New immunoregulatory cells and methods for their production

Abstract

To provide methods for preparing novel immunoregulatory macrophage cells which are useful in the treatment of different immunological and non-immunological diseases and conditions; resulting macrophage cells; and pharmaceutical compositions comprising macrophage cells.SOLUTION: A method for preparing an immunoregulatory macrophage cell is provided, the method comprising: (a) isolating CD14 positive monocytes from a blood sample of a subject; (b) culturing the monocytes in a gas-permeable bag in a culture medium containing (i) M-CSF and / or GM-CSF, and (ii) a CD16 ligand; (c) contacting the cells with IFN-γ; and (d) obtaining the immunoregulatory macrophage cell from the culture medium.SELECTED DRAWING: Figure 10

Description

Technical Field

[0001] The present invention relates to novel immunomodulatory macrophage cells useful for treating a variety of immunological and non-immunological diseases and conditions. The cells are characterized by specific markers and patterns of activity that distinguish them from other cells. The present invention further provides a method for generating immunomodulatory macrophage cells from blood monocytes. In still other aspects, the present invention relates to pharmaceutical compositions comprising the immunomodulatory macrophage cells of the present invention or fractions thereof at the subcellular level. A method for generating fractions of the immunomodulatory macrophage cells of the present invention at the subcellular level is also provided.

Background Art

[0002] Transferring immunomodulatory cells from a tolerant donor to an intolerant recipient as a means of establishing tolerance in the recipient is a technique well known in experimental immunology, but its clinical application has only recently received serious attention [1]. At present, several immunomodulatory cell types, including regulatory T cells [2], tolerogenic dendritic cells [3] and regulatory macrophages [4], are reaching the preclinical development stage, which will allow them to be investigated in early trials as immunosuppressive agents.

[0003] A wide range of immunological conditions, including autoimmune diseases, chronic inflammatory disorders, graft-versus-host disease (GVHD) and transplant rejection mediated by T cells and B cells, may be treatable by cell-based immunomodulatory therapies. In these conditions, cell-based immunomodulatory therapies have the potential to reduce or even eliminate the need for systemic immunosuppressive or anti-inflammatory therapies and thereby rescue the patient from their associated complications. Because immunotolerance of the type supported by regulatory cells is dominant and self-sustaining, there is a possibility that cell-based immunomodulatory therapies will provide treatment options in diseases that would otherwise require long-term systemic immunosuppressive or anti-inflammatory therapies.

[0004] One particularly promising candidate cell type for use as an adjunct immunosuppressant in transplantation is the immunoregulatory macrophage (referred to herein and in the literature as "Mreg"). Mreg cells reflect a distinct state of macrophage differentiation and are distinguished from macrophages in other activated states by their robust phenotype and potent T cell inhibitor function [5]. Human Mregs potently suppress T cell proliferation by mitogen stimulation in vitro, which may be due to interferon (IFN)γ-inducible indoleamine 2,3-dioxygenase activity and contact-dependent activated T cell elimination. In addition, Mregs promote the development of activated induced regulatory T cells, which in turn suppress the proliferation of effector T cells and the maturation of dendritic cells. Thus, it is hypothesized that when Mregs are administered to a recipient, an immunoregulatory feed-forward loop is initiated, resulting in long-term immune tolerance of the allograft or prevention of immune pathology. In a series of case studies and two early-phase trials, Mreg-containing cell preparations were administered to a total of 19 kidney transplant recipients as a form of adjunct immunosuppressive treatment [5]–[9]. These pilot studies clearly demonstrate the validity of this technology for solid organ transplantation.

[0005] Another two living kidney transplant recipients are currently being treated with a more purified donor-derived Mreg at approximately 8.0×10 6 cells / kg [5]. These two patients have now been more than 6 years post-transplant, and renal function has been stable in their low-dose tacrolimus monotherapy as their sole maintenance immunosuppression. Further trials of Mreg therapy in living kidney transplantation are now under regulatory approval within the framework of the ONE Study (Clinicaltrials.gov: NCT02085629). This trial aims to treat 16 patients with donor-derived Mreg cells at a dose of 2.5×10 6 –7.5×10 6 cells / kg body weight, with the assistance of 500 mg / day of mycophenolate mofetil 7 days prior to surgery.

[0006] Despite the great progress made in recent years in the field of immunomodulatory cells, there remains a need for regulatory cells that can be used for therapeutic purposes, for example, to induce immune tolerance of foreign grafts in a recipient, and for methods of producing these cells in the most efficient manner. In particular, there is a need for cell-based therapies that can reduce the commonly used immunosuppressive medications, which are typically associated with high toxicity to patients.

Summary of the Invention

[0007] The present invention provides a novel class of Mreg cells that are significantly different from previously described Mregs. A modified method for producing Mreg cells was unexpectedly found to give rise to a novel class of Mreg cells. Specifically, the inventors found that when monocytes used to produce Mreg cells were cultured in a gas-permeable bag instead of a culture flask, Mreg cells with immunomodulatory properties that make them highly suitable for cell-based therapy approaches were obtained. These cells are referred to herein as "Mreg-bc" to distinguish them from known Mregs.

[0008] Thus, in a first aspect, the present invention relates to a method of producing a novel class of macrophages, comprising culturing monocytes from a blood sample of a subject in a gas-permeable bag in the presence of M-CSF / GM-CSF, CD16 ligand (e.g., immunoglobulin), and IFN-γ.

[0009] In a second aspect, the present invention relates to a novel class of Mreg cells, namely Mreg-bc cells, that can be obtained by the method referred to in the first aspect of the present invention. Mreg-bc cells have a unique phenotype not seen in the prior art. Mreg-bc cells mediate biological activities that are specific to this cell type and result in useful therapeutic properties not described in the prior art.

[0010] In a third aspect, the present invention relates to a pharmaceutical composition comprising Mreg-bc cells according to the second aspect of the present invention or a fraction below the cellular level of said cells. The pharmaceutical composition containing the novel cell type of the present invention may further contain additional active ingredients or excipients as required.

[0011] In a fourth aspect, the present invention relates to the use of Mreg-bc cells according to the second aspect of the present invention, a fraction below the cellular level thereof, or a pharmaceutical composition according to the third aspect of the present invention for therapeutic purposes, particularly for the suppression of harmful immune reactions.

[0012] In a fifth aspect, the present invention relates to a method for producing a fraction below the cellular level of Mreg-bc cells according to the second aspect of the present invention by decomposing Mreg-bc cells under appropriate conditions.

[0013] Finally, in a sixth aspect, the present invention relates to a method for producing immunoregulatory T cells by culturing T cells from a blood sample of a subject together with Mreg-bc cells according to the second aspect of the present invention or a fraction below the cellular level thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0014]

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Mode for Carrying Out the Invention

[0015] According to the present invention, Mreg-bc cells are derived from human CD14+ blood monocytes. To elicit the characteristic biological properties of Mreg-bc cells, monocytes are treated with a specific combination of growth factors, cytokines, and receptor ligands. The cells obtained by the method of the present invention are characterized by a unique phenotype that distinguishes them from blood monocytes, other classes of monocyte-derived macrophages, monocyte-derived dendritic cells, and other inhibitory bone marrow monocyte cell products described in the prior art.

[0016] Thus, in a first aspect, the present invention is a method for generating a novel class of immunomodulatory macrophage cells, comprising: (a) isolating CD14-positive monocytes from a blood sample of a subject; (b) culturing the monocytes in a gas-permeable bag 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 immunomodulatory macrophage cells from the medium and related to a method comprising.

[0017] The method of the present invention uses blood monocytes as starting materials. Although it would be preferable to use the method of the present invention to produce Mreg-bc cells from human blood monocytes, the present invention is not limited to the differentiation of cells derived from humans. In fact, the present invention is also applicable to other types of non-human cells, especially vertebrate cells, such as non-human primate or porcine cells. Thus, the present invention makes an important contribution to the field of xenograft transplantation medicine.

[0018] According to a preferred embodiment, the method of the present invention is used to differentiate CD14-positive monocytes of a human donor into Mreg-bc. The monocytes serving as the starting material for the method of the present invention are obtained from the peripheral blood of a human donor. The donor can be a healthy subject or a patient suffering from one or more diseases. In one embodiment, the monocyte donor is the recipient of the intended differentiated Mreg-bc cells (autologous approach). In another embodiment, the monocyte donor is a person separate from the recipient of the intended differentiated Mreg-bc cells (allogeneic approach). In the latter case, the donor and the recipient may or may not be genetically related. In another embodiment, the monocyte donor is a person separate from the recipient of the intended differentiated Mreg-bc cells but is also the donor of other cells, tissues or organs transplanted into the same recipient. The preferred relationship between the donor and the recipient depends on the clinical use. The use of autologous Mreg-bc cells can help avoid certain adverse reactions. Therefore, it is preferable to use autologous Mreg-bc cells in the case of regenerative therapy or anti-inflammatory therapy. In the context of transplantation, it is preferable to use donor-derived Mreg-bc cells as immunosuppressive therapy because donor antigen-expressing cells are more effective than recipient-derived cells

[11] .

[0019] For the enrichment of monocytes from peripheral blood, various methods are known in the art, and each of these methods can be used in the context of the present invention. For example, blood obtained by venipuncture can be treated with an anticoagulant and subsequently separated by using a separation medium, such as Ficoll-Paque Plus. For this purpose, the anticoagulant-treated blood sample is layered on the Ficoll-Paque Plus solution and centrifuged, whereby layers containing various cell types are formed. The bottom layer contains red blood cells 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. Monocytes and lymphocytes are found at the interface between plasma and Ficoll-Paque Plus due to their lower density. Enrichment of the mononuclear cell fraction can be achieved by isolation of the layer followed by washing and centrifugation.

[0020] Another conventionally used method for separating mononuclear leukocytes from a blood sample involves a leukapheresis method. The leukapheresis method is a specific 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 the selected leukocyte fraction and returns the remaining blood cells and plasma to the donor. The leukapheresis method is, today, a conventional clinical means for obtaining leukocytes or stem cells from peripheral blood. Various devices that can be used to perform the leukapheresis method in the context of the present invention are available from several manufacturers. For example, there is the COBE® Spectra Apheresis System from Terumo BCT. When performing the leukapheresis method by using the COBE® Spectra Apheresis System, it is preferable to use the operating instruction protocol provided by the manufacturer because it has been found that this protocol yields monocytes of better quality compared to the AutoPBSC protocol.

[0021] The use of a separation medium such as Ficoll-Paque Plus and the use of a leukocyte removal device both result in a cell fraction containing not only monocytes but also lymphocytes. According to the present invention, monocytes can be concentrated and separated from lymphocytes by known methods, such as magnetic bead separation, sorting by flow cytometry, purification, filtration, or plastic adherence, before introducing the cells into the production method of the present invention. However, it is not essential to use a uniform monocyte fraction in the method of the present invention. In fact, the presence of lymphocytes in an amount of 0.1 to 20%, preferably 10 to 20%, in the monocyte fraction can have a beneficial effect on the differentiation of monocytes into regulatory macrophages.

[0022] In one embodiment of the present invention, the monocyte fraction used in the method of the present invention is essentially pure and contains non-monocytic nucleated blood cells, such as lymphocytes or granulocytes, at a rate of less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. To obtain a mononuclear cell preparation enriched in monocytes, peripheral blood mononuclear cells may be contacted with CD14 microbeads that bind, for example, CD14-positive monocytes. In one embodiment, the monocytes in step (a) are isolated by a leukocyte removal method and subsequently subjected to a separation step using a CD14 affinity molecule, preferably a CD14 antibody. Such a purification step greatly reduces the contamination of the starting material by non-monocytes. The reduction of T cell contamination is highly valuable from the perspective of patient safety, as it minimizes the potential risk of donor-to-recipient reactions. In a preferred embodiment of the present invention, the CD14 monocytes used in the method of the present invention are isolated by CliniMACS® technology (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany).

