred embodiment, the microangiopathy or macroangiopathy to be treated according to the present invention is a foot ulcer. As used herein, foot ulcers include diabetic foot ulcers and venous leg ulcers.

[0013] According to the present invention, the macrovascular disorders to be treated are preferably selected from the group of aneurysms, dissections, atherosclerosis, atherothrombosis, peripheral arterial occlusive disease (PAD), intermittent claudication, necrosis and gangrene, vascular malformations, Leriche's syndrome or compression syndromes.

[0014] In practice, the immunomodulatory macrophages are formulated as a pharmaceutical composition. The pharmaceutical composition comprises an effective amount of Mreg cells or subcellular fractions thereof as a first composition. As used herein, an effective amount of Mreg cells administered to a patient is about 1 x 10 per patient body weight to be treated. 4 ~Approx. 1×10 8 / kg, preferably about 1 × 10 5 ~Approx. 1×10 7 / kg, more preferably about 1 x 10 6 / kg, approximately 2 × 10 6 / kg, approximately 3 × 10 6 / kg, approximately 4 × 10 6 / kg, approximately 5 × 10 6 / kg body weight, approximately 6 × 10 6 / kg, approximately 7 × 10 6 / kg or approximately 8 × 10 6 / kg, approximately 1 x 10 6 ~Approx. 9×10 6 / kg range.

[0015] Similarly, when the present invention involves the administration of a subcellular fraction of Mreg cells, the fraction is preferably prepared based on an amount of Mreg cells corresponding to one of the ranges associated with the cell administration described above. As used herein, a subcellular fraction of Mreg cells may include necrotic cell particles, apoptotic cell particles, or exosomes. Cell lysates prepared by treating cells with hypotonic solutions, lysis with detergents or acids, freeze-thawing or heating, sonication, irradiation, mechanical disruption, or long-term storage may also be used. Subcellular fractions may also include cell extracts containing all cellular proteins, membrane proteins, cytoplasmic proteins, and purified MHC molecules.

[0016] Besides Mreg cells or subcellular fractions thereof, the pharmaceutical composition may contain further additives such as buffers, pH adjusters, preservatives, and the like. The nature and amounts of additives included in the pharmaceutical composition will depend on the intended route of administration.

[0017] Generally, a specific administration route is suitable for providing Mreg cells or subcellular fractions thereof to a patient in need of treatment. Preferably, the pharmaceutical compositions of the present invention are formulated for parenteral administration, such as subcutaneous, intramuscular, intravenous, or intradermal administration. In one embodiment, Mreg cells or subcellular fractions thereof, or compositions comprising such cells or fractions, are administered to a patient intravenously, for example, by injection or infusion. Pharmaceutical compositions suitable for intravenous administration by injection or infusion typically include sterile aqueous solutions or suspensions and sterile powders for extemporaneous preparation of sterile solutions or suspensions. Mreg cells or subcellular fractions can be formulated into pharmaceutical compositions by applying conventional methods known in the field of drug formulation. Suitable methods are described, for example, in standard textbooks.

[0018] For administration by injection or infusion, suitable carriers may include physiological saline, bacteriostatic water, Cremophor EL™ (BASF), or phosphate buffered saline (PBS). Carriers can also be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, the maintenance of the required particle size in the case of dispersions, and the use of surfactants. Sterile injectable solutions can be prepared by incorporating the required amount of cells or cellular fractions into an appropriate solvent with one or more of the above-mentioned ingredients, followed by sterile filtration. Generally, suspensions are prepared by incorporating the active compound (i.e., cells or cellular fractions) into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying, which yield a powder of cells or cellular fractions, plus any additional desired ingredients, from the above-mentioned sterile-filtered solution. Pharmaceutical compositions must be stable upon administration and preferably preserved against the contaminating action of microorganisms, such as bacteria and fungi, by including in the composition, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.

[0019] When the pharmaceutical composition is to be injected, the total volume to be injected is 1 to 100 ml, preferably 10 to 50 ml, such as 20 ml, 30 ml, or 40 ml. When the pharmaceutical composition is to be injected, the total volume to be injected is 50 to 500 ml. A volume of 90 ml to 250 ml is particularly preferred, and a volume of 90 ml to 150 ml is even more preferred.

[0020] Mreg cells can be administered to patients in need of treatment using a personalized dosing regimen. For example, when cells or cell fractions are administered to a patient by intravenous infusion, the total amount of Mreg cells or cell fractions administered can be provided in one or more infusions. In a preferred embodiment, Mreg cells or cell fractions are provided to a patient using an infusion set with a 200 μm filter. A suspension containing Mreg cells or cell fractions may be primed with 0.9% NaCl. The suspension may be administered in a single infusion, more preferably in a short infusion of less than 60 minutes, for example, within 60 minutes, 30 minutes, 20 minutes, or 15 minutes. Preferably, a central venous catheter is used to administer the Mreg cell suspension.

[0021] Administration of Mreg cells or cell fractions can be completed simultaneously with or after the administration of other active agents described above. For example, when Mreg cells or cell fractions are administered to patients suffering from microvascular or macrovascular disorders, compounds that combat blood hyperviscosity, such as calcium dobesilate, or compounds that exert capillary strengthening effects, such as naphthazone, can be administered before, simultaneously with, or after administration of Mregs.

[0022] Although Mreg cells obtained by the method described in the first aspect of the invention exhibit a stable phenotype, for safety reasons it is recommended that Mreg cells or subcellular fractions obtained from Mreg cells be administered within 24 hours of collection from the cell culture medium. Preferably, the cells are administered within 20 hours, 16 hours, 12 hours, 8 hours or 4 hours after harvesting the cells from culture.

[0023] In another aspect, the present invention relates to immunomodulatory macrophages for use in methods to promote healing of surgical, traumatic, or other wounds in a subject. Mreg therapy may be used to promote healing of acute or chronic wounds, optionally in combination with conventional management (i.e., irrigation, suturing, and dressings). Wounds may be open or closed. Open wounds may include incisions, lacerations, abrasions, abrasions, penetrating trauma, or puncture wounds. Closed wounds may include crush injuries or hematomas. Incisions may be traumatic or iatrogenic (i.e., surgical incisions). Mregs may be used to promote the survival of autologous or allogeneic skin grafts. Mregs may also be used in combination with conventional management to accelerate healing of burns, which may result from skin exposure to heat, cryogenic temperatures, chemicals, friction, radiation, or electrical current. Descriptions made in connection with the treatment of ulcers apply equally to the treatment of wounds or burns.

