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WO2013041843A1 - Différentiation cellulaire - Google Patents

Différentiation cellulaire Download PDF

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WO2013041843A1
WO2013041843A1 PCT/GB2012/052294 GB2012052294W WO2013041843A1 WO 2013041843 A1 WO2013041843 A1 WO 2013041843A1 GB 2012052294 W GB2012052294 W GB 2012052294W WO 2013041843 A1 WO2013041843 A1 WO 2013041843A1
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cells
lymphocytes
immune
immune cells
culture
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Mark Coles
Bridget GLAYSHER
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The University Of York
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2026—IL-4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2086—IL-13 to IL-16
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N5/0669—Bone marrow stromal cells; Whole bone marrow
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • 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]
    • C12N2501/2304—Interleukin-4 (IL-4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • compositions comprising a combination of ligands that bind and activate the cytokine receptors interleukin 4 receptor [IL4R], tumour necrosis factor receptor [TNFR] and lymphotoxin ⁇ receptor [LT3R] expressed by mesenchymal stem cells which induce differentiation to lymphoid stromal cells; methods to differentiate mesenchymal stem cells into lymphoid stromal cells and methods that use said lymphoid stromal cells in the activation of, for example, T- lymphocytes, B-lymphocytes, Dendritic Cells and Regulatory T Cells; and the use of a system comprising said stromal cells as a means to screen for immune modulators.
  • IL4R interleukin 4 receptor
  • TNFR tumour necrosis factor receptor
  • L3R lymphotoxin ⁇ receptor
  • stem cell represents a generic group of undifferentiated cells that possess the capacity for self-renewal while retaining varying potentials to form differentiated cells and tissues.
  • Stem cells can be pluripotent or multipotent.
  • a pluripotent stem cell is a cell that has the ability to form all tissues found in an intact organism although the pluripotent stem cell cannot form an intact organism.
  • a multipotent cell has a restricted ability to form differentiated cells and tissues.
  • adult stem cells are multipotent stem cells and are the precursor stem cells or lineage restricted stem cells that have the ability to form some cells or tissues and replenish senescing or damaged cells/tissues. Examples of multipotent stem cells include mesenchymal stem cells.
  • MSCs Mesenchymal stem cells or MSCs differentiate into a variety of cell types that include osteoblasts, chondrocytes, myocytes, adipocytes and neurones. Typically MSCs are obtained from bone marrow but can originate from other sources such as adipose tissue.
  • the immune system is in part made up of lymphocytes which are able to recognise specific antigens.
  • B lymphocytes recognise antigens in their native conformation through surface immunoglobulin receptors.
  • T lymphocytes recognise protein antigens that are presented as peptides along with self molecules known as human leukocyte antigen (HLA) in humans, on the surface of antigen presenting cells.
  • HLA human leukocyte antigen
  • T lymphocytes may be subdivided into CD8 + "cytotoxic T lymphocytes", which are able to destroy target cells, and CD4 + "T helper lymphocytes".
  • T helper lymphocytes have a regulatory function and are able to help B lymphocytes to produce specific antibodies, or to help macrophages to kill intracellular pathogens.
  • a further class of immune cells are dendritic cells which function to process antigenic material and present it to T lymphocytes of the immune system. They are primarily present in tissues that are in contact with the external environment, for example, skin, nose, lungs, stomach and intestine. Once activated the dendritic cells migrate to the lymph glands and activate T cells and B cells to initiate an adaptive immune response. Thus, dendritic cells play a key role in host defences and a crucial role in putative anti-cancer immune responses.
  • a yet further class of immune cell is the so called Regulatory T cell which are a sub-population of T cells that function to suppress activation of the immune system.
  • lymphocytes repress the activity of other immune cells and are involved in suppressing an immune response once it has successfully dealt with the invading agent.
  • Regulatory T Cells are also involved in controlling the immune system's recognition of self that may result in autoimmune disease if Regulatory T Cell function is impaired.