[0023] The monocyte fraction isolated by leukocyte removal method and / or other methods can be used directly for differentiation by incubating with M-CSF and / or GM-CSF and CD16 ligand, or it can be stored in an anticoagulant citrate dextrose solution (ACD-A) or autologous plasma supplemented with any other suitable buffer until further use. If the isolated monocyte fraction has to be transported to another location for carrying out the differentiation process, consideration has to be given to initiate cell differentiation by incubation with M-CSF / GM-CSF within 24 hours, preferably within 18 hours, 12 hours, 6 hours, 4 hours or 2 hours from cell isolation, preferably from monocyte isolation. For long-term storage, the monocyte fraction may be resuspended in a suitable cryopreservation solution and stored for a long time at a temperature below 20°C, preferably below 80°C.

[0024] After isolating 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, it is also possible to add M-CSF / GM-CSF and CD16 ligand after a certain period from the start of cell culture. The medium used in step (b) of the above method may be any medium described in the literature as suitable for the culture of monocytes and / or macrophages. Suitable media include, for example, PromoCell macrophage generation medium (PromoCell GmbH, Heidelberg, Germany), Dulbecco's modified Eagle's medium (DMEM), DMEM:F12 mixture, Medium199, 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, such as 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; non-specific immunoglobulins and other plasma proteins. In a preferred embodiment of the present invention, the medium is RPMI-1640 or a medium derived therefrom.

[0025] The medium used to incubate isolated CD14-positive monocytes contains 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 affects the proliferation, differentiation, and survival of monocytes, macrophages, and bone marrow progenitor cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF, also known as CSF2) functions as a cytokine and is a monomeric glycoprotein 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 for purchase from various manufacturers. The selection of M-CSF and / or GM-CSF used in the methods of the present invention will depend on the origin of the monocytes to be differentiated into Mreg-bc cells. For example, if human monocytes are to be differentiated into Mreg-bc using the methods described herein, 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. In a particularly preferred embodiment of the present invention, M-CSF and / or GM-CSF are of human origin, such as recombinant human M-CSF and / or GM-CSF, and the monocytes are human monocytes.

[0026] One skilled in the art would be able to find the amounts of M-CSF and / or GM-CSF suitable for differentiating a high proportion of monocytes into Mreg-bc by conventional methods. Generally, the concentration of M-CSF in the medium of step (b) of the above method ranges from 1 to 100 ng of protein per ml of medium. Pilot 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 of M-CSF contained concentrations below physiological levels by day 2 of culture, whereas cultures using an initial dose of 25 ng / ml of M-CSF maintained concentrations above 10 ng / ml throughout the 7-day culture period. From this, in a preferred embodiment of the present invention, the concentration of M-CSF in the medium ranges from 20 to 75 ng / ml, 20 to 50 ng / ml or 20 to 25 ng / ml. A concentration of at least 25 ng of M-CSF per ml of medium is particularly preferred. The above concentrations preferably refer to recombinant human M-CSF.

[0027] When using GM-CSF instead of M-CSF, the same concentrations as outlined above for M-CSF can be used in the medium. Since GM-CSF appears to be more potent than M-CSF, a concentration of GM-CSF in the range of 0.1 to 100 ng of protein per ml of medium is suggested in the present invention. When using both M-CSF and GM-CSF in the medium, the total concentration of these two growth factors will be within the above ranges, i.e., in the range of 20 to 75 ng / ml, 20 to 50 ng / ml or 20 to 25 ng / ml. It is particularly preferred that the total concentration of M-CSF and GM-CSF is 25 ng of M-CSF per ml of medium.

[0028] The medium used in step (b) of the above method contains CD16 ligand in addition to M-CSF and / or GM-CSF. It has been found that stimulation of the CD16 cell surface receptor on monocytes is necessary to induce their differentiation into Mreg-bc cells. More specifically, from the experiments conducted in the course of the present invention, it has become clear that monocytes grown in a medium supplemented with human AB serum (HABS) develop into Mregs, while monocytes grown in a medium supplemented with fetal calf serum (FCS) do not develop into Mregs. Monocytes grown in an equal mixture of both sera develop the Mreg phenotype. Therefore, HABS clearly contains Mreg-inducing activity (see A and B of Figure 1). Removal of the chloroform-extractable fraction of HABS demonstrated that the Mreg-inducing activity of HABS was mainly present in the chloroform-resistant fraction and was therefore likely to be a protein (see B and C of Figure 1). By size fractionation, it was found that the major protein component of HABS responsible for Mreg-bc development was greater than 100 kDa, leading to the hypothesis that the unknown factor was an immunoglobulin (Ig). HABS from which Ig had been removed using protein A / G sepharose was unable to support the development of the Mreg-bc morphology and DHRS9 mRNA expression (see Figure 1D). Readding purified Ig back to the Ig-depleted serum (or adding IVIg) restored its ability to induce DHRS9 expression (see Figure 1D). Similarly, when monocytes were cultured in FCS supplemented with human Ig, an increase in DHRS9 mRNA expression was observed compared to the control of FCS alone, and a normal Mreg-bc morphology was acquired (see D and E of Figure 1). Monocytes treated with anti-FcγRIII antibody expressed DHRS9 mRNA at a significantly lower level compared to monocytes treated with anti-FcγRI (CD64), anti-FcγRIIa / b (CD32a / b) or control antibody (see Figure 1F) and did not develop the Mreg-bc morphology (see Figure 1G). Blocking FcγRIIb or DC-SIGN alone or both receptors together resulted in DHRS9 +This had no effect on the generation of Mregs (see Fig. 1H). To reinforce the view that FcγRIII is required for Mreg-bc generation, siRNA was used to silence FcγRIII expression (see Fig. 1I). In freshly isolated monocytes cultured in 10% HABS, transient suppression of FCGR3A and FCGR3B transcript expression was achieved, and importantly, FCGR2B expression was not decreased by this manipulation. Knockdown of FcγRIII at the protein level was demonstrated by flow cytometry (35.2% ± 4.4 CD16 + cells with negative control siRNA versus 15.3% ± 3.7; n = 4, p = 0.002 with FCGR3 siRNA). Silencing of FcγRIII expression (but not suppression of MAPK1 expression or treatment with negative control siRNA) resulted in significant downregulation of DHRS9 mRNA expression (see Fig. 1I).

[0029] From the above findings, it was concluded that serum Ig acts to induce the Mreg phenotype via FcγRIII (CD16). The dependence of Mreg differentiation on FcγRIII distinguishes Mregs from other Ig complex-induced macrophages described in the prior art. In particular, the mode of induction distinguishes FcγRIII-induced Mregs from FcγRIIb-induced macrophages, FcγRI-induced macrophages, and macrophages generated in the absence of immunoglobulins described in the prior art.

[0030] Since stimulation of the CD16 cell surface receptor is crucial for differentiation into the desired Mreg-bc phenotype, the method of the present invention involves incubating monocytes with a CD16 ligand in step (b). The ligand that binds to the receptor will preferably be a human or non-human immunoglobulin, more preferably a human immunoglobulin or a fragment thereof. The immunoglobulin fragment can be, for example, the Fc fragment of an immunoglobulin. The immunoglobulin or immunoglobulin fragment is preferably added to serum-free medium. Alternatively, a recombinant protein containing the sequence of an immunoglobulin or immunoglobulin fragment, for example, the sequence of a human immunoglobulin, may be used. In another embodiment, to promote Mreg-bc differentiation, a non-human or human antibody or fragment thereof that specifically binds to CD16 by an antigen recognition domain is used. In yet another embodiment, to promote Mreg-bc differentiation, small molecules are used to stimulate the CD16 signaling pathway.

[0031] In a preferred embodiment, the medium used to generate Mreg-bc cells contains 1-20% human serum or an equivalent amount of specific serum components, such as immunoglobulins. More preferably, the medium is supplemented with 10% serum. When using a serum-containing medium to carry out the method of the present invention, the medium contains 5-15%, preferably 10% human serum. A medium containing 10% human AB serum is particularly preferred. In other words, the serum is preferably added at a concentration of about 0.01-10 mg / ml, preferably about 0.1-1 mg / ml, more preferably about 1 mg / ml. When using immunoglobulins or immunoglobulin fragments as the CD16 ligand, a slightly lower concentration can be used. When immobilizing immunoglobulins or other CD16 ligands on the tissue culture surface, beads or other physical substrates, substantially lower concentrations may be used. It is even more preferred that the human serum, such as AB serum, is derived from male donors. When using serum from female donors, consideration must be given to the fact that the donor is not using progesterone contraceptives or progesterone-estrogen contraceptives. It is further preferred that the above medium does not contain monocytes or Mreg-bc cells, or any intermediate morphological forms, such as antibodies against major histocompatibility molecules.

[0032] It has also been found that the antibodies in the medium did not have any measurable effect on the viability, yield, phenotype or inhibitory function of Mreg-bc produced by the method of the present invention. Therefore, it is preferred that the medium used in step (b) of the method of the present invention does not contain any antibiotics.

[0033] When Mreg-bc cells are intended for use in therapeutic applications (see below) where induction of angiogenesis is desired, the medium used to culture monocytes in step (b) of the method of the present invention may contain toll-like receptor (TLR) ligands, such as lipopolysaccharide (LPS), monophosphoryl lipid A (MPLA) or High Mobility Group Box protein 1 (HMGB1), in addition to M-CSF and / or GM-CSF and CD16 ligand to enhance the production of angiogenic factors such as VEGF-A. The TLR ligand may be added to the medium at a concentration range of 1000 ng / ml to 1 μg / ml, preferably 50 to 500 ng / ml, for example 100 ng / ml, 200 ng / ml, 300 ng / ml, or 400 ng / ml. When adding one or more TLR ligands, the total concentration of these ligands must be within the above range. The TLR ligand may be added at any stage of the manufacturing method. It may be present in the initial medium used to culture monocytes, i.e., on day 0 of the culture, or it may be added at a later stage, such as on day 5, 6 or 7 of the culture. Preferably, the TLR ligand is added simultaneously with the addition of IFN-γ.

[0034] According to the present invention, a method for producing Mreg-bc cells includes culturing monocytes in a gas-permeable bag in the presence of M-CSF / GM-CSF and CD16 ligand. As soon as the monocytes are suspended in an appropriate medium, the cell suspension is transferred to a gas-permeable bag for culture and differentiation. Bags for cell culture can be obtained from various suppliers, such as Miltenyi Biotec GmbH (Bergisch Gladbach, Germany), Thermo Fisher Scientific (Schwerte, Germany), or Merck (Darmstadt, Germany). The bag will be made of a material that allows the adhesion of cultured cells to the inner surface of the culture bag. Bags made of plastic, such as bags made of polyolefin or polyethylene, are preferred.

[0035] The bag has an internal culture area of 1 cm 2 per 1 to 2 × 106 It would be preferable to be designed to allow for a single-cell seeding density. This means that a cell suspension containing 180 × 10 2 individual monocytes is preferably cultured in a bag having an internal surface area that is at least 90 cm 2 and not more than 180 cm 6 . The optimal density of cells in the suspension is preferably about 1 × 10 5 cells / ml to 1 × 10 7 cells / ml, more preferably 1 × 10 6 cells / ml. The ratio of the cell suspension volume to the bag volume is at least 1.0, preferably 0.2, more preferably 0.06, in order to minimize the amount of medium that must be concentrated with Mreg-bc at the end of the culture. This means that a cell suspension of 1 L or less, preferably 600 ml or less, more preferably 180 ml or less, is filled into a 3 L culture bag. In a preferred embodiment of the method of the present invention, the volume of the bag used for culturing monocytes in a medium supplemented with M-CSF / GM-CSF and CD16 ligand is at least 3 L.

