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WO2021162554A1 - Moyens et procédés pour déterminer les propriétés des cellules stromales mésenchymateuses - Google Patents

Moyens et procédés pour déterminer les propriétés des cellules stromales mésenchymateuses Download PDF

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Publication number
WO2021162554A1
WO2021162554A1 PCT/NL2021/050096 NL2021050096W WO2021162554A1 WO 2021162554 A1 WO2021162554 A1 WO 2021162554A1 NL 2021050096 W NL2021050096 W NL 2021050096W WO 2021162554 A1 WO2021162554 A1 WO 2021162554A1
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msc
cells
fold
markers
hla
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PCT/NL2021/050096
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Willem E. Fibbe
Melissa Van Pel
Koen SCHEPERS
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Academisch Ziekenhuis Leiden H.O.D.N. Lumc
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Publication of WO2021162554A1 publication Critical patent/WO2021162554A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2301—Interleukin-1 (IL-1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/24—Interferons [IFN]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/25—Tumour necrosing factors [TNF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
    • C12N2502/1114—T cells

Definitions

  • the invention relates to methods of and kits for determining mesenchymal stromal cell (MSC) properties.
  • MSC mesenchymal stromal cell
  • Such properties include, the capacity to inhibit an immune response. For example proliferation of target cells such as T-cells, B- cells, NK cells and other immune cells and/or monocyte/ macrophage polarization resulting in immune inhibition through the production of soluble mediators and induction of regulatory T -cells (T Regs) through various mechanisms, including soluble factors such as CCL-18 and other factors.
  • T Regs regulatory T -cells
  • the invention also relates to a cell bank comprising MSC having such properties and to use of MSC having such properties,
  • MSC are a heterogeneous population of cells present in various tissues which can differentiate into a variety of cell types, including osteoblasts, chondrocytes, and adipocytes. In addition to being important for the physiological repair of various mesenchymal tissues, including cartilage and bone, MSC play a substantial role in the regulation of blood cell development in the bone marrow.
  • MSC graft- versus -host disease
  • MSC intra-and extracellular molecules
  • Many of these molecules are not expressed at steady state, but are upregulated in response to stimulation with proinflammatory molecules, such as cytokines. Not all inflammatory stimuli result in the same type and level of response. Whereas stimulation with some proinflammatory molecules Induce MSC with anti-inflammatory function, stimulation with others results in the generation of MSC with proinflammatory function.
  • the present invention provides such methods and means to perform such test.
  • the tests as disclosed herein allow for quantitative measurement of the biological activity and as such links to relevant biological properties of the cell therapy product.
  • the means and methods of the invention may serve as a release test in future MSC manufacturing. Presently, no such standard test or assay is available.
  • the invention provides a method comprising:
  • cytokines selected from; interleukin (IL)17A, IL-1 ⁇ , oneostatin M (OSM), interferon g (IFN- ⁇ ), tumor necrosis factor a (TNF- ⁇ ), IL-13 and IL-4;
  • MSC binding molecules that bind to an MSC -specific marker and one or more selected from: the extracellular markers CD 54, CD274, human leukocyte antigen (HLA)-DR, the intracellular markers COX-2 and indoleamine-pyrrole 2,3-dioxygenase (IDO); said method further comprising determining the presence of said MSC-specific marker and the fold increase of the expression of said one or more of CD54, CD274, HLA-DR, COX-2 and IDO markers in response to said one or more cytokines.
  • HLA human leukocyte antigen
  • IDO indoleamine-pyrrole 2,3-dioxygenase
  • the invention also provides a kit for determining the capacity and/or potency of MSC to inhibit an immune response. For example proliferation of target cells such as T-ceils, B-eells, NK cells and other immune cells and/or monocyte/macrophage polarization resulting in immune inhibition through the production of soluble mediators and induction of T Regs, comprising binding molecules that hind to an MSC- specific marker and one or more of CD54, CD274, HLA-DR, COX-2 and IDO.
  • target cells such as T-ceils, B-eells, NK cells and other immune cells and/or monocyte/macrophage polarization resulting in immune inhibition through the production of soluble mediators and induction of T Regs, comprising binding molecules that hind to an MSC- specific marker and one or more of CD54, CD274, HLA-DR, COX-2 and IDO.
  • a cell bank comprising MSC of at least 10 different donors, wherein an MSC-specific marker is present on the MSC of each of said donors, and wherein each of said MSC exhibit a fold increase of the expression of one or more of the markers CD54, CD274, HLA-DR, COX-2 and IDO in response to culture with one or more cytokines selected from; IL-17A, IL-18, OSM, ⁇ RN-g, TNF- ⁇ , IL-13 and IL-4.
  • a method of preventing and/or inhibiting an immune response in a subject comprising administering to said subject MSC of which the presence of an MSC- specific marker and fold increase of the expression of one or more of CD54, CD274, HLA-DR, COX-2 and IDO was determined using a method or a kit as described herein.
  • a method for preventing and/or inhibiting graft versus host disease in a subject comprising administering to said subject MSC of which the presence of an MSC- specific marker and fold increase of the expression of one or more of CD54, CD274, HLA-DR, COX-2 and IDO has been determined using a method and/or kit as described herein.
  • a method for determining the capacity and/or potency of MSC to inhibit an immune response for example proliferation of target ceils such as T-cells, B-ceils.
  • target ceils such as T-cells, B-ceils.
  • cytokines selected from: IL-17A, IL-16, OSM, lFN- ⁇ , TNF- ⁇ , IL-13 and IL-4: - contacting said MSC with binding molecules that bind to an MSC-specific marker and one or more markers selected from:
  • MSC mesenchymal stromal cells
  • cytokines selected from: interleukin (IL)17A, IL-18, oncostatin M (OSM), interferon y ( lFN- ⁇ ), tumor necrosis factor a (TNF- ⁇ ), IL-13 and IL-4; contacting said cultured MSC with binding molecules that bind to an MSC-specific marker and one or more selected from: the extracellular markers CD54, CD274, human leukocyte antigen (HLA)-DR, and the intracellular markers cyclo-oxygenase-2 (COX- 2) and in doleamlne -pyrrole 2,3-dioxygenase (IDO); and determining from the presence of said MSC-specific marker and the expression of said one or more of CD54, CD274, HLA-DR and IDO markers the capacity' and
  • a method for treating a subject in need of MSC based therapy with a population of MSC cells having therapeutic potential comprising: a) validating therapeutic potential of the cell population bye providing MSC; - culturing said MSC in the presence and in the absence of one or more cytokines selected from: interleukin (IL)17A, IL-16, oncostatin M (OSM), interferon g (IFN- g), tumor necrosis factor a ( TNF- ⁇ ), IL-13 and IL-4; contacting said MSC with binding molecules that bind to an MSC-specific marker and one or more selected from: the extracellular markers CD 54, CD274, human leukocyte antigen (HLA)-DR, and the intracellular markers cyclooxygenase' 2 (COX-2) and indoleamine- pyrrole 2, 3 -dioxygenase (IDO); b) determining the presence of said MSC-speeifie marker and the fold increase of the expression of said
  • a method for determining a therapeutic potential of mesenchymal stromal cells (MSC) to inhibit an immune response comprising:
  • cytokines selected from: interleukin (IL)17A, IL-1 ⁇ , oneostatin M (OSM). interferon g (IFN- g), tumor necrosis factor a (TNF- ⁇ ), IL-13 and IL-4;
  • MSC-specific marker binding molecules that bind to an MSC- specific marker and one or more selected from: the extracellular markers CD 54, CD274, human leukocyte antigen (HLA)-DR, and the intracellular markers cyclo- oxygenase-2 (COX-2) and indoleamine-pyrrole 2,3-dioxygenase (IDO); said method further comprising determining the presence of said MSC -specific marker and the fold increase of the expression of said one or more of CD 64, CD274, HLA- DR and IDO markers in response to said one or more cytokines and determining from said presence and said fold-increase the therapeutic potential.
  • HLA human leukocyte antigen
  • COX-2 cyclo- oxygenase-2
  • IDO indoleamine-pyrrole 2,3-dioxygenase
  • MSC cells for use in a method of treatment of a subject that: is exhibiting or is at risk of exhibiting an undesired immune response wherein said MSC cells have been determined to have therapeutic potential with a method as described herein,
  • Described herein is a method comprising: - providing MSC; culturing said MSC in the presence and in the absence of one or more cytokines selected from: IL-17A, EL- IB, OSM, lFN- ⁇ , TNF- ⁇ , IL-13 and IL-4; contacting said MSC with binding mol etudes that bind to an MSC- speeifie marker and one or more selected from: the extracellular markers CD 64, CD274, HLA-DR, the intracellular markers COX-2 and IDO; said method further comprising determining the presence of said MSC-specific marker and the fold increase of the expression of said one or more of CD64, CD274, HLA-DR, COX-2 and IDO markers in response to said one or more cytokines.
  • cytokines selected from: IL-17A, EL- IB, OSM, lFN- ⁇ , TNF- ⁇ , IL-13 and IL-4
  • the disclosure describes a method for determining if MSC have a capacity to inhibit an immune response, for example inhibition of proliferation of target cells such as T-cells, B-cells, NK cells and other immune cells and/or monocyte/macrophage polarization resulting in immune suppression through the production of soluble mediators and induction of regulatory T-cells (T Regs), comprising: providing MSC; - culturing said MSC in the presence and in the absence of one or more cytokines selected from: IL-17A, IL-lB, GSM, lFN- ⁇ , TNF- ⁇ , IL-13 and IL-4; contacting said MSC with binding molecules that bind to an MSC- speeifie marker and one or more selected from: the extracellular markers CD 54, CD274, HLA-DR, the intracellular markers COX- 2 and IDO; said method further comprising determining the presence of said MSC 'Specific marker and the fold increase of the expression of said one or more of CD54, CD274, HLA-DR
  • the invention also provides a method for determining the capacity anti/or potency of mesenchymal stromal cells (MSC) to inhibit an immune response comprising providing MSC; culturing said MSC in the presence of one or more cytokines selected from: interleukin (IL)17A, 1L-18, oncostafcin M (OSM), interferon g ( lFN- ⁇ ), tumor necrosis factor a (TNF- ⁇ ), IL-13 and IL-4; contacting said cultured MSC with binding molecules that hind to an MSC -specific marker and one or more selected from: the extracellular markers CD54, CD274, human leukocyte antigen (HLA)-DR, and the intracellular markers cyclo-oxygenase- 2 (COX- 2) and indoleamine-pyrrole 2,3-dioxygenase (IDO); and determining from the presence of said MSC-specific marker and the expression of said one or more of CD 54, CD274, HLA-DR and IDO markers the
  • a method for testing MSC as described herein comprises the method described herein above directed towards determining if MSC have a capacity to inhibit an immune response.