  • the differentiation of lymphocytes involves interaction with lymphoid stromal cells that produce an array of cytokines and monokines that promote cell differentiation.
  • This disclosure relates to a combination of cytokine receptor ligands and their use in the induction of differentiation of mesenchymal stem cells into lymphoid stromal cells.
  • the receptors are interleukin 4 receptor [IL4R], tumour necrosis factor receptor [TNFR] and lymphotoxin ⁇ receptor [LT3R] expressed typically by adipose mesenchymal stem cells.
  • IL4 is part of the interleukin 2 (IL-2) family which also includes IL-2, I IL-7, IL-9, IL-15 and IL21 .
  • the function of IL4 is varied but includes the stimulation of activated T lymphocyte and B lymphocyte proliferation.
  • IL4R binds both IL4 and IL13.
  • Tumour necrosis factors are a large family of cytokines that are involved in apoptosis. TNF a is arguably the best known member of the TNF family which also includes lymphotoxin ⁇ . Other members of the TNF family which activate TNFR include CD154 [also known as CD40L], CD27L, CD30L and TRAIL. Statements of Invention
  • a composition comprising an effective amount of a combination of cytokine receptor ligands wherein said ligands bind and activate IL4 receptor, tumour necrosis factor receptor and lymphotoxin ⁇ receptor wherein said composition induces the differentiation of mesenchymal stem cells into lymphoid stromal cells.
  • said ligand is a polypeptide that naturally active activates said receptor.
  • said polypeptide is IL4 or IL13.
  • polypeptide is a TNF family member.
  • TNF family member is selected from the group consisting of: TNF a, lymphotoxin ⁇ , CD154, CD27L, CD30L and TRAIL.
  • said TNF family member is TNF a and lymphotoxin ⁇ .
  • said ligand is an agonistic antibody, or active antibody binding fragment thereof, which binds and activates the cytokine receptors IL4R, LT3R and TNFR.
  • said agonistic antibody is a monoclonal antibody.
  • said ligand is an antibody fragment which binds and activates said cytokine receptors IL4R, LT3R and TNFR.
  • Various fragments of antibodies are known in the art, e.g. Fab, Fab 2 , F(ab') 2 , Fv, Fc, Fd, scFvs, etc.
  • a Fab fragment is a multimeric protein consisting of the immunologically active portions of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, covalently coupled together and capable of specifically binding to an antigen.
  • Fab fragments are generated via proteolytic cleavage (with, for example, papain) of an intact immunoglobulin molecule.
  • a Fab 2 fragment comprises two joined Fab fragments. When these two fragments are joined by the immunoglobulin hinge region, a F(ab') 2 fragment results.
  • An Fv fragment is multimeric protein consisting of the immunologically active portions of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region covalently coupled together and capable of specifically binding to an antigen.
  • a fragment could also be a single chain polypeptide containing only one light chain variable region, or a fragment thereof that contains the three CDRs of the light chain variable region, without an associated heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multi specific antibodies formed from antibody fragments, this has for example been described in US patent No 6,248,516.
  • Fv fragments or single region (domain) fragments are typically generated by expression in host cell lines of the relevant identified regions.
  • immunoglobulin or antibody fragments are within the scope of the invention and are described in standard immunology textbooks such as Paul, Fundamental Immunology or Janeway et al. Immunobiology (cited above). Molecular biology now allows direct synthesis (via expression in cells or chemically) of these fragments, as well as synthesis of combinations thereof.
  • a fragment of an antibody or immunoglobulin can also have bispecific function as described above.
  • composition is a cell culture medium comprising said cytokine receptor ligands and additional cell culture components necessary to maintain mammalian cells in culture.
  • a method to differentiation mesenchymal stem cells into lymphoid stromal cells comprising:
  • differentiated lymphoid stromal cells are contacted with a preparation comprising immune cells
  • said immune cells are peripheral blood mononuclear cells [PBMCs].