[0036] After transferring the monocytes to the culture bag, prior to IFN-γ stimulation, the cells are incubated in the bag for at least 3 days in the presence of M-CSF / GM-CSF and CD16 ligand, such as human serum or human immunoglobulin. As used herein, a "1-day" culture period refers to a 24-hour culture. Thus, a "at least 3-day" culture period refers to a culture of 72 hours or more. The optimal period of IFN-γ stimulation is at least 12 hours, preferably 18 hours, more preferably 24 hours. According to the present invention, the total culture period, i.e., the period from introducing the monocytes into the culture bag until collecting Mreg-bc, is at least 4 days, but preferably at least 5 days, at least 6 days, at least 7 days, or at least 8 days. In other words, the total culture period is 4 to 8 days, preferably 6 to 8 days, more preferably 7 days. The monocytes in the culture bag are incubated under conditions that allow them to grow and differentiate into Mreg-bc cells. General conditions for culturing monocytes or macrophages are known to those skilled in the field of cell culture.

[0037] For example, a bag containing a suspension can be transferred to an incubation chamber that allows selection of defined temperature, humidity and CO 2 conditions. Suitable temperature conditions are in the range of 30 to 40 °C, preferably 32 to 38 °C, more preferably 37 to 38 °C, for example 37 °C. The humidity used for culturing is usually in the range of 30 to 70%, preferably 40 to 60%, more preferably 50 to 60%, for example 60% humidity. The incubation chamber may contain 10% or less CO 2 . A content of 5% or less CO 2 , 4% or less CO 2 , 3% or less CO 2 , 2% or less CO 2 , or 1% or less CO 2 is particularly preferred. During incubation, the bag is preferably placed flat on the shelf of the incubation chamber.

[0038] The monocytes in the bag are preferably gently agitated from time to time to allow their semi-adherent adhesion to the lower surface of the culture bag. Preferably, the bag is inverted at least once during the entire culture period to allow their adhesion to the opposite surface of the bag. In another embodiment, the bag is inverted at least twice during the entire culture period. In another embodiment, the bag is inverted at least three or four times during the entire culture period. In yet another embodiment, the bag is inverted every 24 hours during the entire culture period. In another embodiment, the bag is inverted every 36 hours during the entire culture period. In yet another embodiment, the bag is inverted every 48 hours during the entire culture period.

[0039] In step (c) of the method of the present invention, the cells are contacted with the cytokine interferon gamma (IFN-γ). It is known in the art that cytokines can alter the transcription of more than 30 genes, thereby producing various physiological and cellular responses. IFN-γ proteins have been isolated from various species and can be purchased from various manufacturers. The selection of IFN-γ to be used in the method of the present invention will depend on the origin of the monocytes to which the method of the present invention is applied. For example, if the method described herein is used to differentiate human monocytes into Mreg-bc, the IFN-γ to be added will be human IFN-γ, preferably recombinant human IFN-γ. Similarly, if porcine monocytes are used in the differentiation method, the IFN-γ added to the medium will be of porcine origin. In a particularly preferred embodiment of the present invention, IFN-γ is human IFN-γ, more preferably recombinant human IFN-γ.

[0040] Any amount of IFN-γ effective to induce the expression of indoleamine 2,3-dioxygenase (IDO) by monocytes in the culture may be added. Preferably, the amount of IFN-γ to be added to the monocyte culture will be in the range of 5-100 ng / ml, more preferably 10-80 ng / ml, even more preferably 20-50 ng / ml. In the present invention, an amount of IFN-γ of 25 ng per 1 ml of medium is particularly preferred.

[0041] IFN-γ can be added to the medium simultaneously with M-CSF / GM-CSF and the CD16 ligand, which means that the cytokine can be added, for example, when introducing monocytes into the culture bag. In such embodiments, the monocytes differentiated by the method of the present invention will be cultured in the presence of M-CSF / GM-CSF, the CD16 ligand, and IFN-γ throughout the culture period. However, it is preferred that the culture period in the presence of IFN-γ is considerably shorter than the culture period in the presence of M-CSF / GM-CSF, which means that IFN-γ is added only after culturing the cells in the presence of M-CSF / GM-CSF for at least 3 days. In a preferred embodiment, IFN-γ is added after culturing the cells in the presence of M-CSF / GM-CSF for 3 to 6 days. Preferably, the cells are cultured in the presence of M-CSF / GM-CSF for at least 3 days, at least 4 days, at least 5 days, or at least 6 days before adding IFN-γ. In a particularly preferred embodiment, the cells are cultured in the presence of M-CSF / GM-CSF for 3 to 6 days, then IFN-γ is added, and the culture is continued for an additional 18 to 72 hours.

[0042] In a particularly preferred embodiment of the present invention, the differentiated cells are collected on the 7th day, for example, after subjecting them to IFN-γ stimulation for 18 to 24 hours following 6 days of culturing monocytes in a medium containing M-CSF / GM-CSF and the CD16 ligand. When several bags are cultured in parallel, the contents of the bags may be combined at the end of the culture process. The differentiated macrophages may be washed with a buffer suitable for use with macrophages. For example, Ringer's solution or phosphate-buffered saline (PBS), preferably supplemented with 5% human serum albumin, can be used to wash the cells by continuously exchanging the buffer by centrifugation and decantation of the supernatant. In the process of the present invention, it has been found that the use of trypsin does not improve the yield of immunomodulatory macrophages. Therefore, in the present invention, it is preferred not to include the addition of trypsin in the collection step.

[0043] These Mreg-bc cells can be transferred and stored in a blood transfusion bag, a glass drip apparatus, or another closed-system container that enables cell transfer to the treatment facility or beside the patient's bed. For this purpose, the differentiated cells will be suspended in an appropriate storage medium. The storage medium can be, for example, a Ringer's solution preferably supplemented with 5% human serum albumin. In a particularly preferred embodiment, the storage medium is a ready-made medium that is serum-free and / or protein-free. A suitable commercially available ready-made medium is HypoThermosol® FRS (Stemcell Technologies SARL, Cologne, Germany). Preferably, the pH of the medium is 6.5 to 8.0, more preferably 7.0 to 7.5, for example 7.4. The cell suspension must be stored at 4°C to minimize energy consumption and cell attachment. Alternatively, the Mreg-bc cells can be resuspended in a cryopreservation solution and stored in frozen form until final use.

[0044] The phenotypic and functional stability of the differentiated macrophage cells of the present invention depends on the choice of excipients and storage temperature. When resuspended in a Ringer's solution supplemented with human serum albumin, the macrophages of the present invention are stable at 20 to 25°C for up to 24 hours after cell collection. When resuspended in HypoThermosol® FRS, the macrophages can be stored at 2 to 8°C, preferably 4°C, for at least 72 hours after cell collection. If a longer storage period is required, the cells may be subjected to freezing or cryopreservation. Generally, the cells of the present invention have been found to be stable in their immunosuppressive phenotype. Treatment with pro-inflammatory mediators, such as lipopolysaccharide, does not promote the development of an activated phenotype.

[0045] The method for producing Mreg-bc cells of the present invention can be automated by using a GMP-compliant infrastructure that provides an integrated solution for streamlining the workflow of cell processing, for example, according to a general method. The method preferably occurs in a "closed system" that utilizes sealed consumables, custom production of tube sets, buffers, and reagents, multiple inflow lines equipped with sterile filters, optional outflow lines for in-process control, and substantially reduced cleanroom requirements. For example, the infrastructure may be equipped with a cell separation system that enables the separation of monocytes from the leukocyte fraction. The cell separation system must be able to separate monocytes from an apheresis fluid or whole peripheral blood with an initial volume of 100 to 1000 ml. Thereafter, the monocytes contained in the isolated mononuclear leukocyte fraction can be isolated, for example, by magnetic beads that bind to CD14+ cells. These cells are then cultured in an appropriate medium. The infrastructure enables the supply of medium, growth factors, and / or cytokines to the cell culture via multiple inflow ports. At the end of the culture process, the cells are automatically washed, collected, and transferred into an appropriate sterile delivery bag. Custom-produced tube sealants that enable the aseptic sealing of PVC and EVA tubes may be used. The cellular product may be barcoded, and the entire process by the manufacturer may be monitored online for quality control purposes.

[0046] In a second aspect, the present invention relates to a new class of Mreg cells, designated Mreg-bc cells, which can be obtained by the method of the first aspect of the present invention. The cells provided by the present invention are monocyte-derived human macrophages and thus express common leukocyte markers and macrophage lineage markers, in particular CD45, CD11b, CD33, and HLA-DR. Mreg is a group of lineage markers and activation markers - namely, CD14 - / low CD16 - / low CD80 - / low CD86 + CD85h + CD258 +(See Figure 2A) It is distinguished from monocytes, a panel of comparison macrophages (i.e., resting macrophages, M1, M2a, and M2c macrophages), and monocyte-derived DCs. CD85h is expressed in Mregs and monocytes, but its expression is lost in resting macrophages, M1 macrophages, M2a macrophages, M2b (stimulated by Ig complex) macrophages, M2c (treated with dexamethasone) macrophages, and monocyte-derived dendritic cells. CD258 is expressed in Mregs and M2b macrophages, but it is not expressed in monocytes, resting macrophages, M1 macrophages, M2a macrophages, M2c (treated with dexamethasone) macrophages, and monocyte-derived dendritic cells.

[0047] When comparing Mreg-bc, i.e., cells cultured in a bag, with Mregs cultured in a flask under otherwise equivalent conditions, Mreg-bc always expresses lower levels of CD14, CD16, and CD80 than flask-cultured cells. Mreg-bc always expressed higher levels of CD85h and CD258 than flask-cultured Mregs. Mreg-bc can be distinguished from those cultured in a flask by the expression of the markers Clec-9a, CD10, and CD103 (see Figure 2B). Alternatively, in contrast to general Mregs, Mreg-bc does not express (or only expresses at low levels) the markers CD38, CD209, and syndecan-3 (see Figure 2C). Characteristically, regardless of whether they are cultured in a bag or in a flask, all human Mregs express relatively high levels of DHRS9, a retinol dehydrogenase of the SDR family of retinol dehydrogenases (see Figure 3). Regardless of whether they are cultured in a bag or in a flask, single human Mreg cells simultaneously express both indoleamine 2,3-dioxygenase (IDO) and arginase-1 (Arg1), which are not found in other classes of monocyte-derived macrophages described in the prior art (see Figure 4).

[0048] Accordingly, the present invention provides Mreg-bc cells that do not express (or express at low levels) one or more of the markers CD38, CD209, and syndecan-3. As used herein, a cell is negative for a particular surface marker if the fluorescence intensity of the cell, as measured by flow cytometry, is less than the fluorescence intensity at the 99th percentile of the corresponding isotype control stained sample. Preferably, the Mreg-bc cells of the present invention are negative for CD209. The Mreg-bc cells are further either negative for CD38 or express low levels of CD38. During the generation of Mreg-bc cells, the initial population of monocytes downregulates CD38 expression on the cell surface. The downregulation of CD38 during Mreg-bc development can be expressed as the percentage of CD38 expression in Mreg-bc cells on day 7 compared to monocytes on day 0 (d0) of culture. The expression of CD38 is proportional to the difference in mean fluorescence intensity between the isotype control stained cells and the specific CD38 signal. Thus, the downregulation % = 100 - 100×(CD38 d7 -Iso d7 ) / (CD38 d0 -Iso d0 ), where CD38 d7 is the specific signal on day 7; Iso d7 is the isotype control signal on day 7; CD38 d0 is the specific signal on day 0; and Iso d0 is the isotype control signal on day 0. The downregulation of CD38 by Mreg-bc cells can be readily determined using standard flow cytometry methods. According to a preferred embodiment, the expression of CD38 by Mreg-bc cells is downregulated by more than 50%, more preferably more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or more than 99% compared to the initial expression of CD38 by monocytes on day 0 of culture.