  • Inhibition of an immune response can be inhibition of proliferation of target cells such as T-cells, B-cells, NK cells and other immune cells and/or monocyte/maerophage polarization resulting in immune suppression through the production of soluble mediators and induction of T Regs,
  • target cells such as T-cells, B-cells, NK cells and/or monocyte/maerophage polarization.
  • inhibition of proliferation of target cells such as T-cells and B-cells and/or monocyte/maerophage polarization.
  • a method for testing MSC as described herein also refers to a method for determining the potency of MSC to inhibit an immune response.
  • Inhibition of an immune response can be inhibition of proliferation of target cells such as T-cells, B-ceils, NK cells and other immune cells and/or monocyte/macrophage polarization resulting in immune suppression through the production of soluble mediators and induction of T Regs.
  • target cells such as T-cells, B-ceils, NK cells and other immune cells and/or monocyte/macrophage polarization resulting in immune suppression through the production of soluble mediators and induction of T Regs.
  • target ceils such as T-eells, B- cells, NK cells and/or monocyte/macrophage polarization.
  • inhibition of proliferation of target cells such as T-cells and B-cells and/or monocyte/macrophage polarization.
  • the capacity and the potency are measures for the quality of MSC for use in anti-inflammatory and regenerative settings, MSC batches that meet the criteria set herein are suitable for administration to a human with the purpose of reducing an undesired inflammatory immune response and/or enhancing regeneration in the thus transplanted human host.
  • HLA-DR, COX- 2 and IDO markers in response to culture with one or more cytokines as disclosed herein indicate that such MSC have a high potency and/or capacity to inhibit an immune response when administered to a subject.
  • MSC with such potency or capacity are very suitable for administration to subjects in need thereof,
  • MSC mesenchymal stromal cells. These stromal cells are sometimes also referred to as mesenchymal stem cells, marrow stromal cells or multipotent mesenchymal stromal cells etc, MSC are adherent multipotent stromal cells that as a population can differentiate into a variety of cell types, including osteoblasts ⁇ hone cells), chondrocytes ⁇ cartilage cells), myocytes (muscle cells) and adipocytes (fat cells) . MSC have a capacity for self- renewal while maintaining their multipotency, MSC have an effect on innate and specific immune cells, MSC can produce a range of molecules having immunomodulatory effects.
  • the cells can be derived from various regions of the body such as for instance, bone marrow, umbilical cord, adipose tissue, amniotic fluid, and molar cells.
  • MSC from different sources typically exhibit similar functional properties, although the magnitude of the effects may vary.
  • MSC have an effect on immune cells such as macrophages, neutrophils, NK cells, mast cells and dendritic cells in innate immunity. MSC are able to migrate to the site of injury where they can exhibit an anti-inflammatory effect or modulate immune responses using intermediate cells such a macrophages.
  • an anti-inflammatory effect is preferably estimated on the basis of a capacity to inhibit immune responses, such as inhibition of proliferation of target cells such as T-cells, R- eelis, NK cells and other immune cells and/or monoeyte/macrophage polarization resulting in immune suppression through the production of soluble mediators and induction of T Regs,
  • MSC-specific marker such a marker can be: CD73, CD90, CD 105, CD29, HLA-ABC, CD 166, CD146, CD44, CD 140a,
  • CD 140b These markers are well known in the art. Antibodies and other binding molecules with which these markers can be visualized are commercially' available.
  • CD 140b is used as an MSC- specific marker.
  • CD 90, or CD 105 is used as an MSC- specific marker.
  • CD 73 is used as an MSC-specifle marker.
  • MSC -specific markers are also markers that specifically detect subsets of MSCs. Examples of such subset markers are CD 13, MSCA-1, CD 56, CD271, CD362 and Stro-1. Other MSC markers will likely be developed further and such are also included in the invention.
  • the expression is of course measured and/or fold increase determined for the same binding molecules that the MSC are contacted with.
  • MSC contacted with binding molecules for one or more markers selected from: the extracellular markers CD64, CD274, HLA-DE, the intracellular markers COX-2 and IDO it is preferred that the MSC are contacted with binding molecules for two or more of the markers, preferably 3 or more of the markers, more preferably 4 or more of the markers. Increasing the number of markers increases the accuracy of the determination.
  • the markers are selected from CD54, CD274, HLA-DR, COX-2 and IDO.
  • markers CD54, CD274, HLA-DE and IDO Preferably from markers CD54, CD274, HLA-DE and IDO. It is preferred that the markers at least comprise CD54 and IDO, preferably at least CD 54, IDO and CD274, more preferably at least CD54, IDO, CD274 and HLA-DR,
  • the MSC are contacted with binding molecules for two or more markers selected from: the extracellular markers CD54, CD274, HLA-DR.
  • the intracellular markers COX-2 and IDO Preferably with binding molecules for three or more markers selected from: the extracellular markers CD54, CD274, HLA-DE, the intracellular markers COX-2 and IDO.
  • binding molecules for all of the markers CD54, CD274, HLA-DR, COX-2 and IDO are contacted with binding molecules for two or more markers selected from: the extracellular markers CD54, CD274, HLA-DR.
  • the MSC are contacted with binding molecules for two or more markers selected from: the extracellular markers CD54, CD274, HLA-DR, the intracellular marker IDO.
  • binding molecules for three or more markers selected from: the extracellular markers CD54, CD274, HLA-DR, the intracellular marker IDO Preferably with binding molecules for the four markers selected from: the extracellular markers CD54, CD274, HLA-DE, and the intracellular marker IDO.
  • the MSC-speeifie marker is present on at least 90 % of the cells and the fold increase of expression of CD54 is at least 10- fold; of CD274 is at least 8-fold; of HLA-DR is at least 8-fold preferably at least 25-fold, of COX-2 is at least 3-fold and/or of IDO is at least 5-faid.
  • Biological activities of MSC include, but are not limited to, inhibition of T-cell proliferation and differentiation of monocytes to DC, Where herein reference is made to the terra “inhibit” in relation to MSC, the term refers to the capacity of MSC to prevent or decrease the extent of a process or action.
  • T- ceils are a type of lymphocyte which develop in the thymus gland and play a central role in the adaptive immune response, T-cells have an important role in controlling and shaping the immune response by providing a variety of immune- related functions. Proliferation of T-cells is associated with increased immune-related activity . Methods of inducing T-eell proliferation are known in the art. An exemplary method is described in the examples, Regulatory T-cells, or T Regs are also known as suppressor T-cells, They are a subpopulation of T-cells that modulate the immune system, prevent auto-immune disease, and maintain tolerance to self-antigens.
  • B-cel!s are a type of lymphocyte that secretes antibodies. They are part of the adaptive immune response. In addition, B-eelis can also present antigens and secrete cytokines.
  • NK-eelis are a type of cytotoxic lymphocytes, that is importan t for the immune system. They derive from the same progenitor cell as T- and B-cells, but are part of the innate immune system. NK-cells are best known for killing viral!y infected cells, and detecting and controlling early signs of cancer.
  • Monocytes are a type of leukocyte which can polarize towards DC or macrophages under inflammatory conditions.
  • An exemplary method for polarizing monocytes to DC is described in the examples.
  • DC are antigen-presenting cells of the mammalian immune system. Their main function is to process antigen material and present it on the cell surface to T-cells, resulting in T-cell activation and/or proliferation.
  • Macrophages are specialized cells involved in the detection, phagocytosis and destruction of e.g. microorganisms and dead cells,
  • MSC can he provided freshly isolated from tissue.
  • MSC can be provided from an existing culture.
  • MSC are preferably obtained from frozen/eryopreserved storage. After thawing and washing such cells can be used directly or first be cultured.
  • a method as described herein comprises providing MSC; culturing said MSC in the presence and in the absence of one or more cytokines selected from: IL-17A, IL-lB, OSM, 1FN-Y, TNF- ⁇ , IL-13 and IL-4; contacting said MSC with binding molecules that bind to an MSC-sspecific marker and one or more selected from: the extracellular markers CD54, CD274, HLA-DR, the intracellular markers COX-2, and IDO; and determining the presence of said MSC- specific marker and the fold increase of the expression of said one or more of CD54, C-D274, HLA-DR, COX-2 and IDO markers in response to said one or more cytokines.
  • cytokines selected from: IL-17A, IL-lB, OSM, 1FN-Y, TNF- ⁇ , IL-13 and IL-4
  • Cytokines are a category of small proteins that are important in cell signaling. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Cytokines have been shown to be involved in autocrine, paracrine and endocrine signaling as immune-modulating agents. They act through receptors, and have an effect on the immune system; cytokines can modulate the balance between humoral and cell-based immune responses, and can regulate the maturation, growth, and responsiveness of particular cell populations. They are important in health and disease, specifically In host responses to infection, immune responses, inflammation, trauma, sepsis, cancer, and reproduction.
  • a proinflammatory cytokine is a cytokine with an inflammatory stimulatory effect
  • a proinflammatory cytokine is often secreted from cells that promote inflammation, like (helper) T-cells
  • Inflammatory cytokines have pleiotropic effects that together have an upregulating effect on inflammatory reactions.