  • said immune cells are selected from the group consisting of: T- lymphocytes, [either or both CD8 + T lymphocytes or CD4 + T lymphocytes] B lymphocytes, Dendritic Cells, T Regulatory Cells, innate lymphoid cells or Natural Killer Cells [NK cells].
  • T- lymphocytes [either or both CD8 + T lymphocytes or CD4 + T lymphocytes] B lymphocytes, Dendritic Cells, T Regulatory Cells, innate lymphoid cells or Natural Killer Cells [NK cells].
  • said mesenchymal stem cells and/or said immune cells are autologous.
  • said mesenchymal stem cells and/or said immune cells are allogenic.
  • said mesenchymal stem cells and/or said immune cells are heterologous.
  • said mesenchymal stem cells are derived from adipose tissue.
  • said cell culture comprises or consists essentially of a collagen based cell support.
  • said collagen based cell support is Matrigel®.
  • Alternative cell support matrices to grow cells in 3D include calcium alginate or other hydrogels or using other extracellular matrix components such as laminin or combinations of laminin and collagen. According to a further aspect of the invention there is provided a method for the expansion of activated immune cells comprising:
  • said immune cells are activated CD8 + T lymphocytes and/or activated CD4 + T lymphocytes.
  • said immune cells are activated B lymphocytes.
  • said immune cells are activated T Regulatory Cells, Dendritic Cells or NK Cells.
  • said method includes two or more cells selected from the group consisting of: activated CD8 + T lymphocytes, activated CD4 + lymphocytes, T Regulatory Cells, Dendritic cells, innate lymphoid cells and NK Cells.
  • said subject is a human cancer subject.
  • cancer refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth.
  • the term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • cancer includes malignancies of the various organ systems, such as those affecting, for example, lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumours, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • carcinoma is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary.
  • carcinosarcomas also includes carcinosarcomas, e.g., which include malignant tumours composed of carcinomatous and sarcomatous tissues.
  • An "adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • sarcoma is art recognized and refers to malignant tumors of mesenchymal derivation.
  • said cancer is melanoma.
  • said cancer is prostate cancer.
  • said expanded immune cells are autologous or allogenic and are administered to a subject in need of treatment.
  • said cell culture comprises or consists essentially of a collagen based cell support.
  • said collagen based cell support is Matrigel®.
  • said human antibodies or selected antibody is administered to a human subject.
  • said subject is a human cancer subject.
  • said cell culture comprises or consists essentially of a collagen based cell support.
  • said collagen based cell support is Matrigel®.
  • a method for the production of a hybridoma cell line comprising:
  • said preparation comprises or consists essentially of a collagen based cell support.
  • said collagen based cell support is Matrigel®.
  • a method to screen for immune regulatory agents comprising:
  • said screening method includes the steps of: collating the activity data in (ii) above; converting the collated data into a data analysable form; and optionally providing an output for the analysed data.
  • a number of methods are known which image and extract information concerning the spatial and temporal changes occurring in cells expressing, for example fluorescent molecules and other markers of gene expression.
  • US5, 989,835 and US09/031 ,271 both of which are incorporated by reference, disclose optical systems for determining the distribution or activity of fluorescent reporter molecules in cells for screening large numbers of agents for biological activity. The systems disclosed in the above patents also describe a computerised method for processing, storing and displaying the data generated.
  • agents require preparing arrays of cells for the handling of cells and the administration of agents.
  • Assay devices include standard multiwell microtitre plates with formats such as 6, 12, 48, 96 and 384 wells which are typically used for compatibility with automated loading and robotic handling systems.
  • high throughput screens use homogeneous mixtures of agents with an indicator compound which is either converted or modified resulting in the production of a signal.
  • the signal is measured by suitable means (for example detection of fluorescence emission, optical density, or radioactivity) followed by integration of the signals from each well containing the cells, agent and indicator compound.