[0049] Similarly, while generating Mreg-bc cells, an initial population of monocytes acquires low-level syndecan-3 expression on the cell surface, whereas Mreg cells cultured in a flask acquire higher-level syndecan-3 expression on the cell surface. Thus, the differentiated Mreg-bc cells obtained by the method of the present invention do not express the marker syndecan-3 or express the marker at only a relatively low level. The expression of syndecan-3 by Mreg-bc cells can be represented in relation to the syndecan-3 expression in flask-cultured Mreg. The expression of syndecan-3 is proportional to the difference in mean fluorescence intensity between isotype control-stained cells and specific syndecan-3 signals. Thus, the expression % = (syndecan-3 Mreg-bc -Iso Mreg-bc ) / (syndecan-3 flask - Iso flask), where syndecan-3 Mreg-bc is the specific signal of syndecan-3-stained Mreg-bc cells on day 7; Iso Mreg-bc is the signal of isotype control-stained Mreg-bc cells on day 7; syndecan-3 flask is the specific signal of syndecan-3-stained flask-cultured Mreg cells on day 7; and Iso flask is the signal of isotype control-stained flask-cultured Mreg cells on day 7. The relative expression of syndecan-3 by Mreg-bc cells and flask-cultured Mreg can be readily determined using standard flow cytometry methods. According to a preferred embodiment, the relative expression of syndecan-3 by Mreg-bc cells when compared to flask-cultured Mreg (expressed as a percentage) is less than 50%, more preferably less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1%.

[0050] Preferably, the Mreg-bc cells of the present invention express at least one, more preferably both, of the markers CD85h and CD258. Mreg-bc The cells preferably express one or more of the markers Clec-9a, CD103, and CD10. In other words, the present invention provides Mreg-bc cells that express one or more of the markers Clec-9, CD103, and CD10. Preferably, the cells express at least one, more preferably both, of the markers CD85h and CD258. The Mreg-bc cells further preferably do not express (or express only at a relatively low level one or more of these markers) one or more of the markers CD38, CD209, and syndecan-3. In a particularly preferred embodiment, the Mreg-bc cells provided herein do not express the markers CD38, CD209, and syndecan-3, and at the same time express the markers CD85h, CD258, Clec-9, CD103, and CD10. Thus, the Mreg-bc cells provided by the method of the present invention are macrophages that can be represented by one of the following marker patterns: (1). CD45 + , CD85h + , CD38 - / low ; (2). CD45 + , CD85h + , CD209 - / low ; (3). CD45+, CD85h+, syndecan 3 - / low ; (4). CD45 + , CD258 + , CD38 - / low ; (5). CD45 + , CD258 + , CD209 - / low ; (6). CD45+, CD258 + , syndecan 3 - / low ; (7). CD45 + , CD85h + , CD258 + , CD38 - / low ; (8). CD45 +, CD85h + , CD258 + , CD209 - / low ; (9). CD45+, CD85h+, CD258 + , Syndecan 3 - / low ; (10). CD45 + , CD85h + , CD258 + , CD38 - / low , CD209 - / low ; (11). CD45 + , CD85h + , CD258 + , CD38 - / low , Syndecan 3 - / low ; (12). CD45 + , CD85h + , CD258 + , CD209 - / low , Syndecan 3 - / low ; (13). CD45 + , CD85h + , CD258 + , CD38 - / low , CD209 - / low , Syndecan 3 - / low ; (14). CD33 + , CD85h + , CD38 - / low ; (15). CD33 + , CD85h + , CD209 - / low ; (16). CD33+, CD85h+, Syndecan 3 - / low ; (17). CD33+ CD258 + , CD38 - / low ; (18). CD33 + , CD258 + , CD209 - / low ; (19). CD33+, CD258 + , Syndecan 3 - / low ; (20). CD33 + 、CD85h + 、CD258 + 、CD38 - / low ; (21). CD33 + 、CD85h + 、CD258 + 、CD209 - / low ; (22). CD33+, CD85h+, CD258 + 、Syndecan 3 - / low ; (23). CD33 + 、CD85h + 、CD258 + 、CD38 - / low 、CD209 - / low ; (24). CD33 + 、CD85h + 、CD258 + 、CD38 - / low 、Syndecan 3 - / low ; (25). CD33 + 、CD85h + 、CD258 + 、CD209 - / low 、Syndecan 3 - / low ; (26). CD33 + 、CD85h + 、CD258 + 、CD38 - / low 、CD209 - / low 、Syndecan 3 - / low ; (27). CD11b+ CD85h + 、CD38 - / low ; (28). CD11b+ CD85h + 、CD209 - / low ; (29). CD11b+, CD85h+, Syndecan 3 - / low ; (30). CD11b + 、CD258 + 、CD38 - / low ; (31). CD11b + 、CD258 + 、CD209 - / low ; (32). CD11b+, CD258 + 、Syndecan 3 - / low ; (33). CD11b + 、CD85h + 、CD258 + 、CD38 - / low ; (34). CD11b + 、CD85h + 、CD258 + 、CD209 - / low ; (35). CD11b+, CD85h+, CD258 + 、Syndecan 3 - / low ; (36). CD11b + 、CD85h + 、CD258 + 、CD38 - / low 、CD209 - / low ; (37). CD11b + CD85h + 、CD258 + 、CD38 - / low 、Syndecan 3 - / low ; (38). CD11b + 、CD85h + 、CD258 + 、CD209 - / low 、Syndecan 3 - / low ; (39). CD11b + 、CD85h + 、CD258 + 、CD38 - / low 、CD209 - / low 、Syndecan 3 - / low ; (40). CD45 + 、CD11b + 、CD85h + 、CD38 - / low ; (41). CD45 + 、CD11b +, CD85h + , CD209 - / low ; (42). CD45 + , CD11b+, CD85h+, syndecan 3 - / low ; (43). CD45 + , CD11b + , CD258 + , CD38 - / low ; (44). CD45 + , CD11b + , CD258 + , CD209 - / low ; (45). CD45 + , CD11b+, CD258 + , syndecan 3 - / low ; (46). CD45 + , CD11b + , CD85h + , CD258 + , CD38 - / low ; (47). CD45 + , CD11b + , CD85h + , CD258 + , CD209 - / low ; (48). CD45 + , CD11b+, CD85h+, CD258 + , syndecan 3 - / low ; (49). CD45 + , CD11b + , CD85h + , CD258 + , CD38 - / low , CD209 - / low ; (50). CD45 + , CD11b + , CD85h + , CD258 + , CD38 - / low , syndecan 3 - / low ; (51). CD45 + , CD11b+ , CD85h + , CD258 + , CD209 - / low , Syndecan-3 - / low ; (52). CD45 + CD11b + CD85h + CD258 + CD209 - / low Clec-9a + (53). CD45 + , CD11b + , CD85h + , CD258 + , CD38 - / low , CD209 - / low , Syndecan-3 - / low 。

[0051] It is particularly preferred that Mreg-bc cells do not express CD34 or do not express it to a significant extent. CD34 is a marker commonly used for hematopoietic stem cells and progenitor cells in clinical hematology. The Mreg-bc cells obtained by the method of the present invention preferably express CD34 in less than 30%, more preferably less than 20%, 15%, 10%, 5% or 1% after 7 days of culture. In one embodiment of the present invention, the macrophage cells of the present invention are derived from a human subject, i.e., of human origin.

[0052] The marker profile of Mreg-bc cells can be easily determined using standard flow cytometry methods. Methods and reagents useful for determining cell surface markers are widely described in the literature. Preferably, the marker phenotype of the Mreg-bc cells of the present invention is determined as described in the Examples section.

[0053] In the present invention, it has been found that the transition from monocytes to regulatory macrophages occurs gradually. During cell generation, the initial population of CD14+ monocytes gradually loses CD14 expression on the cell surface. Thus, in another preferred embodiment, the differentiated Mreg-bc cells obtained from the method of the present invention do not express or express to a significant extent the marker CD14 characteristic of the monocyte lineage. The downregulation of CD14 during Mreg-bc development can be represented as the percentage of CD14 expression in Mreg-bc on day 7 compared to monocytes on day 0 of culture. The expression of CD14 is proportional to the difference in fluorescence intensity between isotype control-stained cells and specific CD14 signals. Thus, the downregulation % = 100 - 100×(CD14 d7 -Iso d7 ) / (CD14 d0 -Iso d0 ), where CD14 d7 is the specific signal on day 7; Iso d7 is the isotype control signal on day 7; CD14 d0 is the specific signal on day 0; Iso d0 is the isotype control signal on day 0. The downregulation of CD14 by Mreg-bc cells can be easily determined using standard flow cytometry methods. Preferably, during the differentiation process from monocytes to Mreg-bc, the expression of CD14 is downregulated by more than 25%, preferably more than 50%, 60%, 70%, 80%, 90%, and more preferably more than 95%.

[0054] Mreg-bc cells are particularly suitable for use for therapeutic purposes, as will be described in more detail below. Conceptually, Mreg-bc therapy is a gain-of-function therapy in the sense that administration of Mreg-bc cells with immunosuppressive, anti-inflammatory or tissue repair functions will complement the lack of those cellular functions in the recipient. By applying appropriate dosages in appropriate amounts, it will be possible to restore or override the above activities in the recipient. In transplantation models and autoimmune models, Mreg-bc treatment has a therapeutic effect that persists in the recipient beyond their own lifespan. This permanent effect can be explained by the effect of Mreg-bc treatment on recipient T cells. Administration of Mreg-bc cells can affect the recipient T cell response in three complementary ways. (a) Mreg-bc cells interact directly with recipient T cells, resulting in specific T cell depletion or conversion to activated induced regulatory T cells (iTreg). (b) Mreg-bc cells modify the behavior of recipient dendritic cells by direct interaction or release of anti-inflammatory mediators. One important function of Mreg-bc cells may be to die in a properly self-regulated environment and transfer antigens to recipient dendritic cells, which in turn specifically suppress recipient T cells. (c) Mreg-bc cells or fractions thereof at the subcellular level exert active or passive non-specific suppression by release of soluble mediators that can act directly or act through recipient bone marrow monocytes.

[0055] In addition to their T cell inhibitory effects, the Mreg-bc cells of the present invention exhibit further characteristic properties that make them valuable for therapeutic use. As shown in Example 6, the Mreg-bc cells of the present invention secrete biologically relevant amounts of vascular endothelial growth factor (VEGF-A) and other angiogenesis-promoting mediators upon stimulation with toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS), monophosphoryl lipid A (MPLA), or High Mobility Group Box protein 1 (HMGB1). As a result, the Mreg-bc cells of the present invention are suitable for treating diseases and conditions where induction of angiogenesis is desired, such as ischemic diseases and conditions.

[0056] In a fourth aspect, the present invention relates to a pharmaceutical composition comprising the Mreg-bc cells of the second aspect of the present invention or a fraction thereof at the subcellular level. The pharmaceutical composition will comprise, as a first component, an effective amount of the Mreg-bc cells of the present invention or a fraction thereof at the subcellular level. As used herein, the effective amount of Mreg-bc cells to be administered to a patient is from about 1×10 4 to about 1×10 8 / kg body weight, preferably from about 1×10 5 to about 1×10 7 / kg body weight, more preferably from about 1×10 6 to about 9×10 6 / kg body weight, for example, about 1×10 6 / kg, about 2×10 6 / kg, about 3×10 6 / kg, about 4×10 6 / kg, about 5×10 6 / kg, about 6×10 6 / kg, about 7×10 6 / kg or about 8×10 6per kg body weight. Similarly, if the present invention involves administration of fractions below the cellular level of the Mreg-bc cells of the present invention, these fractions are preferably prepared based on the amount of Mreg-bc cells corresponding to one of the ranges described above for cell administration. As used herein, fractions below the cellular level of Mreg-bc cells may include necrotic cell particles, apoptotic cell particles, or exosomes containing the major histocompatibility (MHC) molecules of the cells. Cell lysates prepared by treating cells with a hypotonic solution, lysis using a detergent or acid, freeze-thawing or heating, sonication, irradiation, mechanical disruption, or long-term storage may also be used. Fractions below the cellular level may include cell extracts containing whole cell proteins, membrane proteins, cytoplasmic proteins, or purified MHC molecules.