  • Proinflammatory cytokines include, but are not limited to: IL-1 ⁇ , IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, 1L17A, IL-18, TNF- ⁇ , lFN- ⁇ , granulocyte- macrophage colony stimulating factor (GM-CSF), GSM, MCP-1, monocyte chemoattractant protein- 1
  • MCP-1 Chemokine C-C motif ligand 5 (CCL5)/regulated on activation, normal T-eeil expressed and secreted (RANTES), macrophage inflammatory protein (MIP-lu),
  • the cytokines IL-17A, IL-1 ⁇ , OSM, lFN- ⁇ , TNF- ⁇ , IL-13 and IL-4 are well known in the art and suitable sources of cytokines are available to the skilled person.
  • the cytokines are typically used in saturating amounts which for the indicated cytokines amounts to a concentration in the range of 0.1 ng/ml to 100 ng/uil. Suitable concentrations for the cytokines are indicated in the examples and are typically in the range of 5-60 ng/ml.
  • TNF- ⁇ is preferably present in a concentration of at least lng/ml.
  • lFN- ⁇ is preferably present in a concentration of at least 0.33 ng/ml.
  • IL-17A is preferably present in a concentration of at least 50 ng/ml.
  • IL-1 ⁇ is preferably present in a concentration of at least 1 ng/ml.
  • OSM is preferably present in a concentration of at least 20 ng/ml.
  • IL-13 is preferably present in a concentration of at least 20 ng/ml.
  • IL-4 is preferably present in a concentration of at least 20 ng/ml.
  • the amount of it used can be adjusted as is typically done by the person skilled in the art.
  • the particular activity (quality) of a certain batch of cytokine, antibody or other binding molecule can suitably be determined on arrival of the batch.
  • IL-17A, IL-18, OSM, lFN- ⁇ , TNF-o, IL-13 and IL-4 alone or combinations thereof can be used in the culture of MSC as described herein.
  • the eytokine(s) used in the culture are one or more of IL-17A, IL-1 ⁇ , OSM, IFN-Y, TNF- ⁇ and IL-13; preferably one or more of IL-17A, IL-1 ⁇ and OSM; or preferably one or more of lFN- ⁇ , TNF- ⁇ or IL-13.
  • a combination of two or more of the mentioned cytokines is used, preferably 3, 4, 5, 6 or all 7 of said cytokines.
  • a combination of 6 is used, preferably of the cytokines L-17A, IL-1 ⁇ , OSM, lFN- ⁇ , TNF- ⁇ and IL-13.
  • a combination of 3 is used.
  • the combination of three is IL- G7A, IL-1 ⁇ and GSM,
  • the combination of three is lFN- ⁇ , TNF- ⁇ or IL-13.
  • the combination of two is IFN-Y and TNF- ⁇ .
  • Suitable cultures with one cytokine are cultures with lFN- ⁇ or with IL-16,
  • the cytoklneis) used in a method of the invention is/are lFN- ⁇ , IL-1 ⁇ , or the combination of lFN- ⁇ and TNF- ⁇ .
  • the one cytokine or combination of cytokines is used that routinely gives a suitable and preferably the highest difference in expression of CD54, CD274, HLA-DE, COX-2 and/or IDO between the two cultures of a typical MSC preparation.
  • This typical preparation can be a standardized preparation of MSC or model cells or model cell lines that are a good tell-tale for the effect of the cytokines on MSC.
  • the combination of cytokines is determined by measuring the cytokines present in the MSC recipient.
  • the markers CD54, CD274, HLA-DR, COX-2 and IDO are well known in the aid. Antibodies and other binding molecules with which these markers can be visualized are commercially available.
  • the MSC that are to be tested are preferably a representative sample of a larger batch of MSC of which the functionality is to be predicted.
  • the culturing in the presence or absence of certain cytokine(s) is typically done by culturing two samples of the same batch of MSC separately in different cultures, one with and one without the mentioned eytokine(s).
  • the cultures are typically performed in parallel but not necessarily so.
  • the culture without a cytokine can also be a historical reference. For instance of a different batch of MSC cultured in the absence of said cytokine(s).
  • Cultures with and without cytokines are typically essentially the same hut for the presence of the cytokine.
  • the representative MSC sample is split into two or more aliquots and one aliquot is cultured in the absence of the indicated eytokine(s) and another aliquot is cultured in the presence of the indicated cytokine(s), A difference in staining of the MSC of the two cultures is indicative for the functionality of the cells.
  • MSC are preferably ⁇ cultured at 37°C and 5% C02. Methods for culturing MSC are described in the examples section. Effects of stimuli, such as cytokines, on MSC may be risible directly after culture or after an incubation period.
  • Cytokines are a broad category of small proteins that are important in inducing signaling in cells. Cytokines have been shown to he involved in autocrine, paracrine and endocrine signaling as immune- modulating agents. Cytokines include ehemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. They act through cell surface receptors, and are especially important in the immune system ; cytokines modulate the balance between humoral immunity and cell based immune responses, and regulate the maturation, growth, and responsiveness of particular cell populations.
  • a proinflammatory cytokine is a type of signaling molecule that is secreted from immune cells that promote inflammation, like (helper) T 'Cells
  • Inflammatory cytokines play an important role in mediating the innate immune response and are involved in the upregulation of inflammatory reactions
  • Proinflammatory cytokines include, but are not limited to: IL-1 ⁇ , IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, IL17A, IL-18, TNF- ⁇ , lFN- ⁇ , granulocyte-macrophage colony stimulating factor (GM-CSF), OSM, MCP-1, CCLo/RANTES, MIP-la, MIPlB, CD40L.
  • a method for testing MSC as described herein preferably comprises providing MSC: optionally thawing said MSC when said MSC are retrieved from storage; and culturing said MSC in presence and in the absence of one or more of the mentioned cytokines IL-17A, IL-18, OSM, lFN- ⁇ , TNF- ⁇ , IL-13 and IL-4.
  • the method preferably further comprises contacting said MSC with binding molecules that bind to an MSC- speeific marker and one or more selected from; the extracellular markers CD 64, CD274, HLA-DR, the intracellular markers COX- 2 and IDO.
  • Contacting said MSC with binding molecules preferably comprises: contacting said MSC with binding molecules that bind to an MSC-speelfie marker, and one or more selected from: the extracellular markers CD 54, CD274, HLA-DR; washing and permeabilizing said MSC; and contacting said MSC with a binding molecule that hinds said intracellular markers COX- 2 and/or IDO,
  • the capacity and/or potency of said MSC is determined on the basis of the expression of an MSC-speciflc marker on said cells, and the fold Increase in expression of the markers CD54, CD274, HLA-DR, COX-2 and/or IDO of MSC when cultured in the presence of the cytokine(s) when compared to the absence thereof.
  • the MSC are said to have the capacity when said MSC- specific marker is present on at least 90 % of the ceils and the fold increase of expression of CD54 is at least 3-fold, preferably at least 5 fold and more preferably at least 10-fold for CD54; is preferably at least 2-fold, preferably at least 4-fold and more preferably at least 8-fold for CD274; is preferably at least 5-fold, preferably at least 8 fold and more preferably at least 16-fold and more preferably at least 25-fold for HLA-DR, is preferably at least 2-fold, and more preferably at least 3-fold for COX-2, and/or is preferably at least 3- fold and more preferably at least 5-fold for IDO.
  • MSC are said to have the capacity when said MSC -specific marker Is present on at least 90 % of the cells and the fold increase of expression of CD54 is at least 10-fold; of CD274 is at least 8-fold; of HLA-DR is at least 8-fold preferably at least 25-fold, of COX-2 is at least 3-fold and/or of IDO is at least 5-fold.
  • the MSC are said to have the potency when said MSC-specific marker is present on at least 90 % of the ceils and the fold increase of expression of CD54 is at least 3-fold, preferably at least 5 fold and more preferably at least 10-fold for CD54; is preferably at least 2-fold, preferably at least 4-fold and more preferably at least 8- fold for CD274; is preferably at least 5-fold, preferably at least 8 fold and more preferably at least 16-fold and more preferably at least 25-fold for HLA-DR, is preferably at least 2-fold, and more preferably at least 3-fold for COX-2, and/or is preferably at least 3- fold, and more preferably at least d-fold for IDO.
  • the MSC are said to have the potency when said MSC-specific marker is present on at least 90 % of the cells and the fold increase of expression of CD54 is at least 10-fold; of CD274 is at least 8-fold; of HLA-DR is at least 8-fold preferably at least 25-fold, of COX-2 is at least 3-fold and/or of IDO is at least 5-fold,
  • the invention provides a method for determining the capacity and/or potency of mesenchymal stromal cells (MSC) to inhibit an immune response comprising providing MSC; culturing said MSC in the presence of one or more cytokines selected from : interleukin (IL)17A, lL-18, oneostatin M (OSM), interferon g ( lFN- ⁇ ), tumor necrosis factor a (TNF- ⁇ ), IL-13 and IL- 4; contacting said cultured MSC with binding molecules that hind to an MSC- specific marker and one or more selected from: the extracellular markers CD54, CD274, human leukocyte antigen (HLA)-DR, and the intracellular markers cyclo-oxygenase- 2 (COX- 2) and indoleamine-pyrroie 2,3-dioxygenase (IDO); and determining from the presence of said MSC-specific marker and the expression of said one or more of CD 54, CD274, HLA-DR and I
  • the reference is a parallel sample of the same MSC culture in the absence of said one or more cytokines.
  • the comparison of the reference and the cytokine cultured sample preferably yields a score for the relative increase of the expression of said one or more of CD 54, CD274, HLA-DR and IDO markers in said cytokine cultured sample.
  • the score is preferably a fold-increase of expression of one or more of said markers in the cytokine cultured sample when compared to a reference MSC population cultured in the absence of said cytokines.