  • agent includes any small molecule, antibody, polypeptide, peptide, aptamer, antisense or small inhibitory RNA. These can be an agonist or an antagonist.
  • Agents are typically molecular immune modulators that for example act as adjuvants.
  • said cell culture comprises or consists essentially of a collagen based cell support.
  • said collagen based cell support is Matrigel®.
  • a cell culture vessel comprising differentiation lymphoid stromal cells formed by the method according to the invention.
  • said cell culture vessel further comprises immune cells.
  • said cell culture comprises or consists essentially of a collagen based cell support.
  • said collagen based cell support is Matrigel®.
  • Vessel is defined as any means suitable to contain the above described cell culture. Typically, examples of such a vessel is a petri dish; cell culture bottle or flask; multiwell culture dishes.
  • “vessel” includes bioreactors for large scale culture of cells.
  • Bioreactors are known in the art and provide means for the large scale production of cells.
  • Chen et al (Stem Cells (2006) 24(9): 2052-2059) describes a rotary bioreactor adapted for the expansion of human mesenchymal stem cells.
  • Commercially available rotary bioreactors can be purchased from http://www.synthecon.com.
  • Microfluidic devices are also within the scope of the invention for the analysis of differentiation and immune cell function.
  • the method relies on a combination cytokine ligands and a suitable cell culture medium which allows the mesenchymal cells to proliferate, differentiate and form a 3-D lymphoid stroma which modulates immune cell function.
  • the 3-D system is able to support growth and activity of immune cells hereindisclosed to provide a structure which reflects the in vivo state.
  • the system is invaluable for the study of immune cell function. It will provide a tool for use in the identification of agents effective at promoting or inhibiting the function of immune cells.
  • Figure 1 Expression of lymphoid stromal cell markers ICAM-1 , VCAM-1 and podoplanin in ADSC treated with various combinations of cytokines. (MFI, mean fluorescence intensity);
  • Figure 2 Expression of lymphoid stromal cell markers ICAM-1 and VCAM-1 in ADSC treated with various combinations of cytokines. (MFI, mean fluorescence intensity);
  • FIG. 3 ADSC were co-cultured with either mature B or T lymphocytes leading to distinct chemokine expression patterns. (RQ, relative quantity);
  • Figure 4 Expression of ICAM-1 , VCAM-1 and chemokines in cell line HS.5 and Resto15 (RQ, relative quantity);
  • Figure 5 Chemokine expression in ADSC treated with cytokines, co-cultured with lymphocytes or both treated and co-cultured. (RQ, relative quantity);
  • FIG. 6 Expression of markers associated with a T cell 'exhaustion' phenotype in T cells activated in the presence or absence of ADSC (MFI, mean fluorescence intensity); and
  • FIG. 7 ADSC in 3D culture (left) and in 3D co-culture with B cells (right). Cells stained with anti-human ICAM-1 (green), Phalloidin (red) and DAPI (blue). Materials and methods
  • ADSC Human adipose-derived stem cells
  • Human lymphocytes were isolated from peripheral blood apheresis cones (NHS Blood and Transplant, Leeds, UK) using RosetteSep human B or T cell enrichment cocktails (StemCell Technologies, Manchester, UK).
  • Murine ADSC were derived from collagenase digested fat pads.
  • ADSC were treated with recombinant TNFa (10 ng/ml; Peprotech, London, UK), IL-4 (50ng/ml; Peprotech) and lymphotoxin ⁇ 2 (100ng/ml R&D Systems, Abingdon, UK) and left for 3 days.
  • Treated cells were retreated and co-cultured with either B-lymphocytes or T-lymphocytes for 7 days.
  • ADSC were subcultured and retreated every 3-4 days during this time.
  • T-lymphocytes were activated using anti-CD3 and anti-CD28 conjugated to microbeads (Invitrogen). T cells and beads were cultured in a 1 :1 ratio in the presence of IL-2 (50ng/ml; Peprotech). Preparation of Collagen Based Cell Support.