[0057] In addition to cells or fractions below the cellular level of the cells, the pharmaceutical composition can further contain excipients, such as buffers, pH adjusters, preservatives, etc. The nature and amount of the excipients included in the pharmaceutical composition of the present invention will depend on the intended route of administration. Generally, various routes of administration are available for providing the Mreg-bc cells of the present invention or fractions below their cellular level to a patient in need of treatment. Preferably, the pharmaceutical composition of the present invention will be formulated for parenteral administration, such as subcutaneous, intramuscular, intravenous, or intradermal administration. Particularly preferably, the Mreg-bc cells or fractions below their cellular level, or compositions containing the above cells or fractions, are administered to the patient by intravenous administration.

[0058] Formulating the Mreg-bc cells of the present invention or fractions below their cellular level into a pharmaceutical composition can be accomplished by applying conventional methods known in the field of pharmaceutical formulation. Suitable methods are described, for example, in standard textbooks. Pharmaceutical compositions suitable for intravenous administration by injection or infusion usually include sterile solutions or suspensions and sterile powders for extemporaneously preparing sterile aqueous solutions or suspensions. Compositions intended for injection must be sterile and should be fluid to permit easy handling in a syringe or infusion bag.

[0059] The composition should be stable under the administration conditions, and for example, it is preferably protected from the contaminating effects of microorganisms such as bacteria and fungi by including parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. in the composition. In the case of intravenous administration, suitable carriers may include physiological saline, bacteriostatic water, Cremophor EL (trademark) (BASF), or phosphate buffered saline (PBS). The carrier may be a solvent or a dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and lipid polyethylene glycols, etc.), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by using coating agents such as lecithin, maintaining the required particle size in the case of dispersions, and using surfactants. Sterile injectable solutions can be prepared by incorporating the required amount of cells or subcellular fractions together with one or more of the above components in a suitable solvent and then subjecting it to sterile filtration. Generally, suspensions are prepared by incorporating the active compound, i.e., cells or subcellular fractions thereof, into a sterile vehicle containing a base dispersion medium and other required components from the above. In the case of sterile powders for preparing sterile injectable solutions, the preparation methods are vacuum drying and lyophilization, which result in a powder obtained by combining cells or subcellular fractions thereof with any desired additional components with that obtained from its pre-sterile filtered solution.

[0060] Compositions intended for infusion or injection will have a volume of 50 - 500 mL, in which case a volume of 90 ml - 250 ml is particularly preferred, and a volume of 90 ml - 150 ml is even more preferred.

[0061] Mreg-bc cells can be administered to patients in need of treatment according to various dosing schedules. For example, when administering cells or cell fractions to a patient by intravenous infusion, the dose to be administered The total amount of Mreg-bc cells or Mreg-bc cell fractions can be supplied by one or more infusions. In a preferred embodiment, the Mreg-bc cells or cell fractions are supplied to the patient by a set of infusion devices equipped with a 200 μm filter. The suspension containing Mreg-bc cells or Mreg-bc cell fractions may be initially stimulated with 0.9% NaCl. The suspension may be administered as a single infusion, more preferably, it may be administered by a short-term infusion of less than 60 minutes, for example, within 60 minutes, within 30 minutes, within 20 minutes or within 15 minutes. It is preferred to use a central venous catheter to administer the Mreg-bc suspension.

[0062] The administration of Mreg-bc cells or cell fractions can be accompanied by the administration of other active agents before, simultaneously, or after. For example, when administering the Mreg-bc cells or cell fractions of the present invention to prevent the immune response of a patient who has received an organ transplant, an immunosuppressive drug may be administered together with the cells or cell fractions of the present invention. Examples of immunosuppressive drugs conventionally used in the field of transplantation medicine include, but are not limited to, cyclosporine A (CSA), tacrolimus, azathioprine (AZA), mycophenolate mofetil, rapamycin and steroids (STE). Generally, the presence of an immunosuppressive drug in the recipient's blood does not affect the effectiveness of the cells or cell fractions of the present invention.

[0063] The Mreg-bc cells obtained from the method described in the first aspect of the present invention exhibit a stable phenotype, but the Mreg-bc cells or fractions below the cell level obtained from Mreg-bc cells are recommended to be administered within 24 hours after collecting them from the cell culture for safety reasons. Preferably, the cells are administered within 20 hours, within 16 hours, within 12 hours, within 8 hours or within 4 hours after collecting the cells from the culture.

[0064] In a fourth aspect, the present invention relates to the therapeutic use of Mreg-bc cells according to the second aspect of the present invention, or fractions thereof at the cellular level or below, or the composition according to the third aspect of the present invention. As shown elsewhere herein, the Mreg-bc cells provided by the present invention have numerous pharmacological properties that make them highly suitable for use in immunosuppressive therapy, anti-inflammatory therapy or tissue repair therapy, such as immunosuppressive properties, immunomodulatory properties, angiogenesis properties and anti-inflammatory properties. For example, the artificially induced Mreg-bc cells of the present invention are T cell inhibitory and mediate the active removal of activated T cells. Thus, the cells are highly suitable for use as an adjunct immunosuppressive therapy in various immune-mediated diseases, such as organ transplantation.

[0065] Accordingly, in one embodiment of the present invention, the Mreg-bc cells according to the second aspect of the present invention, or fractions thereof at the cellular level or below, or the pharmaceutical composition according to the third aspect of the present invention are used in a method for suppressing transplant rejection and / or prolonging the graft survival period in a subject receiving a graft. Thus, the present invention provides a method for suppressing transplant rejection and / or prolonging the graft survival period in a subject receiving a graft, the method comprising (i) administering an effective amount of the Mreg-bc cells according to the second aspect of the present invention, or fractions thereof at the cellular level or below, or (ii) administering the pharmaceutical composition according to the third aspect of the present invention. Preferably, the graft is an organ, tissue or cell graft. The type of organ transplanted is not limited by the present invention, but is preferably a kidney, liver, heart, lung or pancreas. Particularly preferably, the organ transplanted into the recipient is a human organ.

[0066] When the Mreg-bc of the present invention is used in the case where the graft is a tissue graft rather than an organ graft, it can also be used to suppress transplant rejection and / or extend the graft survival period. Also in this case, the tissue transplanted into the recipient is not particularly limited. The rejection of any tissue derived from an allogeneic donor in the recipient can be prevented or improved by the Mreg-bc of the present invention. The tissue to be transplanted is preferably human tissue, for example, intestine, cornea, skin, composite tissue, bone marrow, or islet tissue.

[0067] The Mreg-bc produced according to the method of the present invention can further assist in the introduction of cell grafts into the recipient by suppressing the immune response in the recipient. When the graft is a cell graft, the nature of the cells to be transplanted is generally not limited, but the cells to be transplanted are preferably selected from the group consisting of adult stem cell grafts, isolated hepatocyte grafts, or leukocyte cell grafts. The Mreg-bc cells of the present invention can further produce soluble factors that promote the homing and engraftment of adult stem cells, such as cathelicidin. In a preferred embodiment of the present invention, the Mreg-bc cells are used to promote the engraftment of hematopoietic stem cells (HSCs) after bone marrow transplantation or HSC transplantation.

[0068] To suppress transplant rejection in a recipient and induce acceptance of an allogeneic organ, tissue or cell graft, the Mreg-bc cells of the present invention or a pharmaceutical composition containing Mreg-bc cells or a fraction below the cellular level thereof can be intravenously administered by injection or infusion as described above. The injection or infusion can be given either preoperatively or postoperatively. When Mreg-bc cells are administered preoperatively, they will be administered to the recipient preoperatively at least once, preferably twice, more preferably three times. Mreg-bc is preferably administered to the recipient within one week before surgery, for example, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day before surgery. When Mreg-bc cells are administered postoperatively, the first administration will preferably be given within 24 hours after surgery, more preferably within 36 hours, 48 hours, 60 hours, 72 hours after surgery. Alternatively, in a transplant recipient who is stably immunosuppressed, Mreg-bc therapy can be administered at any time after transplantation. Alternatively, Mreg-bc can be administered to a transplant recipient suffering from acute or chronic transplant rejection. Mreg-bc can then eliminate the T cell response of the recipient's immune system to the graft and can persist in the recipient's body (particularly the spleen, liver, lung and bone marrow) for a long enough time to confer long-term transplant acceptance on the recipient.

[0069] When using the Mreg-bc of the present invention to suppress transplant rejection or extend the graft survival period in a subject receiving a graft, the graft will typically be an allogeneic graft, i.e., a graft derived from a donor who is genetically different but belongs to the same species as the recipient. In this case, Mreg-bc cells are generated based on blood monocytes obtained from the above donor. Monocytes can be obtained from a living donor or a deceased donor. In the case of a deceased donor, i.e., a cadaver, the donor's body is usually perfused with a perfusion medium by aortic cannulation for organ preservation. Venous blood can be removed from the body and collected to produce Mreg-bc according to the methods described herein. Alternatively, Mreg-bc can also be produced from bone marrow mononuclear cells isolated from the donor's spleen. When applying Mreg-bc produced from a dead donor postoperatively, rejection of the transplanted organ can be prevented by administering immunosuppressive drugs customarily used for this purpose during organ transplantation.

[0070] In another embodiment, in a method for promoting or maintaining the effect of a medicament based on engraftment or regulatory T cells, Mreg-bc cells according to the second aspect of the present invention, or a fraction thereof below the cellular level, or a pharmaceutical composition according to the third aspect of the present invention are used. Accordingly, the present invention further relates to a method for promoting or maintaining the effect of a medicament based on engraftment or regulatory T cells in a subject, comprising (i) administering an effective amount of Mreg-bc cells according to the second aspect of the present invention or a fraction thereof below the cellular level, or (ii) administering a pharmaceutical composition according to the third aspect of the present invention.

[0071] In addition to immunomodulatory and immunosuppressive properties, the Mreg-bc cells of the present invention are chronically inflammatory It has anti-inflammatory properties that enable disabling the immune process. Thus, the Mreg-bc cells provided herein are also useful for treating diseases or disorders characterized by an unregulated immune state or an excessive inflammatory response, particularly chronic inflammatory diseases. Such diseases or disorders include, for example, autoimmune diseases, inflammatory diseases, and allergic reactions.

[0072] Thus, in yet another embodiment, the Mreg-bc cells according to the second aspect of the invention or fractions thereof at the cellular level or below, or the pharmaceutical composition according to the third aspect of the invention, are used in a method for treating or preventing an autoimmune disease, an inflammatory disease, or an allergic reaction.

[0073] When using Mreg-bc to treat an autoimmune disease, the disease can be (a) mainly mediated by T cells, (b) mainly mediated by antibodies, or (c) mainly mediated by other cellular components of the immune system. The disease can be localized or systemic autoimmune symptoms. The types of autoimmune symptoms treated by Mreg-bc therapy are not limited by the present invention, but include, for example, systemic lupus erythematosus (SLE), scleroderma, Sjogren's syndrome, polymyositis, dermatomyositis, and other systemic autoimmune symptoms; rheumatoid arthritis (RA), juvenile rheumatoid arthritis, and other inflammatory arthritides; ulcerative colitis, Crohn's disease, and other inflammatory bowel diseases; autoimmune hepatitis, primary biliary cirrhosis, and other autoimmune liver diseases; cutaneous small-vessel vasculitis, granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, Behcet's disease, thromboangiitis obliterans, Kawasaki disease, and other large, medium, or small-vessel vasculitides with an autoimmune etiology; multiple sclerosis (MS) and neuroimmunological disorders; type I diabetes, autoimmune thyroid dysfunction, autoimmune pituitary dysfunction, and other autoimmune endocrinological disorders; hemolytic anemia, thrombocytopenic purpura, and other autoimmune disorders of the blood and bone marrow; psoriasis, pemphigus vulgaris, pemphigoid, and other autoimmune skin symptoms.