  • Said MSC-specific marker is preferably present on at least 90 % of the cells and the fold increase of expression of CD 54 is at least 3 -fold, preferably at least 5 fold and more preferably at least 10-fold for CD 54; is preferably at least 2 -fold, preferably at least 4-fold and more preferably at least 8-fold for CD274; is preferably at least 5 -fold, preferably at least 8 fold and more preferably at least 16-fold and more preferably at least 25-fold for HLA-DR, Is preferably at least 2-fold, and more preferably at least 3- fold for COX-2, and/or is preferably at least 3-fold, and more preferably at least 5-fold for IDO.
  • the Invention further provides a kit comprising binding molecules that bind to an MSC-specific marker and one or more markers selected from: CD 54, CD274, HLA- DR, COX-2 and IDO,
  • the kit comprises binding molecules that hind to an MSC-specific marker, CD54, CD274, HLA-DR, COX-2 and IDO.
  • the kit comprises binding molecules that bind to CD73, CD54, CD274, HLA-DR, COX-2 and IDO.
  • the kit is useful in a method as described herein. Such a kit is useful in methods for determining if MSC have a capacity to inhibit an immune response.
  • the kit Is also useful in a method for determining the potency of MSC to Inhibit an immune response.
  • inhibition of proliferation of target cells such as T-cells, B-eelis, NK cells and other immune cells and/or monocyte/macrophage polarization resulting in immune suppression through the production of soluble mediators and induction of T Regs.
  • the kit is also useful in a method to predict recipient response to administration of said MSC.
  • the kit preferably further comprises one or more cytokines selected from: IL17A, IL-18, OSM, IFN-y, TNF- ⁇ , IL-13, IL-4.
  • cytokines selected from: IL17A, IL-18, OSM, IFN-y, TNF- ⁇ , IL-13, IL-4.
  • the preferred number of cytokines and the preferred single or combinations of cytokines are as described elsewhere herein.
  • a binding molecule as defined herein is a proteinaceous binding molecule.
  • the binding molecule is typically a peptide, a cyclic or bieyclic peptide of up to and including 20 amino acids or a polypeptide having more than 20 amino acid residues.
  • Proteinaceous binding molecules Often these include one or more complete or derivative antibody variable domains.
  • Non-limiting examples are single chain Fv-fragments, monobodies, VHH, Fab -fragments.
  • Derivative variable domains can be artificial or naturally evolved derivatives both belonging to the class of proteins that have the immunoglobulin fold. Examples of non-immunoglobulin fold containing proteinaceous binding moieties are the avimers initially developed by Amgen.
  • a binding molecule can be any type of molecule that is capable of specifically binding to the indicated markers.
  • Binding molecules as disclosed herein are preferably bivalent monospecific binding molecules. Binding molecules preferably comprise at least a variable domain of an antibody.
  • the binding molecules are antibodies, preferably monoclonal antibodies, i.e. bivalent monospecific antibodies.
  • Antibodies are well known, well defined molecules of which the production and the use in labelling of cells has matured to the extent that such use has become routine and robust, a characteristic that is highly desired in a method for testing MSC as described herein, A feature of such a test is that it is robust and easily incorporated in the procedure of a new lab without typically requiring extensive optimization. Such robustness is especially important when quantitative results are desired.
  • HLA-DR, COX-2 and IDO markers is detected using antibodies.
  • the presence of a MSC-specific marker can be detected using any specific binding molecule.
  • the presence of said MSC-specific marker is detected with an antibody against an MSC-specific marker
  • Binding molecules such as antibodies are preferably labelled with fluo.rochro.mos that are suitable for parallel flow cytometric analysis.
  • the parallel flow cytometric analysis is preferably one wherein the signals of said fiuoroehromes can be acquired independently .
  • the invention further provides a cell bank comprising MSC of at least 10 different donors, wherein an MSC -specific marker is present on the MSC of each of said donors, and wherein each of said MSC exhibit a fold increase of the expression of one or more of the markers CD54, CD274, HLA-DE, COX-2 and IDO in response to one or more cytokines selected from: 1L-17A, IL-18, QSM, lFN-g, TNF- ⁇ , IL-13 and 1L- 4,
  • the cell bank is preferably one wherein the presence and/or the fold increase in expression of said markers of said MSC was determined using a method of testing MSC as described herein or a kit as described herein.
  • the invention further provides a method of preventing and/or inhibiting an immune response in a subject, the method comprising administering to said subject MSC of which the expression of and MSC- specific marker and fold increase of the expression of one or more of the markers CD54, CD274, HLA-DE, COX-2 and IDO was determined using a method and/or a kit as described herein.
  • the subject is exhibiting or is at risk of exhibiting an undesired immune response.
  • the subject is exhibiting inflammatory bowel disease.
  • the subject is the recipient of an allogeneic cell or organ transplant.
  • the subject is diagnosed with GvHD.
  • a method for preventing and/or inhibiting graft versus host disease in a subject comprising administering to said subject MSC of which the presence and fold increase of the expression of said markers has been determined using a method and/or a kit as described herein.
  • Also provided is a method comprising: providing MSC; culturing said MSC in the presence and in the absence of one or more cytokines selected from: 1L-17A, IL-1B, OSM, lFN-g, TNF- ⁇ , IL-13 and 1L-4; contacting said MSC with binding molecules that bind to an MSC- specific marker and one or more selected from: the extracellular markers CD54, CD274, H LA-DR, the intracellular marker COX-2 and IDO; said method further comprising determining the expression of said MSC- specific marker and the fold increase of the expression of said one or more of CD 54, CD274, HLA-DE. COX-2 and IDO markers in response to said one or more cytokines.
  • the fold increase as referred to in a method of treatment or population for use in a method of treatment as described herein is preferably at least 3-fold, preferably at least 5 fold and more preferably at least 10-fold for CD54; is preferably at least 2-fold, preferably at least 4-fold and more preferably at least 8-fold for CD274; is preferably at least 5-fold, preferably at least 8 fold and more preferably at least 16-fold and more preferably at least 25-fold for HLA-DR, is preferably at least 2-fold, and more preferably at least 3-fold for COX-2, and/or is preferably at least 3-fold, and more preferably at least 5-fold for IDO.
  • the fold increase of expression is preferably at least: 10-fold for CD54; 8-fold for CD274; 8-fold and preferably 26-fold for HLA-DR, 3-fold for COX-2, and/or 5-fold for IDO.
  • the disclosure provides a method comprising: providing MSC; optionally thawing said MSC, when said MSC are provided frozen; culturing said MSC in presence and in the absence of one or more cytokines selected from: IL-17A, IL-1 ⁇ , OSM, lFN- ⁇ , TNF- ⁇ , IL-13 and IL-4; contacting said MSC with binding molecules that bind to an MSC- specific marker and one or more selected from: the extracellular markers CD54, CD274, HLA-DR, the intracellular markers COX-2 and IDO; said method further comprising determining the presence of said MSC-specific marker and the fold increase of the expression of said one or more of CD54, CD274, HLA-DR, COX-2 and IDO in response to said one or more cytokines.
  • the disclosure provides a method comprising: providing MSC; culturing said MSC in presence and in the absence of one or more cytokines selected from:
  • cytokines are present in a saturating amount, preferably in a concentration ranging from Q.lng/ml to lOOng/ml, contacting said MSC with binding molecules that bind to an MSC- specific marker and one or more selected from: the extracellular markers CD54, CD274, HLA-DR, the intracellular markers COX-2 and IDO; said method further comprising determining the presence of said MSC-specific marker and the fold increase of the expression of said one or more of CD54, CD274, HLA-DR, COX- 2 and IDO in response to said one or more cytokines.
  • the disclosure provides a method comprising: providing MSC; culturing said MSC in presence and in the absence of IL-lti lFN- ⁇ , or IFN-Y and TNF- ⁇ ; wherein said cytokines are present in a saturating amount, preferably in a concentration ranging from Q.lng/ml to lOOng/ml, more preferably IL- 1B in a concentration of Ing/ml, IFN-y in a concentration of lOng/ml, or lFN- ⁇ in a concentration of lOng/ml and TNF- ⁇ in a concentration of Ing/ml.
  • MSC-specific marker binds to an MSC- specific marker and one or more selected from: the extracellular markers CD54, CD274, HLA-DR, the intracellular markers COX- 2 and IDO; said .method further comprising determining the presence of said MSC- specific marker and fold increase of the expression of said one or more of CDS4, CD274, HLA- DR, COX-2 and IDO in response to said one or more cytokines.
  • antibody refers to an immunoglobulin molecule that is typically composed of two identical parrs of polypeptide chains, each pair having one "heavy” (H) chain and one "light” (L) chain.
  • Human light chains are classified as kappa (K) and lambda (l).
  • Heavy chains are classified as mu. delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • HCVR heavy chain variable region
  • the heavy chain constant regions of IgD, IgG, and IgA are comprised of three domains, CHI, CH2 and CHS, and the heavy chain constant regions of IgM and IgE are comprised of four domains, CHI, CH2, CH3, and CH4.
  • Each light chain is comprised of a light chain variable region ⁇ abbreviated herein as LCVS or VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells).
  • VH and VL regions can he further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR),
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDEs and four FRs, arranged from the amino- terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4.
  • the variable regions of the light and heavy chain together form the antibody binding site and defines the specificity for the epitope.
  • binding molecules are labeled with fiuoroehromes.
  • Fiuoroehromes suitable for labeling binding molecules and detecting markers bound by labeled binding molecules are known in the art.
  • fiuoroehromes used in a method or present in a kit as disclosed herein enable flow cytometric detection of binding molecules bound to their target.
  • binding molecules of a method anti/or kit as disclosed herein are fluorescent! ⁇ 7 labeled.
  • binding molecules that bind to the same marker are labeled with the same fluoroehrome.
  • groups of binding molecule that bind different markers are labeled with distinct fiuoroehromes.
  • fiuorescently labeled binding molecules of a method and/or kit as disclosed herein are fiuorescently labeled antibodies.