[0024] The preparation of Mregs for use in the methods of the present invention has been widely described in the literature. Mreg cells are derived from human CD14+ blood monocytes. To induce the distinctive biological properties of Mreg cells, monocytes are treated with specific combinations of growth factors, cytokines, and receptor ligands. Cells obtained by this process are characterized by a special phenotype that distinguishes them from blood monocytes, monocyte-derived macrophages of other species, monocyte-derived dendritic cells, and other suppressive myelomonocytic cell products.

[0025] A suitable process for preparing Mregs is: (a) isolating CD14-positive monocytes from a blood sample of a subject; (b) culturing monocytes in a medium containing (i) M-CSF and / or GM-CSF and (ii) a ligand for CD16; (c) contacting the cells with IFN-γ; (d) obtaining Mreg cells from the culture medium.

[0026] The method uses blood monocytes as a starting material. Preferably, the method is used to generate Mreg cells from human blood monocytes, but the method is not limited to the differentiation of human-derived cells. In fact, it is also applicable to non-human cells of other species, particularly vertebrate cells, such as non-human primate cells or porcine cells.

[0027] This method may be used to differentiate CD14-positive monocytes from a human donor into Mregs. The monocytes that serve as the starting material for the method of the present invention are obtained from the peripheral blood of a human donor. The donor may be a healthy subject or a patient suffering from one or more diseases. The monocyte donor may be the intended recipient of the differentiated Mreg cells (autologous approach). Alternatively, the monocyte donor may be a different person from the intended recipient of the differentiated Mreg cells (allogeneic approach). In the latter case, the donor and recipient may or may not be genetically related. The preferred relationship between the donor and recipient varies depending on the intended clinical application. The use of autologous Mreg cells may help prevent certain side effects. Therefore, the use of autologous Mreg cells is usually preferred.

[0028] Various methods for enriching mononuclear cells from peripheral blood are known in the art, and each of these methods can be used in conjunction with the above-described preparation method. For example, blood obtained by venipuncture can be treated with an anticoagulant, followed by separation using a separation medium such as Ficoll-Paque Plus. Thus, the anticoagulated blood sample is layered on top of a Ficoll-Paque Plus solution and centrifuged, resulting in the formation of layers containing various cell types. The bottom layer contains red blood cells that have been aggregated and sedimented by the Ficoll-Paque Plus reagent. The layer immediately above the red blood cell layer contains, for the most part, granulocytes that have migrated through the Ficoll-Paque Plus layer. Due to their lower density, monocytes and lymphocytes are found at the interface between the plasma and Ficoll-Paque Plus. Enrichment of the mononuclear cell fraction can be achieved by isolating the layers and subsequent washing and centrifugation.

[0029] Another commonly used method for separating mononuclear leukocytes from blood samples is leukapheresis. Leukapheresis is a unique type of apheresis in which leukocytes are obtained from peripheral blood in a continuous process based on their relative density. In this procedure, the subject's blood is passed through a special centrifuge device that collects a selected fraction of leukocytes and returns the remaining blood cells and plasma to the donor. Leukapheresis is now a routine clinical procedure for obtaining leukocytes or stem cells from peripheral blood. Various devices that can be used to perform leukapheresis in connection with the present invention are available from several manufacturers, such as the Spectra Optia® apheresis system from Terumo BCT. When performing leukapheresis using the COBE® Spectra apheresis system, it is preferable to use the protocol in the operating instructions provided by the manufacturer, as this protocol has been found to produce better quality monocytes than the AutoPBSC protocol.

[0030] Both the use of a separation medium such as Ficoll-Paque Plus and the use of a leukapheresis device result in a cell fraction containing not only monocytes but also lymphocytes. According to the present invention, monocytes can be enriched and separated from lymphocytes by known methods, such as magnetic bead separation, flow cytometric sorting, clarification, filtration, or plastic adhesion, before the cells are introduced into the Mreg preparation method. However, the use of a uniform monocyte fraction is not essential in the Mreg preparation method. In fact, the presence of lymphocytes in an amount of 0.1-20%, preferably 10-20%, in the monocyte fraction can have a positive effect on the differentiation of monocytes into Mregs.

[0031] To obtain a mononuclear cell preparation enriched in monocytes, peripheral blood mononuclear cells may be contacted with, for example, CD14 microbeads that bind CD14-positive monocytes. The monocytes in step (a) of the above method may be isolated by leukapheresis and then subjected to a separation step using a CD14 affinity molecule. The CD14 affinity molecule is preferably a CD14 antibody. Such a purification step significantly reduces contamination of the starting material with non-monocytes. Reducing T cell contamination is highly valuable from the perspective of patient safety, as it minimizes the potential risk of donor-versus-recipient reactions. The isolation of CD14 monocytes can be assisted by automated isolation systems. For example, the CD14 monocytes used in the methods of the present invention can be isolated using CliniMACS® Technology (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany).

[0032] The monocyte fraction isolated by leukapheresis and / or other methods can be used directly for differentiation by incubation with M-CSF and / or GM-CSF and CD16 ligand, or it can be stored in autologous plasma supplemented with anticoagulant citrate dextrose solution (ACD-A) or any other suitable buffer until further use. If the isolated monocyte fraction must be transported to another site for differentiation processing, care must be taken to initiate cell differentiation by incubation with M-CSF / GM-CSF within 24 hours of cell isolation, preferably within 18, 12, 6, 4, or 2 hours of monocyte isolation. For long-term storage, the monocyte fraction may be resuspended in an appropriate cryopreservation solution and stored for long periods at temperatures below 20°C, preferably below 80°C.