[0074] Mreg-bc cells are also effective for treating acute or chronic inflammatory diseases and diseases having pathophysiologically important inflammatory components. The inflammatory diseases to be treated can be either localized or systemic. Types of inflammatory diseases or conditions that benefit from treatment with Mreg-bc include, but are not limited to, arterial occlusive diseases such as peripheral arterial occlusive disease (pAOD), severe ischemic limbs, arteriosclerosis, cerebral infarction, myocardial infarction, renal infarction, intestinal infarction, angina pectoris, and other conditions caused by arterial occlusion or stenosis; microvascular angina, also known as cardiac syndrome X; inflammation associated with systemic metabolic disorders including type II diabetes and obesity-related metabolic syndrome; and skin diseases including eczema. Preferably, the inflammatory disease to be treated is one characterized by chronic inflammation of the intima of the arterial wall, such as myocardial infarction, stroke, severe ischemic limb vasculitis, and pAOD.

[0075] When treatment of an allergic reaction is desired, the allergic reaction is preferably selected from the group consisting of asthma, eczema, allergic rhinitis, angioedema, drug allergy, and mastocytosis.

[0076] Treatment of pAOD is particularly preferred. pAOD is known to be highly debilitating and is generally seen as a condition where amputation is the only treatment option in patients not suitable for revascularization due to either the degree or location of arterial occlusion or significant co-existing diseases. Amputation remains an ultimate treatment and is associated with a relatively high mortality rate, and only a small number of patients later regain full mobility. In the course of the present invention, Mreg-bc cells obtained by the methods described herein have been found to have angiogenic properties. Mreg-bc actively promotes neovascularization, i.e., the formation of new blood vessels, by basal and stimulated expression of angiogenic growth factors such as VEGF, FIGF (VEGF-D), PDGFB, and MDK. Specifically, Mreg-bc cells produce high levels of vascular endothelial growth factor (VEGF) when stimulated with TLR4 ligand. Specifically, in the present invention, it has been found that Mreg-bc induces the expression of vascular endothelial growth factor C (VEGF-C). In the literature, it has been reported that VEGF-C is an angiogenic factor that effectively stimulates neovascularization in vivo

[16] .

[0077] In one embodiment, Mreg-bc cells are intramuscularly or subcutaneously injected into the ischemic limb. In the ischemic tissue, Mreg-bc cells will inevitably be exposed to bacterial components and necrotic tissue components (such as HMGB1) that act as TLR4 agonists. Therefore, Mreg-bc can be used to promote tissue regeneration by local secretion of angiogenesis-promoting growth factors. In another embodiment, Mreg-bc cells may be stimulated with TLR ligands in vitro during the manufacturing process to ensure high-level production of VEGF by them. Examples of the TLR ligands include, but are not limited to, lipopolysaccharide (LPS) or monophosphoryl lipid A (MPLA). The pAOD treated with Mreg-bc of the present invention can be any grade or category of pAOD. For example, the pAOD can be grade I pAOD, classification 1-4, or grade II-IV pAOD.

[0078] The Mreg-bc of the present invention has angiogenesis properties, and their use in other diseases or conditions that require neovascularization is contemplated in the present invention. Accordingly, the present invention further relates to a method of inducing angiogenesis or vasculogenesis in hypoxic tissue, a method of promoting tissue repair processes by participating in tissue remodeling, tissue regeneration, prevention or reduction of fibrosis, a method of reducing ischemic pain, or a method of avoiding major limb amputation, which involves (i) administering an effective amount of Mreg-bc cells according to the second aspect of the present invention or a fraction thereof at the cellular level or (ii) administering a pharmaceutical composition according to the third aspect of the present invention. Therefore, the present invention relates to a method of inducing angiogenesis or vasculogenesis in hypoxic tissue, a method of promoting tissue repair processes by participating in tissue remodeling, tissue regeneration, prevention or reduction of fibrosis, a method of reducing ischemic pain, or a method of avoiding major limb amputation, the method comprising (i) administering an effective amount of Mreg-bc cells according to the second aspect of the present invention or a fraction thereof at the cellular level or (ii) administering a pharmaceutical composition according to the third aspect of the present invention.

[0079] Treatment of autoimmune diseases, inflammatory diseases or allergic reactions can be achieved by either Mreg-bc derived from monocytes that are allogeneic to the patient as described above in the context of transplantation applications or monocytes that are autologous to the patient in need of treatment. Where possible, treatment of autoimmune diseases, inflammatory diseases or allergic reactions will be carried out using autologous monocytes. For this purpose, Mreg-bc can be administered intravenously, with or without concomitant local intramuscular injection.

[0080] In yet another embodiment, the Mreg-bc cells according to the second aspect of the present invention, or a fraction thereof at the subcellular level, or the pharmaceutical composition according to the third aspect of the present invention, are used as a vehicle for delivering gene therapy. Accordingly, the present invention relates to a method for delivering gene therapy, comprising: (i) administering an effective amount of the Mreg-bc cells according to the second aspect of the present invention, which contain a transgene; or (ii) administering a pharmaceutical composition containing the Mreg-bc cells according to the second aspect of the present invention, which contain a transgene.

[0081] According to a fifth aspect, the present invention relates to a process for producing a fraction at the subcellular level of immunomodulatory macrophage cells, comprising: (a) providing the immunomodulatory macrophages described in connection with the first aspect of the present invention; (b) decomposing the immunomodulatory macrophage cells to provide a fraction at the subcellular level; (c) obtaining a fraction at the subcellular level and related to a process comprising.

[0082] The Mreg-bc cells of the present invention can be decomposed according to conventional methods. For example, the cells can be lysed by treating the Mreg-bc cells with a hypotonic solution, a detergent or an acid. Alternatively, the cells can be decomposed by freeze-thawing or heating, sonication, irradiation, mechanical disruption or long-term storage. In the final step of the method, fractions at the subcellular level of the cells, such as a total protein fraction, a membrane protein fraction, a cytoplasmic protein fraction, etc., are obtained. These fractions can be used in place of live Mreg-bc cells for the above therapeutic purposes. Alternatively, the fraction can be further purified to isolate a specific protein, such as an MHC protein.

[0083] Accordingly, according to a sixth aspect, the present invention relates to a process for producing immunomodulatory T cells, comprising: (a) obtaining T cells from a subject; (b) culturing the T cells with the immunomodulatory macrophage cells according to any one of claims 14 to 18 or a fraction thereof at the cellular level or below; (c) obtaining immunomodulatory T cells from the culture medium relates to a process comprising.

[0084] As described elsewhere herein, T cells cultured with Mreg-bc inhibit T cell proliferation. Thus, the immunomodulatory T cells obtained from the above method can be used alone or in combination with the Mreg-bc cells of the present invention for the treatment of any of the diseases or disorders described elsewhere herein. In the first step, T cells are obtained from a blood sample of the subject. The cells can be obtained, for example, from a blood sample or an apheresis fluid, or from the subject's tissue, such as bone marrow or spleen. The cells can be obtained by conventional methods, such as by venipuncture in the case of cells from blood. The T cells used in the above method will be, for example, CD3+ T cells or a subset thereof. CD3+ T cells can be purified or enriched by conventional methods, such as magnetic microbead separation or flow cytometry sorting, before culturing with Mreg-bc cells.

[0085] Thereafter, the T cells are contacted with the Mreg-bc of the present invention. The cells can be contacted at various Mreg:Treg ratios. For example, the cell fractions can be contacted at an Mreg:Treg ratio of 1:5 to 5:1, preferably 1:2 to 2:1. More preferably, the Mreg:Treg ratio is about 1:1. Various media can be used for the co-culture method. The medium can be the one described above in the context of the method for producing Mreg-bc cells. In a preferred embodiment, the medium is X-vivo10 from Lonza. The medium can further contain additional additives, such as M-CSF and / or GM-CSF, preferably human recombinant M-CSF and / or GM-CSF. The amount of M-CSF and / or GM-CSF will be in the range mentioned elsewhere in this specification, for example 5 to 100 ng / ml, preferably 20 to 25 ng / ml. The medium can further contain other additives, such as Glutamax, in an amount of 1 to 5 mM, preferably 2 mM.

[0086] The cells will be co-cultured for 1 to 8 days, preferably at least 3 days, at least 4 days, or at least 5 days. After a predetermined culture period, the T cells are isolated again by concentrating the cells with respect to Tregs. If necessary, the cells can be further formulated as a pharmaceutical. + CD25 + TIGIT + FoxP3 + The cells can be re-isolated by concentrating the cells with respect to Tregs. If necessary, the cells can be further formulated as a pharmaceutical.

[0087] According to a seventh aspect, the present invention relates to a method for detecting immunomodulatory macrophage cells, comprising: (a) providing a sample containing macrophage cells; (b) detecting the presence and / or expression of the DHRS9 protein and / or the DHRS9 gene in the sample, wherein the presence and / or expression of the DHRS9 protein and / or the DHRS9 gene indicates that the sample contains immunomodulatory macrophage cells.

[0088] The method can be used to distinguish immunomodulatory macrophage cells (Mreg) from other activated macrophages, such as monocyte-derived macrophages (Mφ), such as resting Mφ, LPS+IFNγ-stimulated Mφ, IL-4-stimulated Mφ, and immunoglobulin (Ig)-stimulated Mφ. Since the expression of DHRS9 was only observed in Mreg, this marker can be used to identify a population of macrophages containing Mreg among heterogeneous populations of macrophages, such as Mreg and at least one of the following macrophage species: resting Mφ, LPS+IFNγ-stimulated Mφ, IL-4-stimulated Mφ, and immunoglobulin (Ig)-stimulated Mφ. Detection of the DHRS9 marker can be achieved by flow cytometry using standard antibodies against the DHRS9 polypeptide or fragments thereof. The expression of DHRS9 can be detected by PCR, RT-PCR, real-time PCR, and other conventional methods.

[0089] In an eighth aspect, the invention provides a method for isolating immunomodulatory macrophage cells from a heterogeneous population of macrophages, comprising: (a) providing a heterogeneous population of macrophages; (b) isolating the immunomodulatory macrophage cells by their affinity for a molecule that specifically binds to the DHRS9 protein and providing a method comprising the above steps.

[0090] For example, in one embodiment, immunomodulatory macrophage cells within a heterogeneous population of macrophages can be isolated by binding them to an antibody directed against DHRS9. Such an antibody may be monoclonal or polyclonal in origin. In a preferred embodiment, the anti-DHRS9 antibody can be immobilized on a solid phase, such as the bottom of a microtiter plate well. The macrophage population is incubated in the well to allow binding of the anti-DHRS9 antibody to DHRS9 present on the surface of the immunomodulatory macrophage cells. After washing away the unbound macrophages, a uniform population of immunomodulatory macrophage cells is obtained. In another embodiment, the anti-DHRS9 antibody can be immobilized on the surface of magnetic beads. The beads are incubated with the macrophage population to allow binding of the antibody to DHRS9. After separating the beads from the solution containing the macrophage population, a uniform population of immunomodulatory macrophage cells is obtained.

[0091] Accordingly, in yet another aspect, the present invention relates to the use of a molecule that specifically binds to DHRS9, particularly an anti-DHRS9 antibody, for the detection or isolation of immunomodulatory macrophage cells.

Example

[0092] Mreg-bc cells were produced in accordance with current GMP principles regarding the manufacture of sterile pharmaceuticals. At each processing step, care was taken to protect the product, materials, and equipment from contaminants and impurities.

[0093] Example 1: Preparation of Mreg-bc cells Healthy human donors were subjected to leukapheresis to collect peripheral blood mononuclear cells (PBMCs) for use as starting material for Mreg-bc generation. All donors were sensitized Screening was performed for relevant disease markers, including infectious diseases. Donors were re-screened for the same disease markers on the day of leukapheresis. Leukapheresis was performed using a Terumo BCT Cobe Spectra device or equivalent.

[0094] 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 into a bag filled with PBS / EDTA buffer containing 0.5% human serum albumin (HSA). The cells were washed once before labeling with CliniMACS® CD14 reagent according to the manufacturer's instructions. The labeled cell suspension was connected to a sterile tubing set and attached to the CliniMACS® device for isolation of CD14+ monocytes by magnetic separation. The positively isolated CD14+ monocyte fraction was washed with medium to remove the CliniMACS® separation buffer.