  • Collagen I was extracted from rats tail tendons by teasing apart the tendons, dissolving them in 0.5 M acetic acid, stirring slowly for 26-36 hours, then spinning down at 20000 g, 4 ⁇ C for 60 min. The supernatant was retained and stored at 4 °C. This was then lyophilised for at least three days using a Lyotrap lyophiliser (LTE, Oldham, UK), giving a dry white solid which was stored at room temperature. Collagen stock solutions were prepared by dissolving specific mass fractions of collagen solid in 0.02 M acetic acid. Collagen stock was diluted in DMEM (Sigma-Aldrich, Gillingham, UK), to create a working solution of 3mg/ml collagen. The pH was neutralised with 1 M NaOH.
  • Varying numbers of ADSC and/or lymphocytes were spun down and resuspended in collagen working solution. This was then either put into the wells of a chambered coverslip (pretreated with 0.1 % glutaraldehyde for 30 mins to prevent collagen contraction) or dotted onto the surface of a petri dish. After gellation of the collagen (1 hr at 15 Q C) cell culture medium was added and cultures were left for varying amounts of time 9up to 21 days) with the medium being changed at 3-4 day intervals.
  • ADSC treated with cytokines for 3 days They were then placed in 0.6mg/ml collagen gel and allowed to spread out overnight. B cells were then added to the top of the gel. After 3 days of co-culture cells were fixed with 4% PFA and stained with anti-human ICAM-1 (eBioscience, Hatfield, UK), DAPI and phalloidin. Images were taken using an LSM 710 confocal microscope (Zeiss, Jena, Germany).
  • ADSCs After 3 days of treatment with TNF, IL-4 and lymphotoxin, ADSCs showed an increase in the expression of lymph node stromal markers ICAM-1 , VCAM-1 and podoplanin (Fig 1 ). Using IL-13 in place of IL-4 had a similar effect (Fig 2).
  • ADSC which had been treated with TNF, IL-4 and lymphotoxin for three days were co- cultured with either B-lymphocytes or T-lymphocytes for an additional 7 days.
  • ADSC co- cultured with B- lymphocytes showed an increase in the expression of CXCL13 and a decrease in both CCL19 and CCL21 expression (Fig 3).
  • ADSC co-cultured with T-lymphocytes showed a decrease in CXCL13 expression and an increase in both CCL19 and CCL21 expression (Fig 3).
  • Human bone marrow stromal cell line HS-5 and human tonsil stromal cell line Resto15 were treated with TNF, IL-4 and lymphotoxin for three days and then co-cultured with either B-lymphocytes or T-lymphocytes for an additional 7 days. There was no up- regulation of ICAM-1 or VCAM-1 in response to the cytokine treatment nor was there a pattern of chemokine expression follow that found when ADSC were used (Fig 4). This demonstrates that the lymphoid stromal phenotype cannot be induced in stromal cell lines.
  • ADSC were treated with TNF, IL-4 and lymphotoxin for 10d.
  • Chemokine mRNA levels from these cells were compared with mRNA from ADSC which had been treated with TNF, IL-4 and lymphotoxin for three days were co-cultured with either B-lymphocytes or T-lymphocytes for 7 days. High levels of chemokine expression were found only in ADSC which had been both treated and co-cultured. Treatment or lymphocyte co- culture alone was not sufficient to induce high levels of chemokine expression (Fig 5).
  • T cells were activated in the presence or absence of treated ADSC. Expression of CD27 decreases and PD-1 increases with 'exhaustion'. T cells activated in the presence of ADSC were found to have higher CD27 and lower PD-1 expression when compared to T cells activated in the absence of ADSC (Fig 6).
  • ADSC were co-cultured with B cells in collagen gels for 3 days.
  • the ADSC spread out to form a 3D network and the B cells migrated into the gels and formed clusters around the ADSC (Fig 7).