[0033] After isolating the monocytes, the cells are incubated in the presence of M-CSF / GM-CSF and CD16 ligand. For example, the cells may be suspended in a medium containing M-CSF and / or GM-CSF and CD16 ligand. Alternatively, M-CSF / GM-CSF and CD16 ligand may be added some time after the initiation of cell culture. The medium used in step (b) of the above method may be any medium described in the literature as suitable for use in culturing monocytes and / or macrophages. Suitable media include, for example, PromoCell Macrophage Generation Medium (PromoCell GmbH, Heidelberg, Germany), Dulbecco's Modified Eagle Medium (DMEM), DMEM:F12 mixture, Medium 199, or RPMI-1640 medium. The medium is preferably a chemically defined medium. In addition to M-CSF / GM-CSF, the medium may contain other factors that promote the survival and differentiation of Mregs, including growth factors and cytokines such as epidermal growth factor (EGF) or IL-4; fatty acids, cholesterol and other lipids; vitamins, transferrin and trace elements; insulin, glucocorticoids; cholecalciferol or ergocalciferol and other hormones; nonspecific immunoglobulins and other plasma proteins. In a preferred embodiment of the present invention, the medium is RPMI-1640 or a medium derived therefrom.

[0034] The medium used to incubate isolated CD14-positive monocytes may contain macrophage colony-stimulating factor (M-CSF, also known as CSF1), granulocyte-macrophage colony-stimulating factor (GM-CSF), or both. M-CSF is known in the art as a hematopoietic growth factor that influences the proliferation, differentiation, and survival of monocytes, macrophages, and myeloid progenitor cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF, also known as CSF2) is a monomeric glycoprotein that functions as a cytokine and is secreted by macrophages, T cells, mast cells, NK cells, epithelial cells, and fibroblasts. M-CSF and GM-CSF proteins from various species have been described and are available commercially from various manufacturers. The choice of M-CSF and / or GM-CSF will depend on the origin of the monocytes to be differentiated into Mreg cells. For example, if human monocytes are differentiated into Mregs using the process described above, the medium used will contain human M-CSF and / or human GM-CSF, preferably recombinant human M-CSF and / or recombinant human GM-CSF. Similarly, if porcine monocytes are used in the differentiation method, the M-CSF and / or GM-CSF added to the medium will be of porcine origin.

[0035] Typically, the concentration of M-CSF in the medium in step (b) of the above method ranges from 1 to 100 ng protein per ml of medium. Time course experiments measuring the amount of M-CSF in the medium revealed that M-CSF was consumed or degraded over time, such that cultures using an initial dose of 5 ng / ml contained subphysiological concentrations by the second day of culture. In contrast, cultures using an initial dose of 25 ng / ml maintained concentrations above 10 ng / ml throughout the 7-day culture period. Consequently, the M-CSF concentration in the medium typically ranges from 20 to 75 ng / ml, such as 20 to 25 ng / ml. If GM-CSF is used instead of M-CSF, the same concentrations outlined above for M-CSF can be used in the medium. Because GM-CSF appears to be more potent than M-CSF, concentrations of 0.1 to 100 ng protein per ml of medium are suggested. When both M-CSF and GM-CSF are used in the culture medium, the total concentration of these two growth factors is within the range mentioned above, ie, 20-75 ng / ml.

[0036] The medium used in step (b) of the above method contains CD16 ligand in addition to M-CSF and / or GM-CSF. Stimulation of the CD16 cell surface receptor on monocytes is necessary to induce their differentiation into Mreg cells. Stimulation of the CD16 cell surface receptor can be achieved by adding human or non-human immunoglobulins, more preferably human immunoglobulins or fragments thereof. The immunoglobulin fragment can be, for example, an Fc fragment of an immunoglobulin. It is believed that immunoglobulins activate through FcγRIII (CD16) and induce the Mreg phenotype. A simple way to achieve CD16 ligand stimulation is to add human serum to the medium. Consequently, the medium used to generate Mreg cells can contain 1-20% human AB serum.

[0037] If Mreg cells are intended for therapeutic use in which induction of angiogenesis is desired, the medium used to culture monocytes in step (b) of the method of the present invention can contain, in addition to M-CSF / GM-CSF and CD16 ligand, a Toll-like receptor (TLR) ligand, such as lipopolysaccharide (LPS), monophosphoryl lipid A (MPLA), or high-mobility group box protein 1 (HMGB1), to enhance the production of angiogenic factors such as VEGF-A. TLR ligands can be added to the medium at concentrations ranging from 1000 ng / ml to 1 μg / ml. The TLR ligand can be added at any stage of the production method. It can be present in the initial medium used to culture the monocytes, i.e., on day 0 of culture, or it can be added at a later stage, such as on day 5, 6, or 7 of culture. Preferably, the TLR ligand is added simultaneously with the addition of IFN-γ.

[0038] In step (c) of the method of the invention, the cells are contacted with the cytokine interferon gamma (IFN-γ). The choice of IFN-γ used in the method of the invention depends on the origin of the monocytes subjected to the method of the invention. If human monocytes are differentiated into Mregs, the IFN-γ added will usually be recombinant human IFN-γ. The amount of IFN-γ added to the monocyte culture ranges from 5 to 100 ng / ml. An amount of 25 ng IFN-γ per ml of medium is particularly suitable.

[0039] IFN-γ can be added to the culture medium simultaneously with M-CSF / GM-CSF and CD16 ligand. This means that the cytokine can be added, for example, at the beginning of monocyte culture. In such methods, monocytes differentiated by the methods of the present invention are cultured in the presence of M-CSF / GM-CSF, CD16 ligand, and IFN-γ throughout the entire culture period. However, typically, the culture period in the presence of IFN-γ is significantly shorter than the culture period in the presence of M-CSF / GM-CSF. This means that IFN-γ is added after the cells have been cultured in the presence of M-CSF / GM-CSF for 3 days, and the culture in the presence of IFN-γ continues for another 18 to 72 hours.

[0040] Particularly good results were obtained when cells were cultured in the presence of M-CSF / GM-CSF and CD16 ligand for 6 days, pulsed with IFN-γ for 18–24 hours, and then harvested on day 7. Differentiated macrophages may be washed with a buffer suitable for use with macrophages, such as phosphate-buffered saline (PBS) supplemented with 5% human serum albumin. Mreg cells can be transferred and stored in an infusion bag, glass infusion device, or another closed container that is transportable.

