WO2003022885A1 - Chimiokines - Google Patents

Chimiokines Download PDF

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Publication number
WO2003022885A1
WO2003022885A1 PCT/GB2002/004083 GB0204083W WO03022885A1 WO 2003022885 A1 WO2003022885 A1 WO 2003022885A1 GB 0204083 W GB0204083 W GB 0204083W WO 03022885 A1 WO03022885 A1 WO 03022885A1
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cells
chemokine
chemokines
apcs
regulatory
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PCT/GB2002/004083
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Alex Betz
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Medical Research Council
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    • 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
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • 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
    • C12N2501/21—Chemokines, e.g. MIP-1, MIP-2, RANTES, MCP, PF-4
    • 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

Definitions

  • the present invention relates to chemokines, compositions and methods effective to modulate the immune response in a mammal, via the recruitment of regulatory T-cells to antigen presenting cells (APCs).
  • the present invention relates to modulating an immune response by increasing or decreasing or otherwise altering the functional activity of any one or more of the chemokines CCLl, CCL4, CCL3, CCL2, and XCLl.
  • Uses of chemokines, compositions and methods of the present invention are also described.
  • Chemokines (chemoattractant cytokines) were initially identified as cytokines profoundly induced by pro-inflammatory stimuli that control the recruitment of leukocytes into inflammatory foci. They are now emerging as important regulators of cellular trafficking in development and homeostasis. In addition, the chemokines also seem to be critical for the coordinated movement of dendritic cells and lymphocytes necessary to generate long lasting antigen specific immunity.
  • chemokine superfamily unrecognised 10 years ago, now has over 40 different members, classified into different subfamilies on the basis of conserved structural features. Most chemokines contain four cysteine residues per monomer which are crosslinked Cysl-> Cys3 and Cys2->Cys4. The backbone of the chemokine molecule consists of beta strands while the N and C termini of the protein appear to have a less ordered structure. All chemokines for which the structure has been determined exist as non-covalently-linked dimers in solution.
  • the arrangement of the cysteine residues within the chemokine protein sequence forms the basis for the structural classification of chemokines.
  • Chemokines in which the Cl and C2 cysteine residues are separated by a single amino acid are the CXC chemokines; examples include IL-8 and PF4.
  • CC chemokines the Cl and C2 cysteine residues are adjacent, examples include RANTES, MCP-1 (CCL2) and eotaxin.
  • the T cell chemoattractant chemokine lymphotactin (XCLl) is the sole C chemokine with only one cysteine residue in the N terminal half of the protein.
  • the only described CXXX3C chemokine is fractalkine, which has three amino acids separating Cl and C2.
  • Fractalkine is also unusual in being the only chemokine that exists both as a membrane bound protein with the CXXX3C chemokine domain sitting on top of a mucin stalk and as a cleaved soluble molecule (Bazan et al.1997).
  • chemokine superfamily has been made on the basis of amino acid sequence comparisons. For instance, a subgroup of CXC chemokines shares a three amino acid motif ELR (Glu-Leu-Arg) immediately preoceeding the first cysteine residue. Most of these ELR CXC chemokines bind the chemokine receptor CXCR2 and can act as neutrophil chemoattractants. A number of recently described CC chemokines have six rather than the more usual four cysteine residues and some have an extended amino terminus relative to other CC chemokines. The six-cysteine CC chemokine family includes the human chemokines 1-309 (CCLl) and Secondary Lymphoid organ Chemokine (SLC).
  • CCLl human chemokines 1-309
  • SLC Secondary Lymphoid organ Chemokine
  • Chemokines mediate their effects on cells via specific cell surface receptors that contain seven transmembrane spanning regions(7TM receptors). Chemokine receptors are coupled to G proteins that transmit intracellular signals via linked phosphodiesterases. Upon binding their cognate chemokine ligands, chemokine receptors initiate changes in the intracellular concentrations of calcium and cAMP. Chemokine receptors also transmit signals to the small GTP binding proteins of the Ras and Rho families leading to rearrangement of actin bundles, pseudopod formation and cell migration.