  • Fiuorescently labeled antibodies can be commercially obtained from various companies, including, but not limited to: Bee ton Dickinson (BD), eBiosciences, Bio legend, Dako, Beckman Coulter, CYTOGNOS, Cal tag, Pharmlngen, Exbio, Sanquin, R&D systems, and Invitrogen,
  • MSC are contacted with a saturating amount of fiuorescently labeled binding molecules or antibodies.
  • the fiuoroehromes are selected for brightness, limited spectral overlap and limited need for compensation, stability, etc. (see: Kalina et al. Leukemia 2012; 26: 1986).
  • all fluorochromes of a method and/or kit as disclosed herein can he acquired independently from each other.
  • Fluorochromes suitable for a method and/or kit of the disclosure include, but are not limited to: pacific blue (PacB), brilliant violet 421 (BV421), Horizon V450, pacific orange (PacO), Horizon V60Q (HVoQQ), BV6I0, Khrome orange (KO), 00515, Horizon BB515, fluorescein isothiocyanate (FITC), Alexa488, phyeoerythrin (PE), peridinin chlorophyl protein/eyanine 5.6 (PerCP-CyS.5), PerCP, PE Texas Red, phycoerythrin/cyanine?
  • PE-Cy7 allophycocyanine
  • API allophycocyanine
  • AFC-H7 allophycocyanine/hilite 7
  • fluorescently labeled binding molecules of a method and/or kit as described herein comprise the fluorochromes PE, APC, APC-H7, BV421, PE- Cy7.
  • a method and/or kit as described herein comprises PE- labeled antibodies that bind to CD54, APC-laheled antibodies that bind to CD274,
  • the procedures and methods for testing MSC as described herein are preferably performed using standardized flow -cytometer settings, above the electronic noise and target values for the photomultiplier tube (PMT) set using rainbow beads. Multicolor immunostaining and flow -cytometry is preferably performed according to the so-called Euro Flow protocols as described by Van Dongen et al. (Leukemia 2012; 26: 1908) and by Kalina et al.
  • a method as described herein comprises the use of software for data integration and multidimensional analysis of flow cytometry files.
  • the combinations of fluorescently labeled binding molecules as disclosed can be used in combination with commercially available software tools.
  • a method and/or kit of the disclosure is suitable for acquisition of fluorescence according to the standards of the Euro Flow consortium.
  • standards include, but are not limited to: flow cytometry acquisition settings, antibody concentration, antibodies, fluorochromes, and combinations of fluorochromes.
  • the disclosure provides a method comprising: providing MSC; culturing said MSC in presence and in the absence of one or more cytokines selected from: IL-17A, IL-16, OSM, IEN-g, TNF- ⁇ , IL-13 and IL-4: - contacting said MSC with antibodies that bind to an MSC -specific marker and one or more selected from: the extracellular markers CDS4, CD274, HLA- DR, the intracellular markers COX-2 and IDO; said method further comprising determining the presence of said MSC-specific marker and fold increase of the expression of said one or more of CD54, CD274, HLA- DR, COX-2 and IDO in response to said one or more cytokines.
  • marker refers to proteins expressed by ceils, which can serve to help identify and classify cells, Extracellular .markers are present on the outside of the cell, intracellular markers are present on the inside of the cell. Some markers are unique for cells types, others are more widely expressed. Specific combinations of markers can predict certain capacities of cells.
  • Markers suitable for, or that can he added to the method include, but are not limited to: CD la, CD2, CDS, CD4, CDS, CD6, CD7, CD8a, CD9, GDI la, CD lib, CD 11c, CD 14, CD16, CD 17, GDIS, CD19, CD20, CD21, CD22, CD23, CD24, CD2o, CD27, CD28, CD29, CD30, CD31, CD33, CD34, CD3S, CD36, CD37, CD38, CD40, CD41, CD42b, CD43, CD44, CD46, CD47, CD48, CD49d, CD50, CD51, CDS2, CDS3, CDo4, CD65, CD56, CD58, CD59, CD61, CD62L, CD62P, CD63, CD66a, CD 66b, CD66c, CD66d/e, CD69, CD71, CD72, CD73, CD79a, CD80, CD90, CD9S, CD97, CD98, CD99, CD 105,
  • the disclosure provides a method comprising: providing MSC; culturing said MSC in presence and in the absence of one or more cytokines selected from: IL-I7A, IL-1 ⁇ , OSM, lFN- ⁇ , TNF- ⁇ , IL-13 and IL-4; contacting said MSC with antibodies that bind to an MSC- specific marker and two or more selected from: the extracellular markers CD54, CD274, HLA- DR, the intracellular markers COX-2 and IDO; said method further comprising determining the presence of said MSC-specific marker and fold increase of the expression of said two or more of CD54, CD274, HLA- DR, COX-2 and IDO in response to said one or more cytokines.
  • cytokines selected from: IL-I7A, IL-1 ⁇ , OSM, lFN- ⁇ , TNF- ⁇ , IL-13 and IL-4
  • the disclosure provides a method comprising: providing MSC; culturing said MSC in presence and in the absence of one or more cytokines selected from: IL-17A, IL-1 ⁇ , OSM, IFN-y, TNF- ⁇ , IL-13 and IL-4; contacting said MSC with binding molecules that bind to an MSC specific extracellular markers and three or more of the extracellular markers CD 54, CD274, HLA-DR and the intracellular markers COX-2 and IDO said method further comprising determining the presence of said MSC-specific marker and fold increase of the expression of said three or more of CD54, CD274,
  • HLA-DR HLA-DR
  • COX-2 IDO in response to said one or more cytokines.
  • the disclosure provides a method comprising: providing MSC; - culturing said MSC in presence and in the absence of one or more cytokines selected from: IL-I7A, IL-16, OSM, lFN- ⁇ , TNF- ⁇ , IL-13 and IL-4; contacting said MSC with binding molecules that bind to an MSC specific extracellular markers and CD54, CD274, HLA-DR and the intracellular markers COX- 2 and/or IDO said method further comprising determining the presence of said MSC-specific marker and fold increase of the expression of CD54, CD274, HLA-DR, COX-2 and/or IDO in response to said one or more cytokines.
  • cytokines selected from: IL-I7A, IL-16, OSM, lFN- ⁇ , TNF- ⁇ , IL-13 and IL-4
  • a method wherein an intracellular marker is contacted with binding molecules comprises additional steps of washing, fixing, and permeabilizing the ceils.
  • the binding molecules that bind to extracellular markers are washed away first.
  • MSC are fixed.
  • the MSC are permeabiiized, where after the cells are contacted with binding molecules that bind to intracellular markers.
  • the disclosure provides a method comprising: providing MSC; culturing said MSC in presence and in the absence of one or more cytokines selected from: IL-17A, IL-lfi OSM, lFN- ⁇ , TNF- ⁇ , IL-13 and IL-4; - contacting said MSC with binding molecules that bind to an MSC- specific marker and one or more selected from: the extracellular markers CD54, CD274, HLA-DR washing said MSC permeabilizing said MSC - contacting said MSC with binding molecules that bind to the intracellular markers COX-2 and/or IDO; said method further comprising determining the presence of said MSC-specific marker and fold increase of the expression of said two or more of CD54, CD274, HLA- DR, COX-2 and IDO in response to said one or more cytokines.
  • cytokines selected from: IL-17A, IL-lfi OSM, lFN- ⁇ , TNF- ⁇ , IL-13 and IL-4
  • the disclosure provides a method comprising: providing MSC; culturing said MSC in presence and in the absence of one or more cytokines selected from: IL-17A, IL-1 ⁇ , OSM, lFN- ⁇ , TNF- ⁇ , IL-13 and IL-4; contacting said MSC with binding molecules that bind to an MSC- specifie marker and two or more selected from: the extracellular markers CD 54, CD274, HLA-DR, and: washing said MSC; - permeabilizing said MSC; contacting said MSC with binding molecules that bind to the intracellular markers COX-2 and/or IDO; said method further comprising determining the presence of said MSC-specific marker and fold increase of the expression of three or more of CD54, CD274, HLA-DR, COX-2 and IDO in response to said one or more cytokines.
  • cytokines selected from: IL-17A, IL-1 ⁇ , OSM, lFN- ⁇ , TNF- ⁇ , IL-13 and IL-4
  • the disclosure provides a method comprising: providing MSC; culturing said MSC in presence and in the absence of one or more cytokines selected from: IL-17A, 1L-1B, OSM, IRN-g, TNR-a, IL-13 and 1L-4; contacting said MSC with binding molecules that bind to an MSC- specific marker and extracellular markers CD 51.
  • determining refers to the action of ascertaining or establishing by research or calculation.
  • a capacity of MSC to inhibit an immune response For example inhibit proliferation of target cells such as T-ceils, B-eelis, NK cells and other immune cells and/or monoeyte/macrophage polarization resulting in immune suppression through the production of soluble mediators and induction of T Regs is determined on basis of the presence of an MSC-specific marker on the MSC and the fold increase of the expression of one or more other markers in response to one or more cytokines.
  • presence of a marker
  • fold increase the term refers to a ratio.
  • the fold increase indicates the number of times the presence of a marker on MSC has increased as a result of contacting MSC with one or more cytokines.
  • presence of a marker is deducted from the presence of a binding molecule bound to that marker.
  • binding .molecule is labelled with a iluorochrome.
  • the fold increase in presence of a marker is typically calculated by dividing the fluorescent signal of the marker on MSC after culturing in presence of one or more cytokines, by the fluorescent signal of the marker on MSC after culturing in absence of cytokines.
  • Fold increase can also be referred to as fold induction.
  • expression of a .marker
  • the reference is to the amount of the marker protein present in or on the cell at the time of analysis. This is also referred to as the level of the m arker or protein.
  • Fold increase in the expression and/or the level refers to the ratio of the expression and/or level determined using one condition and the expression anchor level determined in another condition.