[0095] Subsequently, the monocyte density in the cell culture medium was adjusted to 10 6 cells / ml. CD14+ monocytes were collected for analysis by flow cytometry as in-process control. In process-related calculations, cell numbers were determined using an automated blood counter with the WBC parameter as the total white blood cell count. The viability of all cell types was evaluated by flow cytometry.

[0096] The isolated CD14+ monocytes were resuspended at a density of 10 6 cells / ml in RPMI medium supplemented with 10% male-only human AB serum (heat-inactivated together), 2 mM GlutaMAX™, and 25 ng / ml recombinant human monocyte colony-stimulating factor (M-CSF).

[0097] This monocyte suspension was dispensed into Miltenyi® cell differentiation bags such that each bag was seeded at 1×10 6 cells / cm 2 inner surface area. For culture, at 36 - 38 °C, 5 ± 1% CO2 、The differentiation bags were placed flat on the shelves in an incubator set at a humidity of 60% or more. Monocytes were allowed to sediment on the lower surface of the culture bag over a period of one day. On the first day, the bag was inverted to attach the monocytes to the opposite side. The culture was maintained in the incubator for an additional five days.

[0098] To induce the final differentiation from monocytes to Mreg-bc and to induce indoleamine 2,3-dioxygenase (IDO) expression, the monocytes were stimulated with the addition of 25 ng / ml of IFN-γ. After the addition of IFN-γ, the differentiation bag was inverted once again. Then the bag was incubated at 36 - 38 °C, 5 ± 1% CO 2 for 18 - 24 hours at a humidity of 60% or more.

[0099] On the seventh day, the differentiated Mreg-bc were collected. Cells from all parallel culture bags were pooled and washed prior to phenotypic and functional analysis.

[0100] Example 2: Evaluation of the phenotypic characteristics of Mreg-bc The phenotype of Mreg-bc obtained by the method of Example 1 was analyzed in detail. During culture, macrophages adopted a unique morphology with an epithelial-like form in which the cells were spread out, forming an almost confluent monolayer (see Figure 6A). Individual macrophages were large, densely packed, granular cells with a prominent central body and a thin cytoplasmic skirt that spread symmetrically over the surface of the culture vessel. Ultramorphological examination of macrophages by transmission electron microscopy confirmed the impression of large, flat cells adhering very closely to the underlying surface (see Figure 6B). In most respects, the ultramorphological appearance of the macrophages was typical of activated macrophages: projections extended from the outer periphery and upper surface of the cell; the nucleus was seen to be actively involved with abundant chromatin; and the cytoplasm contained numerous intracellular vesicles, lipid inclusions, and prominent smooth endoplasmic reticulum.

[0101] The cell surface phenotype of Mreg-bc cells was characterized by flow cytometry. To prepare Mreg-bc cells for flow cytometry analysis, cells were harvested and washed once with Ca 2+ / Mg 2+ -free DPBS, and then resuspended in Ca 3 / Mg 2+ -free DPBS containing 1% BSA, 0.02% NaN 2+ 3, and 10% FcR block (Miltenyi) at 1-5×10 5 cells / 100 μl. The samples were then incubated at 4 °C for 15 min. Fluorescent dye-conjugated antibodies were obtained from various manufacturers, contacted with the cell suspension, vortexed, and incubated at 4 °C for 20 min in the dark. After adding 10 μl of 7-AAD, each sample was vortexed briefly and further incubated at 4 °C for 10 min in the dark. Subsequently, the samples were washed twice with cold Ca 2+ / Mg 2+ -free DPBS and resuspended for analysis. Clec-9a signal was enhanced using FASER reagent (Miltenyi) twice according to the manufacturer's instructions. For intracellular staining, cells were first stained for the cell surface antigens described above, and then fixed and permeabilized using a buffer set for intracellular fixation & permeabilization (eBioscience) according to the manufacturer's instructions. Cells were resuspended in permeabilization buffer containing 10% FcR block and then incubated at 4 °C for 15 min in the dark. Fluorescent dye-conjugated antibodies were contacted with the cell suspension, vortexed briefly, and then incubated at 4 °C for 30 min in the dark. Cells were washed twice with permeabilization buffer and resuspended for analysis. Data were acquired on a Canto II flow cytometer (BD Biosciences, Germany) and analyzed using FlowJo 7.6 software (TreeStar, USA) or Kaluza 1.1 software (Beckman Coulter, Germany).

[0102] This flow cytometry analysis showed that the primary human Mreg-bc cells of the present invention were CD14 - / low CD209 - / lowCD16 - / low CD80 - / low CD86 + CD10 + / - CD103 + / - CD38 - / low CD85h + CD258 + Syndecan-3 - / low Clec-9a + was shown to exhibit the phenotype (Figure 2).

[0103] Example 3: Specificity of the Mreg phenotype A panel of macrophage populations was generated to clarify the phenotypic relationship between Mreg and other activated-state macrophages known in the art for comparison with Mreg. From the perspectives of morphology, cell surface marker expression, cytokine production, and overall gene expression profiles, Mreg could be easily distinguished from all these other macrophage populations by their characteristic morphology (see Figure 6C) and their prominent cell surface phenotype (see Figure 6D). Specifically, Mreg was found to be unique in that it downregulates CD14 and lacks the expression of cell surface CD16, TLR2, and CD163.

[0104] The panel of Mreg and comparison macrophages was also distinguished by their cytokine and chemokine production profiles. Mreg constantly produces very small amounts of TNF-α and IL-6 and does not secrete detectable amounts of IL-12p40. Mreg expresses detectable levels of TGF-β and large amounts of IL-1Ra, but the expression of IL-10 is significantly less than that of other macrophage populations. This cytokine secretion profile is relatively stably maintained after exposure to IFN-γ and LPS.

[0105] To identify markers exclusively expressed by Mregs, Mregs and IFN-γ-Mφ were generated from peripheral blood leukocytes obtained as a by-product of platelet apheresis from healthy donors according to the previously described method

[10] . Briefly, CD14+ monocytes were isolated from PBMCs prepared by Ficoll by positive selection with anti-CD14 microbeads (Miltenyi, Bergisch-Gladbach), and then, in 6-well Cell+ plates (Sarstedt, Numbrecht), 10% heat-inactivated human AB serum (Lonza), 2 mM Glutamax (Invitrogen, Karlsruhe), 100 U / mL penicillin (Lonza), 100 μg / mL streptomycin (Lonza), and 25 ng / ml rhM-CSF (R&D Systems, Wiesbaden-Nordenstadt) with 0.1% human albumin (CSL-Behring, Hattersheim-am-Main) as a carrier were supplemented in RPMI-1640 (Lonza, Cologne) at 10 cells / cm 5 2 ​They were seeded. On the 6th day of culture, the cells were further stimulated with 25 ng / ml of rhIFN-γ (Chemicon, Billerica, MA) for 18 - 24 hours. IFN-γ-stimulated macrophages (IFN-γ-Mφ) were generated by culturing CD14+ monocytes under the same conditions as for Mreg, except that human serum was replaced with 10% heat-inactivated fetal calf serum (FCS) (Biochrom, Berlin). Macrophages (Mφ) in other defined polarization states were generated from positively isolated CD14+ monocytes according to the protocols adopted from references

[12] -

[15] . Briefly, as follows: for resting Mφ, supplemented with 20% FCS and 100 ng / ml of M-CSF for 7 days; for lipopolysaccharide (LPS)-activated Mφ, supplemented with 20% FCS and 100 ng / ml of M-CSF, and on the 6th day, 100 ng / ml of LPS (Enzo Life Sciences) and 20 ng / ml of IFN-γ were added; for IL-4-stimulated Mφ, supplemented with 20% FCS and 100 ng / ml of M-CSF, and on the 6th day, 20 ng / ml of IL-4 (R&D Systems) was added; for Ig-stimulated Mφ, supplemented with 10% FCS and 100 ng / ml of M-CSF, cells grown in plastic products were pre-coated with human IVIg (PrivigenTM, CSL Behring), and on the 6th day, 100 ng / ml of LPS was added; for glucocorticoid (GC)-stimulated Mφ, supplemented with 20% FCS and 100 ng / ml of M-CSF, and on the 6th day, 10-7 M of dexamethasone (Sigma-Aldrich) was added, in RPMI-based medium containing 100 U / ml of penicillin, 100 μg / ml of streptomycin, and 2 mM of GlutaMAXTM. 5 cells / cm 2 The monocytes were cultured for 7 days in Cell+ plastic products (Sarstedt).

[0106] A series of monoclonal antibodies (mAbs) were generated by vaccinating mice with human Mreg lysates. By screening these mAbs by immunocytochemistry, an mAb clone (ASOT1) was identified that strongly reacts with Mreg but not with other monocyte-derived macrophages (Mφ) including resting Mφ, LPS+IFNγ-stimulated Mφ, IL-4-stimulated Mφ, and immunoglobulin (Ig)-stimulated Mφ (see Fig. 3A). By immunoprecipitating and sequencing its antigen, ASOT1 mAb was shown to recognize DHRS9, a retinol dehydrogenase of the less-studied SDR family (see Fig. 3B). Quantitative PCR confirmed that DHRS9 mRNA expression is limited to Mreg (Fig. 3C). Rabbit polyclonal antibodies generated against the N-terminal epitope of DHRS9 reacted with the approximately 35-kD protein immunoprecipitated by ASOT1 (see Fig. 3D). Since a commercially available monoclonal antibody (clone 3C6) recognizing DHRS9 also reacted with the same protein detected by the rabbit antibody, it can be confidently concluded that both ASOT1 and the rabbit polyclonal antibody recognize DHRS9. Using this rabbit pAb, DHRS9 protein expression was shown to be unique to Mreg (Fig. 3E).

[0107] Using whole-genome expression profiling, a comprehensive insight into the phenotypic relatedness between Mreg and other activated-state macrophages was obtained. Microarray analysis was performed on a panel of nine comparative macrophage species generated in parallel from three separate donors. Genes expressed with a difference of more than 20-fold between any two samples were selected and hierarchically clustered When clustered, the Mreg samples were found to be most similar to the IFN-γ untreated Mreg and LPS-stimulated Mreg compared to any other macrophage samples. This clustering pattern remained stable when all significantly differentially expressed probes were used for analysis. The Mreg samples were more similar to Ig-stimulated M2b macrophages than to other macrophage species, highlighting the importance of Ig stimulation in the development of the Mreg phenotype according to step (c) of embodiment 1. Resting macrophages and IFN-γ-stimulated macrophages were clustered with M2a macrophages and M2c macrophages. The similarity between classically activated M1 macrophages and the comparison macrophage species was lower than the similarity between the comparison macrophages themselves. From these observations, it can be concluded that Mreg is in a unique activation state and is relatively refractory to reprogramming to the M1 phenotype by LPS stimulation.

[0108] The array results of the microarray were consistent with the findings by flow cytometry in so far as CD163, IL-10, and CD14 were not found in the list of downregulated genes that distinguish Mreg from all other comparison macrophages. Among the gene sets uniquely upregulated by Mreg, CD258 (TNFSF14, LIGHT) and CD85H (ILT1, LILRA2) were identified as useful markers for Mreg identity. The expression of CD258 and CD85b by Mreg, rather than by IFN-γ-stimulated macrophages, was confirmed by flow cytometry (Figure 7).

[0109] CD45 + CDllb + CDllc + CD14 - / low CD209 - / low CD16 - / low CD80 - / low CD86 + CD10 + / - CD103 + / - CD38 - / low CD85h + CD258+ Cindecan-3 - / low Clec-9a + DHRS9 + and Arg-1 + and IDO + A group of and is a precise and stable definition of the Mreg phenotype.