[0041] The above preparation process produces immunoregulatory macrophages characterized by the expression of specific marker molecules that distinguish Mreg cells from other regulatory and non-regulatory macrophages. Most importantly, Mregs for use in the methods of the present invention express the markers CD258, DHRS9, and IDO. The combination of these three markers provides a reliable means for unambiguously detecting Mregs and separating them from other macrophages.

[0042] CD258, also known in the literature as LIGHT or TNFSF14, is a secreted protein of the TNF superfamily. The human sequence of CD258 can be found under NCBI Gene_ID8740. IDO stands for indoleamine 2,3-dioxygenase. The human gene sequence encoding this marker can be found under NCBI Gene_ID3620. IDO is also known as IDO1 or INDO. DHRS9 is a retinol dehydrogenase of the SDR family of retinol dehydrogenases. The human gene sequence encoding this marker can be found under NCBI Gene_ID10170.

[0043] In a preferred embodiment of the present invention, the immunomodulatory macrophages for use in the methods of the present invention further express at least one marker selected from the group consisting of TGFβ1 and PAEP. TGFβ1 is transforming growth factor beta and represents a multifunctional cytokine belonging to the transforming growth factor superfamily. The human gene sequence encoding this marker can be found under NCBI Gene_ID 7040. PAEP represents progesterone-related endometrial protein. The human gene sequence encoding this marker can be found under NCBI Gene_ID 5047.

[0044] Because Mregs are macrophages, they also express common macrophage markers, and thus, in a preferred embodiment, Mregs further express at least one macrophage marker selected from the group consisting of CD33, CD11b, and HLA-DR.

[0045] In particularly preferred embodiments, Mreg cells used according to the present invention are of one of the following phenotypes: (1) CD258, DHRS9, IDO; (2)CD258, DHRS9, IDO, TGFβ1; (3)CD258, DHRS9, IDO, PAEP; (4)CD258, DHRS9, IDO, TGFβ1, PAEP; (5)CD258, DHRS9, IDO, TGFβ1, PAEP, CD33; (6)CD258, DHRS9, IDO, TGFβ1, PAEP, CD33, CD11b; (7)CD258, DHRS9, IDO, TGFβ1, PAEP, CD33, CD11b, HLA-DR; (8)CD258, DHRS9, IDO, CD33, CD11b, HLA-DR; (9)CD258, DHRS9, IDO, CD33, CD11b, HLA-DR, TGFβ1; (10)CD258, DHRS9, IDO, CD33, CD11b, HLA-DR, PAEP.

[0046] Here, immunoregulatory macrophages that express CD258, DHRS9, IDO, TGFβ1 and PAEP are particularly preferred.

[0047] In a preferred embodiment, Mregs used in the methods of the invention do not express at least one of the markers Clec-9a, CD10, and CD103. In another preferred embodiment, Mregs used in the methods of the invention do not express all of the markers Clec-9a, CD10, and CD103. In another preferred embodiment, Mregs used in the methods of the invention express at least one of the markers CD38, CD209, and syndecan-3. In yet another preferred embodiment, Mregs used in the methods of the invention express all of the markers CD38, CD209, and syndecan-3. In another preferred embodiment, Mregs used in the methods of the invention do not express at least one of the markers Clec-9a, CD10, and CD103, but do express at least one of the markers CD38, CD209, and syndecan-3.

[0048] The expression of marker can be measured by mRNA or protein amount.In a particularly preferred embodiment, marker is detected by transcription amount.Suitable methods for observing gene expression by transcription amount include those that can quantitatively or semi-quantitatively detect mRNA amount.For example, quantitative RT-PCR (for example, TaqMan™ RT-PCR), real-time RT-PCR, Northern blotting analysis or other methods well known in the art.

[0049] Detection of transcript levels typically requires, as a first step, the isolation of mRNA from the macrophages to be analyzed, e.g., those obtained from a blood sample. Methods for isolating RNA, such as mRNA, are well known in the art and are discussed in detail in the literature (see, e.g., Sambrook et al. (1989), Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, and Ausubel et al. (1994), Current Protocols in Molecular Biology, Current Protocols Publishing, New York). Such methods typically involve lysis of cells or tissues obtained from the subject to be tested. Cell lysis may be carried out using a detergent capable of disrupting the plasma membrane of cells. For example, a buffer containing guanidine thiocyanate and / or SDS may be used for cell lysis. This method may include a step in which cellular DNA is enzymatically digested to obtain pure RNA free of trace amounts of DNA that may interfere with further downstream applications, such as monitoring expression levels. Inhibitors of enzymes that lead to RNA degradation may be added to the lysis buffer. Kits for preparing highly pure RNA are available from several manufacturers, including Qiagen, Ambion, Stratagene, Clontech, Invitrogen, Promega, and others.

[0050] RNA isolated from cell or tissue samples using commercially available kits typically contains various species of RNA. Preferably, the RNA obtained from a tissue sample is total RNA, including mRNA, transfer RNA (tRNA), and ribosomal RNA (rRNA). For the methods of the present invention, it is desirable to enrich the mRNA fraction relative to other cellular RNA fractions. Preferably, mRNA is separated from other RNA molecules. Methods for enriching or purifying mRNA are known in the art. For example, because mRNA contains a poly(A) tail at its 3' end, affinity chromatography can be performed using oligo(dT) or poly(U) bound to a solid matrix such as cellulose or a Sephadex™ matrix (see, e.g., Ausubel et al. (1994), Current Protocols in Molecular Biology, Current Protocols Publishing, New York). Poly(A)+ mRNA bound to the affinity matrix can be eluted using 2 mM EDTA / 0.1% SDS.

[0051] One of the commonly used methods for detecting expression by transcriptional abundance is RT-PCR. In this method, an mRNA template is transcribed into cDNA by a reverse transcription (RT) reaction. The reverse transcription of the RNA template is catalyzed by reverse transcriptase, and the reaction is initiated by a specific oligonucleotide primer, or alternatively, an oligo-dT primer. The cDNA is then used as a template for a subsequent PCR reaction. In the subsequent PCR step, the cDNA is amplified by using specific oligonucleotide primers and a polymerase enzyme, such as Taq polymerase. In a preferred embodiment, the RT-PCR reaction is performed as real-time RT-PCR, which allows for the detection and simultaneous quantification of amplified DNA in real time. Quantification occurs either as an absolute copy number or as a relative amount normalized by using additional gene expression products.