  • a capacity of MSC to inhibit an immune response For example inhibition of proliferation of target cells such as T-cells, B-cells, NK cells and other immune cells and/or monocyte/maerophage polarization resulting in immune suppression through the production of soluble mediators and induction of regulatory ⁇ -cells is determined on basis of the presence of an MBC-specIfic marker and the fold increase of one or more of CD.54, CD274, BLA-DR, COX-2 and IDO.
  • the fold increase is at least 2, preferably at least 3-fold, more preferably at least 5.
  • the fold increase of CD54 is at least 2-fold, preferably at least 3-fold, preferably at least 5-fold, more preferably at least 10-fold.
  • the fold increase of CD274 is at least 2-fold, preferably at least 3-fold, preferably at least 5-fold, more preferably at least 8-fold.
  • the fold increase of HLA-DR is at least 2-fold, preferably at least 3-fold, more preferably at least 5-fold, at least 10-fold, at least 20-fold, more preferably at least 25- fold.
  • the fold increase of COX-2 is at least 2-fold, preferably at least 3-fold, in a further embodiment the fold increase of IDO is at least 2 -fold, preferably at least 3-fold, preferably at least 6-fold.
  • the disclosure provides a method suitable for determining if MSC have a capacity to inhibit an immune response, for example inhibition of proliferation of target cells such as T-cells, B-cells, NK cells and other immune cells and/or monocyte/maerophage polarization resulting in immune suppression through the production of soluble mediators and induction of regulatory T-cells comprising: providing MSC; - culturing said MSC in presence and in the absence of one or more cytokines selected from: IL-17A, IL-1 ⁇ , OSM, IFN-y, TNF- ⁇ , IL-13 and IL-4; contacting said MSC with binding molecules that bind to an MSC- specific marker and one or more selected from: the extracellular markers CD 54,
  • the disclosure provides a method suitable for determining if MSC have a capacity to inhibit an immune response, for example inhibition of proliferation of target cells such as T-cells, B-cells, NK cells and other immune cells and/or monocyte/maerophage polarization resulting in immune suppression through the production of soluble mediators and induction of regulatory T-cells comprising: - providing MSC: culturing said MSC in presence and in the absence of one or more cytokines selected from: IL-17A, 1L-1B, OSM, IRN-g, TNF- ⁇ , IL-13 and IL-4; contacting
  • the disclosure provides a method suitable for determining if MSC have a capacity to inhibit an immune response, for example inhibition of proliferation of target cells such as T-cells, B-cells, NK cells and other immune cells and/or monocyte/maerophage polarization resulting in immune suppression through the production of soluble mediators and induction of regulatory T-cells comprising: providing MSC; culturing said MSC in presence and in the absence of one or more cytokines selected from: IL-17A, IL-1 ⁇ , OSM, lFN- ⁇ , TNF- ⁇ , IL-13 and IL-4; - contacting said MSC with binding molecules that bind to an MSC- specific marker and one or more selected from: the extracellular markers CD 54.
  • target cells such as T-cells, B-cells, NK cells and other immune cells and/or monocyte/maerophage polarization resulting in immune suppression through the production of soluble mediators and induction of regulatory T-cells
  • cytokines selected from: IL
  • CD274, HLA-DR the intracellular markers COX-2 and IDO; said method further comprising determining the presence of said MSC-specifie marker and fold increase of the expression of said one or more of CD54, CD274, HLA- DR, COX-2 and IDO in response to said one or more cytokines, wherein said MSC have said capacity if said one or more markers are increased at least 5-fold,
  • the term ‘'ceil death” refers to the event of a biological cell ceasing to carry out its functions. Cell death may result from various causes, for example apoptosis, programmed cell death, mitotic catastrophe, necrosis, ischemic cell death and/or immunogenic ceil death.
  • the term “cell viability” relates to the capacity of the cell to perform certain functions, such as metabolism, growth, reproduction, some form of responsiveness, and adaptability. Cell death and cell viability can be evaluated by a number of suitable assay’s known to the skilled person. Dye exclusion methods are frequently used as a measure to determine dead cells. Dyes as trypan blue do not easily pass the membrane of living cells hut will enter dead cells as these are not able to maintain the integrity of their cell membrane.
  • a method suitable for determining if MSC have a capacity to inhibit proliferation of T- cells and/or inhibit differentiation of monocytes to DC further comprises contacting MSC with a viability stain and determining viability- of the MSC.
  • MSC are contacted with a dye for exclusion together with binding mol etudes binding to extracellular markers.
  • MSC preparations comprise preferably at least 80%, preferably at least 90%, and more preferably at least 96% viable cells.
  • the disclosure provides a method comprising: - providing MSC; culturing said MSC in presence and in the absence of one or more cytokines selected from: IL-17A, IL-1 ⁇ , OSM, lFN- ⁇ , TNF- ⁇ , EL- 13 and IL-4; contacting said MSC with binding molecules that bind to an MSC- specific marker and one or more selected from: the extracellular markers CD 54, CD274, HLA-DR and a dye for exclusion of dead cells, and the intracellular markers
  • COX-2 and IDO said method further comprising determining the presence of said MSC-specific marker and fold increase of the expression of said one or more of CD54, CD274, HLA- DR, COX-2 and IDO in response to said one or more cytokines and the viability of said MSC.
  • kit refers to a set of articles or equipment needed for a specific purpose.
  • a kit comprises binding molecules that bind to an MSC-specific marker and one or more of CD 54, CD274, HLA-DR, COX-2 and IDO.
  • binding molecules are antibodies.
  • a kit further comprises one or more proinflammatory cytokines, more preferably one or more cytokines selected from: IL17A, IL-18, OSM, IFN-y, TNF- ⁇ , IL-13, IL-4. More preferably lFN- ⁇ ; IL-1 ⁇ ; lFN- ⁇ and TNF- ⁇ ; IL17A,IL-1B and OSM; IL17A, IL-18, OSM, lFN- ⁇ , TNF- ⁇ and IL-13; IL17A, IL-18, OSM, lFN- ⁇ , TNF- ⁇ , IL-13 and IL-4, or; lFN- ⁇ , TNF- ⁇ , IL-13 and IL-4.
  • cytokines more preferably one or more cytokines selected from: IL17A, IL-18, OSM, IFN-y, TNF- ⁇ , IL-13, IL-4. More preferably lFN- ⁇ ; IL-1 ⁇ ; lFN- ⁇ and TNF- ⁇ ; IL17A,IL
  • the term “ceil bank” refers to a facility that stores cells for the purpose of future use in a product or medicinal needs .
  • a cell bank stores cryopreserved cells, Cryopreserved cells are typically stored in vials or freezing bags.
  • the term “vial” refers to a small container, typically cylindrical, suitable for storing cells, including cryopreserved cells.
  • one vial contains MSC of one donor.
  • one vial contains MSC of more than one donor.
  • the term “freezing bag” refers to a bag suitable for storing cells, including cryopreserved cells,
  • one freezing contains MSC of one donor. In a further embodiment, one freezing bag contains MSC of more than one donor.
  • presence and fold increase of markers is determined using a method and/or kit as disclosed herein.
  • the disclosure provides a method for preventing and/or inhibiting an immune response in a subject, comprising administering to a subject
  • MSC of which the presence of an MSC-specific marker and the fold increase of one or more of the markers CD54, CD274, HLA-DR, COX-2 and IDO was determined using a method and/or kit as disclosed herein.
  • the subject is exhibiting or at risk of exhibiting an undesired immune response.
  • Undesired immune responses include, but are not limited to: autoimmune disorders including inflammatory bowel disease, Type 1 diabetes, rheumatoid arthritis and multiple sclerosis, disorders associated with ado-immune responses, including solid organ transplantation and graft versus host disease, and (chronic or acute) inflammatory disorders including pulmonary emphysema, chronic kidney disease osteoarthritis, inflamed skin, brain lesions such as stroke, etc.
  • the subject is exhibiting inflammatory bowel disease.
  • the subject is the recipient of an allogeneic ceil or organ transplant
  • the present disclosure provides a method for preventing and/or inhibiting graft versus host or host versus graft disease in a subject comprising administering to a subject MSC of which the presence of an MSC- specific marker and the fold increase of one or more of the markers CD54, CD274, HLA-DR, COX-2 and IDO was determined using a method and/or kit as disclosed herein.
  • a “subject” is an human or an animal.
  • Subjects include, but are not limited to, mammals such as humans, pig's, ferrets, seals, rabbits, cats, dogs, cows and horses, and birds such as chickens, ducks, geese and turkeys.
  • a subject is a mammal.
  • the subject is a human.
  • the disclosure provides a cell therapy product for use comprising MSC of which the presence of an MSC-specific marker and the fold increase of one or more of the markers CD 54, CD274, HLA-DR, COX- 2 and IDO was determined using a method and/or kit as disclosed herein.
  • a “cell therapy product'’ is a product suitable for cell therapy.
  • ceil therapy this term is intended to refer to grafting, injecting, applying or implanting a cell therapy product into a subject
  • to comprise and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
  • verb “to consist” may be replaced by “to consist essentially of’ meaning that a compound or adjunct compound as defined herein may comprise additional components) than the ones specifically identified, said additional components) not altering the unique characteristic of the invention.
  • treatment refers to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one oi' more symptoms thereof, as described herein.
  • treatment may be administered after one or more symptoms have developed.
  • treatment may be administered in the absence of symptoms.
  • treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.
  • lFN- ⁇ stimulation results in an increased capacity of MSC to inhibit T-eell proliferation
  • IE- 18 stimulation enhances the capacity of MSC to inhibit DC differentiation.
  • Antibody array-based analysis on culture supernatants (left) or cell pellets (right) of unstimulated and IFN-y-stimulaied MSC (n 3 MSC per stimulus). Significantly differentially expressed proteins showing a log2 (fold change) of ⁇ -0.5 or > 0.5 in the supernatant of unstimulated or IFN-y-treated stimulated MSC are depicted.