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Abstract

La présente invention concerne une composition comprenant une combinaison de ligands qui, tout en se liant à eux, rendent actifs des récepteurs de cytokines, en l'occurrence le récepteur de l'interleukine 4 ou "IL4R", le récepteur du facteur de nécrose tumorale ou "TNFR", et le récepteur de la lymphotoxine β ou "LTβR", exprimés par les cellules souches mésenchymateuses qui induisent la différentiation en cellules stromales lymphoïdes et les utilisations desdites cellules stromales lymphoïdes.
PCT/GB2012/052294 2011-09-19 2012-09-18 Différentiation cellulaire WO2013041843A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3127198A (en) 1964-03-31 figure
US5989835A (en) 1997-02-27 1999-11-23 Cellomics, Inc. System for cell-based screening
WO2000027999A2 (fr) * 1998-11-12 2000-05-18 Cell Science Therapeutics, Inc. Production de cellules lymphoides specifiques d'un tissu a partir de cellules souches hematopoietiques se trouvant dans des dispositifs tridimensionnels
US6248516B1 (en) 1988-11-11 2001-06-19 Medical Research Council Single domain ligands, receptors comprising said ligands methods for their production, and use of said ligands and receptors
WO2010021993A1 (fr) * 2008-08-19 2010-02-25 Cytori Therapeutics, Inc. Procédés d'utilisation de cellules issues du tissu adipeux dans le traitement du système lymphatique et d'une maladie maligne
WO2010073119A1 (fr) * 2008-12-23 2010-07-01 Adocia Composition pharmaceutique stable comprenant au moins un anticorps monoclonal et au moins un polysaccharide amphiphile comprenant des substituants hydrophobes

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3127198A (en) 1964-03-31 figure
US6248516B1 (en) 1988-11-11 2001-06-19 Medical Research Council Single domain ligands, receptors comprising said ligands methods for their production, and use of said ligands and receptors
US5989835A (en) 1997-02-27 1999-11-23 Cellomics, Inc. System for cell-based screening
WO2000027999A2 (fr) * 1998-11-12 2000-05-18 Cell Science Therapeutics, Inc. Production de cellules lymphoides specifiques d'un tissu a partir de cellules souches hematopoietiques se trouvant dans des dispositifs tridimensionnels
WO2010021993A1 (fr) * 2008-08-19 2010-02-25 Cytori Therapeutics, Inc. Procédés d'utilisation de cellules issues du tissu adipeux dans le traitement du système lymphatique et d'une maladie maligne
WO2010073119A1 (fr) * 2008-12-23 2010-07-01 Adocia Composition pharmaceutique stable comprenant au moins un anticorps monoclonal et au moins un polysaccharide amphiphile comprenant des substituants hydrophobes

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHEN ET AL., STEM CELLS, vol. 24, no. 9, 2006, pages 2052 - 2059
DA SILVA CLAUDIA LOBATO ET AL: "Dynamic cell-cell interactions between cord blood haematopoietic progenitors and the cellular niche are essential for the expansion of CD34(+), CD34(+)CD38(-) and early lymphoid CD7(+) cells", JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, JOHN WILEY & SONS, US, vol. 4, no. 2, 1 March 2010 (2010-03-01), pages 149 - 158, XP009165271, ISSN: 1932-6254, [retrieved on 20091124] *
DONILLARD; HOFFMAN; SCHWARTZ: "Compendium of Immunology", 1981, article "Basic Facts about Hybridomas"
KOHLER; MILSTEIN, NATURE, vol. 256, 1975, pages 495 - 497
TUMANOV ALEXEI ET AL: "Distinct role of surface lymphotoxin expressed by B cells in the organization of secondary lymphoid tissues", IMMUNITY, CELL PRESS, US, vol. 17, no. 3, 1 September 2002 (2002-09-01), pages 239 - 250, XP009165309, ISSN: 1074-7613 *

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