[0052] In a more preferred embodiment, TaqMan RT-PCR is used to measure the expression levels of markers. TaqMan RT-PCR is a fluorescent-based RT-PCR method that is specific and detects the accumulation of amplification products during PCR. In TaqMan RT-PCR, RNA is first transcribed into cDNA using reverse transcriptase. In the subsequent PCR reaction, a single-stranded oligonucleotide probe is added that is complementary to a 10-60 nucleotide fragment within the DNA template and located between two PCR primers. A fluorophore and a quencher dye are covalently attached to the 5' and 3' ends of the probe, respectively. Alternatively, the quencher dye can be attached to an internal nucleotide, while the fluorophore is attached to the 5' or 3' end of the probe, or vice versa. When the fluorophore is selectively excited, the proximity between the fluorophore and quencher dye attached to the probe interferes with the fluorescence emission from the fluorophore. During DNA synthesis in PCR, the 5' exonuclease activity of Taq polymerase cleaves the oligonucleotide probe hybridized to the template DNA, resulting in steric separation of the fluorophore and quencher dye. Fluorescence is detected during the PCR cycles and is directly proportional to the amount of fluorophore released and the amount of DNA template present during PCR. Any fluorophore-quencher pair known in the art can be used in the methods of the present invention. Examples of suitable fluorophores are FAM (6-carboxyfluorescein), TET (tetrachlorofluorescein), or VIC. A suitable quencher dye is TAMRA (tetramethylrhodamine). The structure and labeling of oligonucleotides used as probes in the TaqMan approach have been described in great detail in the literature. TaqMan reactions can be performed, for example, using an ABI PRISM 7700 system (Perkin-Elmer / Applied Biosystems, Foster City, Calif., USA) or a Lightcycler system (Roche Molecular Biochemicals, Manheim, Germany).

[0053] Microarrays are another tool commonly used in expression profiling. Microarrays refer to an ordered arrangement of spatially resolved probes, such as nucleic acid probes, on a substrate. Such arrays can contain at least one, preferably two or more, oligonucleotides complementary to genes of interest, such as CD258, DHRS9, and IDO genes, thereby hybridizing each gene sequence or its transcript. The substrate is preferably a solid-phase substrate with a surface having multiple probes attached to individual, known locations (spots). The spots on the microarray are usually either printed on the microarray or synthesized by photolithography or inkjet printing. There can be thousands of spots on a typical microarray, and each spot can contain a large number of identical probes, such as nucleic acid fragments or oligonucleotides. Such microarrays typically have a size of 1 cm. 2 In certain embodiments, the array has a density of at least 100 oligonucleotides or fragments per 1 cm. 2 Approximately at least 500, at least 1000, at least 10,000, at least 10 5 Pieces, at least 10 6 Pieces, at least 10 7 The support can have a density of 10 ...

[0054] The primers or probes used in the PCR or RT-PCR reactions referred to herein are designed to enable specific hybridization and subsequent amplification of target sequences within each marker gene, such as the CD258, DHRS9, or IDO gene. Based on the present disclosure, those skilled in the art will be readily able to design oligonucleotide primers and / or probes that can be used to detect the expression of each Treg marker gene. Methods for designing sequence-specific oligonucleotide primers for PCR or RT-PCR are described, for example, in Dieffenbach et al. These primers have been discussed in great detail in scientific literature, such as by Roberts and Dveksler, "PCR Primer," A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 2003. Parameters to be considered when designing PCR primers include, for example, the number of nucleotides, the G / C content of the primer, the melting temperature, the presence of complementary nucleotides that may lead to secondary structures, and the like. Oligonucleotides for use in the methods of the present invention preferably have a length of at least 8 nucleotides, and more preferably at least 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides. Oligonucleotide primers can be prepared by any suitable technique known in the art. For example, oligonucleotide primers may be synthetically derived, for example, by the phosphoramidite method. Apart from oligomers or polymers containing the naturally occurring nucleotide bases adenine, thymine (uridine), cytosine or guanine, the oligonucleotide primers of the invention may also contain modified bases such as 5-methylcytosine, 5-hydroxymethylcytosine and the like.

[0055] Probes and primers for detecting the markers described herein can be designed by conventional methods based on the human genome sequence of each marker. The sequences of these markers are known in the art and can be searched in sequence databases. It will be understood that the probes and primers do not necessarily need to be completely complementary to the genome sequence over their entire length. As long as the probes and primers exhibit specific binding to each mRNA, a limited number of sequence deviations can be tolerated. Typically, the overall sequence identity of the probes and primers to the homologous mRNA molecule is at least about 90%, 95%, 96%, 97%, 98%, 99% or more. Computer programs for measuring the degree of identity between nucleotide sequences are available, for example, in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, USA), including the programs BESTFIT, FASTA, and GAP. This is based on the Smith and Waterman algorithm. These programs can be used with the standard parameters recommended by the manufacturer.

[0056] If high levels of mRNA for markers such as CD258, DHRS9, and IDO are detected, it can be assumed that the cells are Mreg cells. Non-Mreg cells, such as resting macrophages, may also express one or more of the above-listed markers to some extent, but such expression is relatively low. Therefore, according to the present invention, it is preferable to use marker expression by non-Mreg cells, particularly resting macrophages, as a negative control. Measurement of marker expression at RNA levels at least about 50%, at least about 75%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 550%, at least about 600%, at least about 750%, or at least about 1000% higher than the negative control, i.e., the respective amount of resting macrophages, clearly indicates that the cells being tested are Mreg cells.