  • Targets screened with different techniques (as indicated). Arrows indicate downregulated (white) and upregulated (black) genes as identified by mKNA sequencing. Numbers indicate the amount of antibodies used to identify the target. Numbers highlighted in white and black indicate downregulated and upregulated targets, respectively. The first number indicates the amount of antibodies identifying the target as a hit, followed by the number of antibodies that this target was screened for with the indicated technique. The column indicated with “FACS” shows the targets either analyzed (highlighted in grey) or confirmed (highlighted in black) by flow cytometry.
  • Figure 7 Venn-diagram of the overlap in targets that were identified using the different IL-1 ⁇ -based screenings, as well as the FACS-eonfirmed hits. For mRNA hits; only those hits are shown that were also screened for with at least one of the other screening methods.
  • Figure 8
  • Targets and hits observed in IL-16 screening Targets that are screened using different approaches are indicated. Numbers indicate the amount of antibodies that identify the target using each technique. Numbers highlighted in black indicate upregulated targets, in which the first number indicates the amount of antibodies that identified the target as a hit, followed by the number of antibodies that this target was screened for with the indicated technique.
  • the column indicated with "FACS” show the targets that were either screened (highlighted in grey) or confirmed (highlighted in black) by flow cytometry.
  • MSC peripheral blood mononuclear cells
  • PRMC peripheral blood mononuclear cells
  • Y- cell proliferation is presented as percentage proliferation as compared to anti- CD3/CD28 bead stimulated PBMC.
  • IDO and COX-2 inhibitors used were Cay 1058.1. and NS398, respectively.
  • Blockade of IDO and COX-2 with enzyme- inhibiting drugs and their impact on the capacity of MSC to inhibit DC proliferation Purified CD14 + cells were co -cultured with MSC in the presence of GM-CSF and IL-4. Following 6 days of cultures, the ceils were analysed for CD la and CD 14 expression by flow cytometry. This assay was performed in the presence or absence DMSO, IDO inhibitor (INCB-024360) or COX-2 inhibitor (NS398). The percentage of CDla+CDl4- ceils after 6 days of co-culture with CD14+ monocytes is shown.
  • Figure 14 The ratio of fold induction and of the mean fluorescence intensity (MFI) of MSC tested in the potency assay.
  • the numbers refer to the different culture regimens. Each dot represents the ratio of fold induction by dividing the fold induction of experiment 1 by the fold induction obtained in experiment 2 (i.e. the duplo of experiment 1).
  • N 4 MSC tested in 4 experiments.
  • Upregulation of COX-2 was measured by flow cytometry using a PE -labelled anti- COX-2 antibody (clone AS67, BD Pharmingen).
  • MSC mesenchymal cells
  • pro-inflammatory molecules such as cytokines
  • cytokines the effect of different cytokines on two anti-inflammatory capacities of MSC were tested, These two anti-inflammatory capacities were the capacity to inhibit T-cell proliferation, and the capacity to inhibit dendritic ceil (DC) differentiation.
  • MSC were stimulated for different periods of time (as indicated in Figure 1) in the presence or absence of different pro-inflammatory molecules at the following concentrations' Poly (1:C) (20 pg/rnL; from Sigma Aldrich Chernie), IL-18 (1 ng/mL) (R&D Systems Inc.), lFN- ⁇ (10 ng/mL; Peprotech), TNF- ⁇ (1 ng/mL; Peprotech).
  • MSC were plated at 16. ox 10 3 cells/cm 2 and cultured overnight in culture medium after which the culture medium was replaced by medium containing pro- inflammatory molecules.
  • PBMC Human peripheral blood mononuclear cells
  • T-cell-enrlehed PBMC Approximately' Ib.dcIO 3 MSC (either un- or pre-stimulated) and 1.0x10 5 freshly isolated T-cell-enrlehed PBMC were plated in a 96- wells V-shaped plate (Corning Inc.) In a MSCiPBMC ratio of 1:6 in RPMI 1640 culture medium (Gibco #22409-015) supplemented with Glutamax (lx, Gibco #35050-38), T-cell- enriched PBMC were stimulated with human T-cell-activator CD3+/CD2S+ Dynabeads (Thermo Fisher Scientific Inc.) in a celbhead ratio of 5:1.
  • Approximately 3x10 3 MSC ⁇ either un- or pre-stimulated) and 1.0x10 6 freshly isolated monocyte- enriched PBMC were eo -cultured in 6- well plates in RPMI culture medium supplemented with IL-4 (10 ng/mL) and granulocyte-monocyte colony- stimulating factor (GM-CSF) (o ng/mL) (both from Invitrogen Corp.). After 2 days, half of the medium was replaced by cytokine-containing RPMI culture medium (IL-4 and GM-CSF at 20 ng/mL and 10 ng/mL, respectively). After 6 days, cells were analyzed by flow cytometry for expression of CD 14 and CD la.
  • IL-4 ng/mL
  • GM-CSF granulocyte-monocyte colony- stimulating factor
  • MSC pre-stimulated with lFN- ⁇ were found to be most potent in inhibiting T- eell proliferation, followed by MSC pre-stimulated with TNF- ⁇ or IL-16 ( Figure 1, upper panel). Inhibition of dendritic cell differentiation was most potently induced by pre-stimulation with IL-16 ( Figure 1, lower panel).
  • IL-l ⁇ most strongly induces the capacity of MSC to inhibit differentiation from monocytes to DCs.
  • infra -and extracellular molecules have been identified to be implicated in the immunomodulatory action of MSC. Many of these molecules are not expressed at steady state, but tire upregulated in response to stimulation with proinflamraatory molecules, such as cytokines.
  • hmMSC Human hone marrow MSC
  • MSC were stimulated for different periods of time (as indicated) in the presence or absence of different pro-inflammatory molecules at the following concentrations: LPS (200 ng/niL), Poly (I:C) (20 pg/mL; both from Sigma Aldrich Chemie), IL-16 (1 ng/mL) (E&D Systems Inc.), lFN- ⁇ (10 ng/mL is default, 1 and 100 ng/ml when indicated), TNF- ⁇ (1 ng/.mL) (both Peprotech) or both iFN-g and TNF- ⁇ (10 ng/ml IFN- Y and 1 ng/ml TNF- ⁇ ).
  • LPS 200 ng/niL
  • Poly (I:C) 20 pg/mL; both from Sigma Aldrich Chemie
  • IL-16 (1 ng/mL)
  • lFN- ⁇ 10 ng/mL is default, 1 and 100 ng/ml when indicated
  • TNF- ⁇ (1 ng/.mL) (both Peprotech
  • RNA analysis by sequencing MSC were detached using trypsin/EDTA or TrypLE Select, resuspended in RNAlater and stored at -80°C.
  • MSC were detached using trypsin/EDTA or TrypLE Select, resuspended in 350 uL RLT-buffer (Qlagen) containing Rib 6-mereaptoethanol (Sigma Aldrich Cheraie) and stored at -80°C.
  • RLT-buffer Qlagen
  • Rib 6-mereaptoethanol Sigma Aldrich Cheraie
  • antibody array analysis MSC were cult ured in phenol -red free Optimum supplemented with penicillin/ streptomycin (Gibeo).
  • MSC were detached using TrypLE Select and used for co-cultures.
  • RNA Total RNA (mRNA, miRNA and other small RNAs) was isolated using a QIAsymphony using the miRNA CT 400 protocol and an elution volume of 100 m ⁇ The samples were processed using the NEBNext Ultra RNA Library Prep Kit and the protocol “NEBNext Ultra Directional RNA Library Prep Kit for IUumina”(NEB#E7420S/L). Briefly, mRNA was Isolated from total RNA using the oligo-dT magnetic beads. After fragmentation of the mRNA, a cDNA synthesis was performed. This was used for ligation with the sequencing adapters and PCR amplification of the resulting product. The quality' and yield after sample preparation was measured with a Fragment analyzer.
  • the size of the resulting products was consistent with the expected size distribution, i.e. a broad peak between 300-500 bp.
  • Clustering and DNA sequencing using the Illumina cBOT and HiSeqy 4000 was performed according to the manufacturer’s protocols. A concentration of 3.0 nM of DNA was used. Image analysis, base calling, and quality check was performed with the Illumina data analysis pipeline RTA v2,7.7 and Bel2fastq v2.17.
  • the antibody microarrays (ScioCD version 1) were produced and hybridized by Sciomics (Sciomics, Germany). For hybridization, samples were labelled with NHS- esters of the fluorescence dye Dy-649 (Dyomies, Jena, Germany), Samples of unstimulated cells were used as reference sample and labelled with the fluorescent dye Dy- 549 (Dyomies) for competitive dual-color incubations. Scanning of the slides was performed with a PowerSeanner (Tecan) at a resolution of 10 pm, maintaining laser power and the photomultiplier constant. Spot segmentation was performed with GenePix Pro 6.0 software package (Molecular Devices, Sunnyvale, CA, USA).
  • the spot signals were analysed with R-Bioeonductor using a one-factorial linear model fitted with LIMMA package, which resulted in a t-tesfc based on moderated statistics.
  • a specialized invariant Lowess method was applied as described before (C, Schroder et al. Proteoraics Clin, Appl. 2013), In the analyses, duplicate spots were taken into consideration.
  • a one-factorial linear model was fitted with LIMMA resulting in a two-sided t-iesfc based on moderated statistics. Negative fold change values indicate a lower abundance, positive values indicate a higher abundance of the given protein in the sample as compared to the unstimulated condition.
  • MSC were washed with staining buffer (CSM, lx PBS with 0.5% bovine serum albumin and 0.02%» sodium azide, Fluidigm Sciences) and incubated with 1 ml CSM containing 1:500 diluted 500 pM rhodium DNA intercalator (Fluidigm Sciences) for 15 min at room temperature (RT) to stain dead cells.
  • CSM staining buffer
  • lx PBS with 0.5% bovine serum albumin and 0.02%» sodium azide
  • Fluidigm Sciences 1 ml CSM containing 1:500 diluted 500 pM rhodium DNA intercalator (Fluidigm Sciences) for 15 min at room temperature (RT) to stain dead cells.