[0110] Example 4: Generation of activated peripheral blood-derived human Tregs by allogeneic Mreg-bc treatment in NOD / SCID / IL2rγ null Mice The Mreg-bc cells of the present invention were prepared as described in Example 1. Immunodeficient NOD / SCID / IL2rγ null (NSG) mice were reconstituted using human T cells. These mice were either treated with the Mreg-bc cells of the present invention or not (see Figure 8A). Five days after Mreg-bc cell treatment, human T cells were recovered from the spleens of the recipient mice. The T cell population in the Mreg-treated mice was FoxP3 + Tregs and TIGIT + FoxP3 + Tregs were enriched (see Figure 8B). In NSG mice treated with Mreg-bc cells, the serum levels of human IL-10 were significantly higher compared to untreated animals (see Figure 8C). This example demonstrates that human Mreg-bc can directly interact with allogeneic human T cells in vivo to induce Treg development.

[0111] Example 5: Treatment of kidney transplant recipients with Mreg-bc cells before surgery The Mreg-bc cells of the present invention were prepared as described in Example 1. Mreg-bc cells were administered to a 43-year-old promising living donor kidney transplant recipient with end-stage renal failure due to polycystic kidney disease. The Mreg-bc cells were made from monocytes collected from the healthy 62-year-old father of the recipient who later donated a kidney to his son. The donor and recipient had a single mismatch at the HLA-A, -B, and -DR loci.

[0112] A total of 4.75×10 8 individual live Mreg-bc were administered by slow central venous infusion. No side effects were encountered. Specifically, there were no signs of pulmonary vascular occlusion, right heart load, transfusion reaction, allergic reaction, or biochemical impairment. Treatment with Mreg-bc cells did not cause the recipient to produce anti-donor HLA antibodies.

[0113] The recipient's allograft function is now stable and more than 15 months have passed since transplantation. The recipient is currently maintained on a low-dose immunosuppression regimen that includes tacrolimus and MMF. This case illustrates the validity of administering Mreg-bc cells to pre-operative kidney transplant recipients.

[0114] Example 6: Production of angiogenic factors by Mreg-bc cells It was tested whether Mreg-bc cells produce the angiogenic factor VEGF-A when stimulated by monophosphoryl lipid A (MPLA). The setup of this experiment is depicted in Figure 9.

[0115] Under the first test condition, Mreg-bc were grown until day 7 as described in Example 1, including standard stimulation with 25 ng / ml of IFN-γ on day 6. On day 7, Mreg-bc cells were harvested and 0.5×10 6 cells per well were re-seeded in 1 ml of RPMI-1640 + 1% HABS + Pen-Strep + 2 mM GlutaMAX in 24-well plates. These re-seeded Mreg-bc cells were then either stimulated with 1 μg / ml of LPS or not stimulated. In parallel, Mreg-bc cells were harvested and examined by flow cytometry for CD14, CD10, CD16, CD38, CD80, CD86, CD85h, CD103, CD258, CD209, and syndecan-3.

[0116] In the second condition, Mreg-bc cells were additionally stimulated with 100 ng / ml of MPLA on the 6th day of culture, and IFN-γ was added simultaneously. On the 7th day, Mreg-bc cells collected from condition 2 were reseeded and stimulated in the same manner as in condition 1. Furthermore, Mreg-bc cells from condition 2 were analyzed for markers CD14, CD10, CD16, CD38, CD80, CD86, CD85h, CD103, CD258, CD209, and syndecan-3 by flow cytometry.

[0117] In the third condition, Mreg-bc cells were stimulated with 25 ng / ml of IFN-γ on the 6th day as usual. On the 7th day, the cells were further stimulated with 100 ng / ml of MPLA for an additional 24 hours. Then on the 8th day, the cells were collected for analysis in the same manner as in conditions 1 and 2.

[0118] The secretion of VEGF-A by cells from all three conditions was measured by ELISA. The phenotypes of cells from all three conditions were compared by flow cytometry to evaluate the stability of the cell surface phenotype that characterizes Mreg-bc under the test conditions.

[0119] Results: The results of the VEGF-A determination are depicted in Figure 9. Treatment with 100 ng / ml of MPLA on the 6th or 7th day was found to enhance VEGF expression induced by LPS. Treatment with 100 ng / ml of MPLA did not dramatically change the Mreg-bc phenotype within 24 hours, with only a slight upregulation of CD80 expression observed. These examples indicate that MPLA treatment during Mreg-bc culture could be a useful method to enhance the production of VEGF-A by Mreg-bc cells prior to application to patients.

[0120] Example 7: Production of angiogenic factors by Mreg-bc cells It was tested whether Mreg-bc cells produce angiogenesis-related factors. For this purpose, 30×10 from 5 new donors 6Isolated more than from CD14+ monocytes / donors. Medium (RPMI-1640 + 10% HABS + 2 mM GlutaMax + PS + 25 ng / ml recombinant human M-CSF) was prepared for 5 × 500 ml bags. 30 × 10 6 monocytes / bag were filled per donor. On day 6 all bags were stimulated with IFN-γ. On day 7, Mregs were harvested and counted.

[0121] Subsequently, Mreg medium (RPMI-1640 + 10% HABS + 2 mM GlutaMax + PS + 25 ng / ml recombinant human M-CSF) was prepared for subculture. Exactly 15 ml of medium was added to each of 8 × 50 ml tubes. NaCl solution or 10 ng / ml LPS (Enzo) was added as follows and vortexed. TIFF2025081763000002.tif73162

[0122] 1 × 10 6 cells / well of Mregs were seeded into 24-well plates. 1 well per condition and 1 donor was used. TIFF2025081763000003.tif66165

[0123] The cultures were incubated for exactly 48 hours. Supernatants were harvested and purified. Two aliquots of more than 500 μl were prepared. Samples were stored at -80 °C until analyzed by ELISA for VEGF-A, VEGF-C, VEGF-D and TNF-α.

[0124] Results: The results of ELISA are shown in Figure 10. As can be seen from Figure 10A, Mregs in combination with LPS induced VEGF-A, VEGF-C and TNF-α, but not VEGF-D. Under hypertonic conditions, Mregs were induced to express VEGF-C but not VEGF-A, VEGF-D or TNF-α (Figure 10B).

[0125] Literature [1] Geissler EK, Hutchinson JA. Cell therapy as a strategy to minimize maintenance 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 tolerogenicdendritic 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.

[10] Hutchinson JA, Riquelme P, Geissler EK, and Fandrich F. Human regulatory macrophages. Methods Mol. Biol. 2011; 677: 181-192.

[11] Riquelme P, Tomiuk S, Kammler A, Fandrich F, Schlitt HJ, Geissler EK, Hutchinson JA. Mol Ther. 2013; 21(2):409-22.

[12] Martinez FO, Gordon S, Locati M,Mantovani A. J Immunol 2006;177: 7303-7311.

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[14] Sironi M, Martinez FO, D'Ambrosio D et al. J Leukoc Biol 2006;80: 342-349.

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[16] Cao Y, Linden P, Farnebo J, Cao R, Eriksson A, Kumar V, Qi JH, Claesson-Welsh L, Alitalos K, Proc. Natl. Acad. Sci. USA 1998; 95: 14389-14394.

Claims

1. (a) isolating CD14 positive monocytes from a blood sample of a subject; (b) culturing the monocytes in a gas permeable bag in a medium containing (i) M-CSF and / or GM-CSF and (ii) CD16 ligand; (c) contacting the cell with IFN-γ; (d) obtaining the immunomodulatory macrophage cells from the culture medium.

2. A method for producing an immunoregulatory macrophage cell, comprising:

2. The method of claim 1, wherein in step (b) the medium comprises human serum, such as human AB serum.

3. The method according to claim 1 or 2, wherein in step (b) the concentration of said M-CSF and / or GM-CSF is in the range of 5 to 100 ng / ml, preferably 20 to 25 ng / ml.

4. The method of any of claims 1 to 3, wherein in step (b) the monocytes are cultured for at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days prior to IFN-γ stimulation.

5. The method according to any one of claims 1 to 4, wherein the gas permeable bag is made of plastic, preferably polyolefin.

6. The method according to any of claims 1 to 5, wherein in step (c) the concentration of said IFN-γ is in the range of 5 to 100 ng / ml, preferably 20 to 25 ng / ml.

7. Immunoregulatory macrophage cells obtained by the method according to claims 1 to 6.

8. Immunomodulatory macrophage cells that do not express one or more of the following markers: CD38, CD209 and syndecan-3.

9. An immunoregulatory macrophage cell expressing at least one of the markers CD103, CD10 and Clec-9a.

10. 10. The immunomodulatory macrophage cell of claim 8 or 9, further expressing at least one of the markers CD85h and CD258.

11. A pharmaceutical composition comprising the immunoregulatory macrophage cells or subcellular fractions thereof according to any one of claims 7 to 10.

12. An immunomodulatory macrophage cell or a subcellular fraction thereof according to any one of claims 7 to 10 or a pharmaceutical composition according to claim 11 for use in a method for inhibiting transplant rejection and / or prolonging transplant survival in a subject receiving a transplant.

13. 13. The immunomodulatory macrophage cells or subcellular fractions thereof or pharmaceutical composition for use in the method of any of claims 12, wherein the transplant is an allogeneic transplant.

14. The immunomodulatory macrophage cell line according to any one of claims 7 to 10 for use in a method for enhancing or prolonging the effect of a medicinal product based on engraftment or regulatory T cells. A cell or subcellular fraction thereof or a pharmaceutical composition as described in claim 11.

15. An immunoregulatory macrophage cell or a subcellular fraction thereof according to any one of claims 7 to 10 or a pharmaceutical composition according to claim 11 for use in a method for treating or preventing an autoimmune disease, an inflammatory disease or a hypersensitivity reaction.

16. The autoimmune disease may be systemic lupus erythematosus (SLE), scleroderma, Sjogren's syndrome, polymyositis, dermatomyositis and other systemic autoimmune conditions; rheumatoid arthritis (RA), juvenile rheumatoid arthritis and other inflammatory arthritis; ulcerative colitis, Crohn's disease and other inflammatory bowel diseases; autoimmune hepatitis, primary biliary cirrhosis and other autoimmune liver diseases; cutaneous small vessel vasculitis, granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, Behcet's disease, thromboangiitis obliterans, Kawasaki disease, and other autoimmune diseases of that etiology.

16. The immunomodulatory macrophage cells or subcellular fractions thereof or pharmaceutical compositions for use in the method of claim 15, selected from the group consisting of: other large, medium or small vessel vasculitis; multiple sclerosis (MS) and neuroimmunological disorders; type I diabetes, autoimmune thyroid dysfunction, autoimmune pituitary dysfunction and other autoimmune endocrinological disorders; hemolytic anemia, thrombocytopenic purpura and other autoimmune disorders of the blood and bone marrow; psoriasis, pemphigus vulgaris, pemphigoid and other autoimmune skin conditions.

17. 16. The immunomodulatory macrophage cell or subcellular fraction thereof or pharmaceutical composition for use in the method of claim 15, wherein the inflammatory disease is selected from the group consisting of arterial occlusive disease, such as peripheral arterial occlusive disease (pAOD), critical limb ischemia, arteriosclerosis, cerebral infarction, myocardial infarction, renal infarction, intestinal infarction, angina pectoris and other conditions caused by arterial blockage or stenosis; microvascular angina, also known as cardiac syndrome X; inflammation associated with systemic metabolic disorders, including type II diabetes and obesity-related metabolic syndrome; and skin diseases, including eczema.

18. 16. The immunomodulatory macrophage cell or subcellular fraction thereof or pharmaceutical composition for use in the method of claim 15, wherein the hypersensitivity reaction is selected from the group of asthma, eczema, allergic rhinitis, angioedema, drug hypersensitivity and mastocytosis.

19. An immunomodulatory macrophage cell or a subcellular fraction thereof according to any one of claims 7 to 10 or a pharmaceutical composition according to claim 11 for use in a method for promoting tissue repair processes by participating in tissue remodelling, tissue regeneration, angiogenesis, vascular development or preventing / limiting fibrosis.

20. (a) obtaining T cells from a subject using CD3e microbeads; (b) culturing said T cells with an immunomodulatory macrophage cell or a subcellular fraction thereof according to any one of claims 14 to 18; (c) obtaining the immunoregulatory T cells from the culture medium; and A method for producing immunoregulatory T cells, comprising:

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