[0057] In yet another particularly preferred embodiment, the detection of Mreg markers, such as CD258, DHRS9, and IDO, involves detecting marker gene expression at the translational level. This means that the protein amount of each Mreg marker, such as CD258, DHRS9, and IDO, is measured. The amount of Mreg protein can be measured by any suitable method that can specifically detect the marker protein in a biological sample. Protein detection can be based on a molecule that specifically binds to the protein or on separating the protein from other proteins present in the sample. Molecules that specifically bind to Treg marker proteins include antibodies and antibody fragments that have binding activity for Mreg markers. Many antibodies directed against Mreg marker proteins, such as CD258, DHRS9, and IDO, have been prepared and are commercially available. These antibodies or their fragments can be used to detect Mreg marker proteins using immunohistochemical methods, including Western blotting, quantitative Western blotting, enzyme-linked immunosorbent assay (ELISA), polarimetry (quantitative), surface plasmon resonance (SPR), or quantitative electron microscopy. In a particularly preferred embodiment of the present invention, the detection of markers involves ELISA. Other methods that can detect specific binding include, for example, fluorescence resonance energy transfer (FRET). Methods that separate proteins from other components in a biological sample, thereby allowing quantitative detection of Treg marker proteins, include quantitative mass spectrometry, electrophoretic methods such as two-dimensional gel electrophoresis, and chromatographic methods such as size exclusion chromatography or ion exchange chromatography.

[0058] As mentioned above, non-Mreg cells may also produce low amounts of the above-mentioned Mreg markers, such as CD258, DHRS9, and IDO. Therefore, the protein levels of non-Mreg cells, such as non-regulatory macrophages, should also be measured to provide a negative control. If the protein levels for the cells tested reveal higher marker expression than the negative control, it can be concluded that the tested cells are Mreg. Specifically, measuring marker expression at a protein level at least about 50%, at least about 75%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 550%, at least about 600%, at least about 750%, or at least about 1000% higher than the respective levels in the negative control, i.e., non-Mreg cells, such as non-regulatory macrophages, clearly indicates that the tested cells are Mreg cells.

[0059] In an even more preferred embodiment of the present invention, the detection of Mreg markers involves flow cytometry. Flow cytometry is a widely used method for analyzing the expression of cell surface markers and intracellular molecules. Flow cytometry is routinely used for applications such as cell counting, cell sorting, and biomarker profiling. In particular, flow cytometry can be used to determine different cell types within a heterogeneous cell population. Flow cytometry is primarily used to measure the fluorescence intensity generated by fluorescently labeled antibodies that detect markers on the cell surface. It can also be used to detect intracellular markers, but such detection is less desirable because antibodies usually must permeate cells to kill them. This prevents the detection of intracellular markers for cell sorting applications, which aim to preserve a viable, identical cell population.

[0060] In another preferred embodiment, Mreg cells for use in the methods of the present invention have the ability to induce CD25+FoxP3+ iTregs. The ability to induce Tregs can be tested by co-culturing Mregs with allogeneic T cells at a 1:2 ratio for 5 days. [Brief explanation of the drawings]

[0061] [Figure 1] Figure 2 shows the results of treating leg ulcers in patients with peripheral arterial disease (PAD) by injection of 1x107 Mregs. [Figure 2] The phenotype and specialized functional properties of human Mregs are described. [Figure 2A] Strong DHRS9 mRNA expression was detected by RT-qPCR in Mreg but not in comparative macrophage species (n = 6; mean ± SD). [Figure 2B] 1 shows the results of immunoblotting demonstrating that DHRS9 protein expression distinguishes Mregs from control macrophages. [Figure 2C] Shown is the expression of TNFSF14 / CD258 mRNA in a panel of comparative macrophages quantified by microarray analysis (n=3, mean±SD). [Figure 2D] Figure 1 shows cell surface expression of CD258 by human Mregs as detected by flow cytometry. [Figure 2E] An experimental system is illustrated to show that direct interaction of naive CD25-FoxP3-CD4+ T cells with human Mregs leads to their conversion into activated CD25+FoxP3+CD4+ T cells. [Figure 2F] Figure 1 shows iTregs generated from naive CD25-FoxP3-CD4+ T cells by interaction with human Mregs when co-cultured at a ratio of 1:2. [Figure 2G] IDO mRNA expression in a panel of comparative macrophages as quantified by microarray analysis (n=3, mean±SD) is shown. [Figure 2H]Figure 1 shows the expression of IDO by human Mregs detected by intracellular staining and flow cytometry. [Figure 2I] PAEP mRNA expression in a panel of comparative macrophages as quantified by microarray analysis (n=3, mean±SD) is shown. [Figure 2J] We show that neutralization of secreted PAEP protein using a neutralizing antibody against PAEP led to a significant decrease in iTreg generation. [Figure 2K] Shown is the expression of TGFβ1 mRNA in a panel of comparative macrophages quantified by microarray analysis (n=3, mean±SD). [Figure 2L] Figure 2 shows the secretion of TGFβ1 by human Mreg and control macrophages over 24 hours. [Figure 2M] Comparison of Mregs with PCMO on Treg induction is shown. [Example]

[0062] Mreg is manufactured in accordance with current GMP standards for the manufacture of sterile pharmaceuticals. Care is taken throughout the processing steps to protect the product, materials and equipment against contamination and unsanitary conditions.

[0063] Example 1 Preparation of Mregs The first Mreg preparation (" Mreg A ") were prepared based on Hutchinson's modified protocol. Healthy human donors underwent leukapheresis to collect peripheral blood mononuclear cells (PBMCs), which were used as starting material for Mreg generation. All donors were screened for markers of relevant diseases, including infection, for up to 30 days prior to leukapheresis. Donors were rescreened for the same disease markers on the day of leukapheresis. Leukapheresis was performed using a Terumo BCT Cobe Spectra device or similar device. CD14+ monocytes were isolated from the leukapheresis product using a Miltenyi CliniMACS® system according to the manufacturer's instructions.

[0064] The cells were cultured at a density of 35 × 10 in 30 ml of phenol red-free (Lonza)-based RPMI 1640 medium supplemented with 10% human AB serum (Lonza), 2 mM L-glutamine (Lonza), 100 U / ml penicillin, 10 μg / ml streptomycin (Lonza), and recombinant human M-CSF (RSCD Systems) diluted with 0.1% human serum albumin (Aventi) to a final concentration of 5 ng / ml. 6 Single ball / 175cm 2 Plastic-adherent monocytes were added to culture flasks (Cell+T175 flasks; Sarstedt). Cells were cultured for 6 days, with complete medium changes on days 1, 2, and 4. On day 6, cultures were stimulated with 25 ng / ml recombinant human IFN-γ (Imukin; Boehringer Ingelheim). On day 7, the adherent monocyte fraction was collected by trypsin-EDTA treatment (phenol red-free TrpE Express; Invitrogen) followed by careful scraping. Mregs from all flasks were pooled and resuspended in saline solution containing 5% human albumin.