  • RT room temperature
  • Antibody staining reactions were performed in 100 pi final volume.
  • MSC flow cytometry analysis
  • MSC were incubated for 30 minutes at 4°C with antibodies against extracellular markers.
  • the MSC were fixed (fixation/permeabilization buffer and diluent, eBioseience) for 20 minutes at room temperature (RT) and thereafter permeabilized (permeabilization buffer, eBioseience) and stained for the intracellular markers for 25 minutes at RT.
  • eBioseience fixation/permeabilization buffer and diluent
  • eBioseience permeabilized permeabilization buffer
  • eBioseience permeabilization buffer
  • MFI median fluorescent intensity
  • CD9, C-D29 and CD39 were downregulated and CD 140b and CD273 were upregulated by MSC at the protein level following ⁇ RN-g stimulation.
  • CyTOF- experiments identified up regulation of CD54 and downregulation of CD9, CD 103, CD 120a and CD 140a upon IL-1 ⁇ -stimulation ( Figure 6A).
  • qRT PCR we identified upregulation of CNCL9, CXCLIO, CXCL11, TSG-6, IDO, COX-2,
  • the MSC showed a different slope, suggesting that there might be differences between MSC products in the extent to which they respond to cytokine- stimulation with regards to upregulation of molecules and the capacity to modulate T-cell proliferation ⁇ figure 9).
  • Example 3 Determine which of these molecules can be measured by flow cytometry in a reproducible and standardized fashion
  • the target values for the PMTss were set using rainbow beads.
  • the optimal fluorescent channels for visualizing the surface markers and intracellular stains were determined, taking into account the level of auto-fluorescenee of the MSC in a given channel and the expected expression level for a given target molecule.
  • all antibodies were titrated till saturating levels. If chosen antibodies did not reach saturation and/or went out of the linearity range of the flow cytometer, additional antibody clones that recognized the same target and/or other iluoroehrome- conjugates were tested. In total 25 different antibodies and 10 different clones have been evaluated to come to the most optimal antibody panel.
  • MSC flow cytometry analysis to validate rnENA and proteomics based screenings
  • MSC were harvested and stained for flow cytometry analysis, MSC were incubated for 30 minutes at 4°C with antibodies against T-cell surface markers.
  • the MSC were fixed (ilxatiuii/permeabilization buffer and diluent, eBioscience) for 20 minutes at room temperature (RT) and thereafter permeabilized (permeabilizaiion buffer, eBioscience) and stained for the intracellular markers for 25 minutes at RT, When cells were stained for intracellular IL-6, cells were incubated with Golgistop (RD Biosciences) overnight the morning before harvesting the cells.
  • MSC The MSC were analysed by flow cytometry with the BD FACSTM Canto II cytometer (BD Bioseiences). Analysis of the data was performed with FlowJo software version 8.7.1 (Tree Star Inc., Ashland, OR, USA). For single cells the median fluorescent intensity (MFI) was obtained and fold induction was calculated by dividing the MFI of stimulated MSC by the MFI of unstimulated MSC,
  • CD 106 was added to serve as an identifying marker for MSC.
  • Alternatives for CD73 are CD90, CDIGo, CD29, HLA-ABC, CD 166, CD 146, CD44, CD 140a, CD 140b. All details on the antibodies that were evaluated and selected for the final panel are depicted in table 5.
  • Example 4 Optimization of the in vitro MSC stimulation to obtain a robust and standardised assay
  • MSC 63, MSC 67 and MSC 120 were used for both experiments. The experiments were performed on different days.
  • potency of a single MSC was evaluated when cultured in 2 different batches of fetal bovine serum (batches ABB and PAA), The test was performed in quadruplicate; experiments I and 2, and experiments 3 and 4 are duplicates performed on the same day. The experiments 1-2 and 3-4 were performed on different days.
  • MSC are thawed and plated in 2 wells of a 6-well plate, at a density of 160 x10 3 viable cells per well, in 2.5 ml culture medium (DMEM, supplemented with 10% fetal bovine serum, penicillin and streptomycin) and cultured overnight at 37°C, 7% COg.
  • DMEM fetal bovine serum
  • Preprotech 1 ng/ml TNF- ⁇
  • cells are harvested using TrypLE Select and a total of 45,000 cells are stained for 30 minutes (4°C, protected from light) with antibodies directed against CD54, CD274, HLA-DR and CD73 using the clones and fiuoroehromes shown in table 5.
  • the antibodies are diluted in PBS supplemented with 2% (v/v) human albumin.
  • cells are washed in PBS and dead cells stained using the fixable Aqua dead cell stain kit (Thermo Fisher) according to the manufacturer ’ s recommendation.
  • the cells Prior to staining the intracellular molecule IDO, the cells are fixated and permeabilised using the fixation/per meabilization kit obtained from eBioseience according to the manufacturer’s recommendation. Cells are acquired in a Canto II using pre- defined Euroflow MSC- settings.
  • CDS4 expression is dependent on the fetal bovine serum batch used.
  • COX-2 is consistently upregulated upon incubation with JL- Ib for 24h
  • Nine individual MSC products were incubated with IL-1 ⁇ for 24 hours. All MSC products showed an increase in expression of COX-2 in response to IL-1 ⁇ (Figure 17), supporting that expression of COX-2 is consistently increased in MSC in response to IL-1 ⁇ .
  • FIG. 16 shows the results of 81 MSC in total (79 bone marrow-derived MSC and 2 iPSC-derived MSC). Differences in FBS and/or TNF- ⁇ have limited to no influence on the reproducibility. This number of assays allows us to obtain an overview of the variability in fold- induction values that can be obtained per cell marker.
  • iMSC P5 and iMSC P9 Two MSC-lines derived from induced Pluripotent Stem Cells (iPSC) were evaluated in the potency assay: iMSC P5 and iMSC P9, The same standardized protocol was used as for the BM-derived MSC. In table 9, the result of the potency assay is depicted.
  • the markers CD54, IDO. CD274, HLA-DR and CD 73 were measured. COX- 2 was not analyzed in these experiments.
  • CD73 expression was upregulated compared to bone marrow-derived MSC.
  • this marker serves as an MSC -identifying marker and it’s expression is not related to MSC potency.
  • the potency assay can also be used to evaluate iPSC-derlved MSC.
  • MSC soluble pro inflammatory mediators
  • IRNg soluble pro inflammatory mediators
  • IL-1 ⁇ soluble pro inflammatory mediators
  • TNFa soluble pro inflammatory mediators
  • GMP Good Manufacturing Practice
  • MSC products that have been administered to patients with steroid refractory aGvHD after allogeneic stem cell transplantation in the context of a clinical trial, exhibit enhanced expression of the surface- markers CD54, CD274 and HLA-DE and of the intracellular markers IDO and/or COX-2, when exposed in-vitro to pro-inflammatory mediators, including IFNy, IL-18 and TNFa.
  • Results are shown in the table below: Testing MSC products from all 1.4 healthy donors, including multiple products derived from a single donor where applicable; our results show a significant increase in the expression of the intracellular marker IDO and of the extracellular markers CD 64, CD274 and HLA-DR, showing the reproducible and robust immune suppressive potential of these cells,

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Abstract

L'invention concerne des moyens et un procédé pour tester des cellules stromales mésenchymateuses (MSC) pour déterminer la qualité, la puissance et/ou la capacité à inhiber une réponse immunitaire, telles que l'inhibition de la prolifération de cellules cibles telles que des lymphocytes T, des lymphocytes B, des cellules NK et d'autres cellules immunitaires et/ou une polarisation de monocytes/macrophages conduisant à une suppression immunitaire directe par la production de médiateurs solubles et l'induction de lymphocytes T régulateurs (T-reg). De tels procédés peuvent comprendre la fourniture d'une MSC ; la culture de ladite MSC en présence et en l'absence d'une ou de plusieurs cytokines choisies parmi : l'interleukine (IL)17A, l'lL-1 bêta, l'oncostatine M (OSM), l'interféron gamma (IFN-gamma), le facteur de nécrose tumorale alpha (TNF-alpha), l'IL-13 et l'IL -4 ; la mise en contact de ladite MSC avec des molécules de liaison qui sont renvoyées à un marqueur spécifique à la MSC et un ou plusieurs parmi : les marqueurs extracellulaires CD54, CD274, l'antigène leucocytaire humain (HLA)-DR, les marqueurs intracellulaires cyclo-oxygénase-2 (COX -2) et l'indoléamine-pyrrole 2,3-dioxygénase (IDO). Un tel procédé peut en outre comprendre la détermination de ladite qualité, la puissance et/ou la capacité à inhiber sur la base de l'expression dudit marqueur spécifique à la MSC et de l'augmentation de l'expression dudit ou desdits un ou plusieurs parmi CD54, CD274, HLA-DR, COX-2 et IDO en réponse à ladite ou auxdites cytosines. L'invention concerne en outre des bibliothèques de cellules, des kits utiles dans et pour des procédés de traitement utilisant la MSC testée.
PCT/NL2021/050096 2020-02-14 2021-02-12 Moyens et procédés pour déterminer les propriétés des cellules stromales mésenchymateuses WO2021162554A1 (fr)

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WO2014093948A1 (fr) * 2012-12-14 2014-06-19 Rutgers, The State University Of New Jersey Procédés permettant la modulation de l'effet immunorégulateur des cellules souches
EP3527981A2 (fr) * 2016-10-17 2019-08-21 Samsung Life Public Welfare Foundation Procédé pour le tri de cellules souches hautement efficaces pour le traitement de trouble immunitaire

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WO2014093948A1 (fr) * 2012-12-14 2014-06-19 Rutgers, The State University Of New Jersey Procédés permettant la modulation de l'effet immunorégulateur des cellules souches
EP3527981A2 (fr) * 2016-10-17 2019-08-21 Samsung Life Public Welfare Foundation Procédé pour le tri de cellules souches hautement efficaces pour le traitement de trouble immunitaire

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