[0065] The second Mreg preparation ( "Mreg B ") were prepared according to the protocol described in Example 1 of International Application No. PCT / EP2017 / 055839. Healthy human donors underwent leukapheresis to collect peripheral blood mononuclear cells (PBMCs) used as starting material for Mreg generation. All donors were screened for markers of relevant diseases, including infection, for up to 30 days prior to leukapheresis. Donors were rescreened for the same disease markers on the day of leukapheresis. Leukapheresis was performed using a Terumo BCT Cobe Spectra device or similar device.

[0066] CD14+ monocytes were isolated from the leukapheresis product using the Miltenyi CliniMACS® system according to the manufacturer's instructions. Briefly, the leukapheresis product was transferred to a bag filled with PBS / EDTA buffer containing 0.5% human serum albumin (HSA). According to the manufacturer's instructions, the cells were washed once before labeling with the CliniMACS® CD14 reagent. To isolate CD14+ monocytes by magnetic separation, the labeled cell suspension was connected to a sterile tubing set and introduced into the CliniMACS® instrument. The positively isolated CD14+ monocyte fraction was washed with medium to remove the CliniMACS® separation buffer. The cells were cultured at 10°C in RPMI medium supplemented with 10% human male AB serum (stored and heat-inactivated), 2 mM GlutaMAX™, and 25 ng / ml recombinant human monocyte colony-stimulating factor (M-CSF). 6 Isolated CD14+ monocytes were resuspended at a density of 1 x 10 cells / ml. Each bag contained 1 x 10 6 cells / cm 2 This monocyte suspension was dispensed into Miltenyi® cell differentiation bags so that the inner surface area was seeded. For culture, the differentiation bags were placed horizontally on a shelf in an incubator set at 36-38°C, 5±1% CO2, and 60% or higher humidity. After 1 day, the monocytes were allowed to settle on the underside of the culture bag. On day 1, the bag was inverted to allow the monocytes to adhere to the opposite side. Culture was continued in the incubator for an additional 5 days. To induce terminal differentiation of monocytes into Mregs and induce indoleamine 2,3-dioxygenase (IDO) expression, 25 ng / ml IFN-γ was added to stimulate the monocytes. After IFN-γ addition, the differentiation bag was inverted once more. The bag was then incubated for an additional 18-24 hours at 36-38°C, 5±1% CO2, and 60% or higher humidity. Differentiated Mregs were collected on day 7. Cells from all parallel culture bags were pooled and washed prior to phenotypic and functional analysis.

[0067] Example 2: Phenotypic characterization of Mregs The "Mreg A" cells obtained in Example 1 were tested for the expression of various markers. In particular, the expression of various markers potentially having specificity for Mreg was examined by RT-PCR.

[0068] DHRS9 We were able to demonstrate that DHRS9 expression distinguishes between monocyte- and dendritic cell-derived human Mregs. Strong DHRS9 mRNA expression was detected in Mregs but not in control macrophage species (n=6; mean ± SD). See Figure 4A. Immunoblotting with a custom-designed rabbit anti-DHRS9 pAb demonstrated that DHRS9 protein expression distinguishes between control macrophage-derived Mregs. See Figure 4B.

[0069] CD258 CD258 (TNFSF14) was shown to be an informative marker for human Mregs. TNFSF14 mRNA expression was detected in Mregs but not in control macrophages (n=3; mean±SD). See Figure 4C.

[0070] IDO Mregs have been shown to express IDO, whereas control macrophages do not express this marker. IDO expression by Mregs was detected by flow cytometry. See Figure 4D.

[0071] PAEP Although Mregs have been shown to express PAEP, control macrophages do not express this marker. Figure 4E shows that PAEP mRNA is expressed in a panel of control macrophages (n = 3; mean ± SD).

[0072] TGFβ1 Mregs have been shown to express TGFβ1, whereas control macrophages do not express this marker. Figure 4F shows that TGFβ1 mRNA is expressed in a panel of control macrophages (n = 3; mean ± SD).

[0073] Treg induction Human Mregs generated from peripheral blood CD14+ monocytes were co-cultured with allogeneic T cells at a 1:2 ratio for 5 days. T cells were then analyzed by flow cytometry and functional assays. T cells co-cultured with allogeneic Mregs for 5 days were enriched for T cell-suppressive CD25+FoxP3+ iTregs (Figure 4F), which were easily distinguished from CD25+FoxP3- / low polyclonal activated T cells generated by 5 days of stimulation with αCD3 / αCD28 beads. Mregs were compared with PCMO cells. The results are shown in Figure 2M. This demonstrates that the ability to induce Tregs is limited to Mregs.

[0074] Example 3 :Mreg for foot ulcer treatment A 78-year-old male patient with stage IIa peripheral arterial disease (PAD) in the right lower extremity and stage III peripheral arterial disease (PAD) in the left lower extremity had undergone multiple percutaneous transluminal angioplasty (PTA) procedures and bypass surgery of the common femoral artery (AFC) and superficial femoral artery (AFS), along with the left popliteal artery. He presented with severe pain during walking and at rest, especially at night.

[0075] The patient was treated with the Mreg preparation obtained from Example 1 ("Mreg A"). Specifically, 1 x 10 6 Mregs diluted in 12 ml of 5% human serum albumin were administered. 7 Mreg was injected into six injection sites along the outer portion of the ulcer, with 2 ml per injection site. If the muscle tissue under the wound could be reached, the injection was given intramuscularly; otherwise, the injection was given subcutaneously. No further treatment was administered thereafter. The wound was kept sterile with a dry dressing, which was changed daily.

[0076] resultClinical follow-up of the patient showed significant improvement in terms of wound closure over time. Clinically, nighttime pain completely disappeared without any ischemic pain at rest. This improvement was first observed 3 months after Mreg injection. Walking distance improved to over 2 km. Treatment results are shown in Figure 1.

[0077] Example 4 :Mreg for PAD treatment A 64-year-old female patient was diagnosed with diabetes mellitus with an extended arteriopathic component and peripheral artery disease (PAD) in the right lower extremity. She presented with a heel ulcer that exposed the bone. There was no longer any collateral circulation in the superficial femoral artery (AFS) or throughout the lower leg. The lack of connecting vessels in the leg precluded any operable intervention. Transfemoral amputation was considered a last resort.

[0078] The patient received the Mreg preparation obtained in Example 1 (" Mreg A A total of 50,000 Mregs were administered at five individually identified (angiographic) treatment points as described in Example 3. No further treatments were administered thereafter. The wounds were kept sterile and dressings were changed daily.

[0079] result Clinical follow-up of the patient demonstrated significant improvement in that the wound healed two weeks after injection. Three months later, wound healing was complete and circulation in the right leg was compensated. Angiographic evidence also demonstrated the formation of additional collateral circulation. Transfemoral amputation was avoided.

[0080] literature [1] Geissler EK, Hutchinson JA. Cell therapy as a strategy to minimize main tenance immunosuppression in solid organ transplant recipients. Curr Opin Organ Transplant 2013; 18: 408-15. [2] Tang Q, Bluestone JA, Kang SM. CD4(+) Foxp3(+) regulatory T cell therapy in transplantation. J Mol Cell Biol 2012; 4: 11-21. [3] Moreau A, Varey E, Bouchet-Delbos L, et al. Cell therapy using tolerogenic dendritic cells in transplantation. Transplant Res 2012; 1: 13. [4] Broichhausen C, Riquelme P, Geissler EK, et al. Regulatory macrophages as therapeutic targets and therapeutic agent in solid organ transplantation. Curr Opin Organ Transplant 2012; 17: 332-42. [5] Hutchinson JA, Riquelme P, Sawitzki B, et al. Cutting edge: immunological consequences and trafficking of human regulatory macrophages administered to renal transplant recipients. J Immunol 2011; 187: 2072-8. [6] Hutchinson JA, Riquelme P, Brem-Exner BG, et al. Tranplant acceptance- inducing cells as an immune-conditioning therapy in renal transplantation. Transpl Int 2008; 21: 728-41. [7] Hutchinson JA, Brem-Exner BG, Riquelme P, et al. A cell-based approach to the minimization of immunosuppression in renal transplantation. Transpl Int 2008; 21: 742-54. [8] Hutchinson JA, Roelen D, Riquelme P, et al. Preoperative treatment of a pre- sensitized kidney transplant recipient with donor-derived transplant acceptance-inducing cells. Transpl Int 2008; 21: 808-13. [9] Hutchinson JA, Govert F, Riquelme P, et al. Administration of donor-derived transplant acceptance-inducing cells to the recipients of renal transplants from deceased donors is technically feasible. Clin Transplant 2009; 23: 140 -5.

Claims

1. Immunomodulatory macrophages expressing the markers CD258, DHRS9 and IDO for use in a method of treating microvascular or macrovascular disorders of the lower extremities in a subject.

2. An immunomodulatory macrophage expressing the markers CD258, DHRS9 and IDO for use in a method of inducing wound healing in a subject.

3. 3. An immunomodulatory macrophage for use in the method of claim 1 or 2, wherein said macrophage expresses the markers CD258, DHRS9, IDO and TGFβ1.

4. An immunomodulatory macrophage for use in the method of any one of claims 1 to 3, wherein said macrophage expresses the markers CD258, DHRS9, IDO, TGFβ1 and PAEP.

5. An immunomodulatory macrophage for use in the method of any one of claims 1 to 4, wherein the macrophage expresses CD258, DHRS9, IDO, TGFβ1 and PAEP.

6. 6. An immunomodulatory macrophage for use in the method of any one of claims 1 to 5, wherein the macrophage further expresses at least one marker selected from the group consisting of macrophage lineage markers CD33, CD33, CD11b, and HLA-DR.

7. An immunomodulatory macrophage for use in the method according to any one of claims 1 to 6, wherein the microangiopathy or macroangiopathy is a diabetic microangiopathy or macroangiopathy.

8. An immunomodulatory macrophage for use in the method of any one of claims 1 to 7, wherein the microangiopathy or the macroangiopathy is a diabetic foot ulcer or a venous leg ulcer.

9. The method comprises administering 1×10 5 ~1 x 10 7 9. An immunomodulatory macrophage for use in the method of any one of claims 1 to 8, comprising administering macrophages of

10. 10. An immunomodulatory macrophage for use in the method of any one of claims 1 to 9, wherein the method comprises administering the macrophage directly to the ulcer by subcutaneous or intramuscular injection.

11. 10. The immunomodulatory macrophage for use in the method of any one of claims 1 to 9, wherein the immunomodulatory macrophage is selected from the group consisting of vasculitis, arteritis, vascular dysplasia, atrophy alba, scleroderma, Determann's syndrome, diabetic vasculopathy, endarteritis obliterans, erythromelalgia, fibromuscular dysplasia, perforation of the foot, Mönckeberg's medial calcific sclerosis, Osler's disease, compartment syndrome, Paget-von-Schroetter syndrome, Raynaud's disease, and foot ulcers.

12. 12. The immunomodulatory macrophage for use in the method of any one of claims 1 to 11, wherein the macrovascular disorder is selected from the group consisting of aneurysm, dissection, atherosclerosis, atherothrombosis, peripheral arterial occlusive disease (PAD), intermittent claudication, necrosis and gangrene, vascular malformation, Leriche syndrome, or compression syndrome.

13. An immunomodulatory macrophage for use in the method of any one of claims 1 to 12, wherein the subject suffers from diabetes.

14. A pharmaceutical composition for use in a method of treating microvascular or macrovascular disorders of the lower extremities in a subject, comprising immunoregulatory macrophages that express the markers CD258, DHRS9 and IDO.

15. A pharmaceutical composition for use in a method for inducing wound healing in a subject, comprising immunoregulatory macrophages that express the markers CD258, DHRS9 and IDO.

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