  • chemokine receptors appear to be restricted to only limited amount of cell types eg. CCR6 is expressed on immature dendritic cells and some activated T cells. Most cellular chemokine receptors bind their cognate chemokine ligands with affinities in the nanomolar range. Most chemokine receptors have multiple chemokine ligands eg. CCR4 binds Macrophage Derived Chemokine (MDC now called CCL22) and Thymus and Activation Regulated Chemokine (TARC).
  • MDC Macrophage Derived Chemokine
  • TARC Thymus and Activation Regulated Chemokine
  • the Duffy antigen is a 7TM receptor expressed on erythrocytes which binds a wide range of chemokines with low affinity (Kd ⁇ 10-6).
  • the Duffy receptor is thought to act as a clearance receptor for chemokines.
  • Chemokines are involved in the regulation of biological functions as diverse as angiogenesis, inflammation, lymphoid organ development and the orchestration of immune responses. They have been shown to be central to regulating the migration of cells of the immune system through various secondary lymphoid organs. Gene deletion models, in which chemokines or their receptors were targeted, highlight such functions although it is unclear whether the resulting phenotype is a consequence of altered lymphoid organ development or a direct effect on specific cell populations. Chemokines are thought to mediate interaction between T helper cells, effector cells and antigen presenting cells (APC), which are central to the generation of immune responses. The interaction between APC and T cells is a critical determinant of the ensuing immunological response.
  • APC antigen presenting cells
  • the present inventors realised that if the mechanism by which regulatory T-cell recruitment to APCs could be elucidated, then a method for modulating immune responses, and for the treatment or prevention of diseases may be established.
  • the present invention addresses the problem of providing substances, methods and compositions effective to modulate an immune response in a mammal by affecting the recruitment of regulatory T-cells to APCs.
  • APCs are defined here as professional antigen presenting cells. However, the regulatory effect can be on the B cell in the function of an antigen presenting cell or in the function as an effector cell of the B cell mediated immunity.
  • B cells can act as APC in order to recruit T cell help.
  • the present inventors have used gene expression profiling described infra, to study the changes in chemokine expression in response to physiological activation. Using this, and other methods described herein, several chemokines were identified which are involved in the modulation of the immune response via the recruitment of regulatory T-cells to APCs.
  • chemokines described herein can be used to modulate the immune response.
  • the present invention provides a method for modulating the immune response in a vertebrate comprising the step of increasing or decreasing, or otherwise altering, the functional activity of at least one chemokine secreted by antigen presenting cells (APCs) on their activation, and which is capable of acting as a chemoattractant for the recruitment of regulatory T-cells to APCs.
  • APCs antigen presenting cells
  • the present invention provides a method for modulating an immune response in a vertebrate comprising the steps of:
  • step (a) testing one or more chemokines for their ability to be secreted by APCs on their activation, and also which are capable of acting as chemoattractant/s for the recruitment of regulatory T-cells to APCs.
  • step (b) Selecting the one or more chemokines which possess the characteristics defined in step (a),
  • step (c) Modulating the functional activity of the one or more chemokines selected according to step (b) in the vertebrate.
  • chemokine in the context of the present invention includes any cytokine which is involved in immune or and/or pro-inflammatory responses.
  • Suitable antigen presenting cells for use in the method of the present invention include any one or more selected from the group consisting of: B cells, dendritic cells and macrophages.
  • the vertebrate is a mammal.
  • the chemokines secreted by the activated APCs are any one or more selected from the group consisting of: CCLl, CCL4, CCL2, CCL3 and XCLl.
  • the chemokine is CCL4 and/or CCLl.
  • the chemokine is CCL4.
  • the term 'increasing or decreasing the functional activity of at least one chemokine' in the context of the present invention includes within its scope increasing or decreasing the expression and/or intracellular or extracellular distribution, and/or activity of at least one chemokine as described herein.
  • Increasing the expression may occur as a result of increasing mRNA expression, or by increasing gene transcription using methods known to those skilled in the art. Those skilled will appreciate that there are many suitable methods to increase or decrease the expression of a nucleic acid sequence encoding a chemokine as herein described.
  • chemokines may be increased or decreased by increasing or decreasing the levels of chemokine mRNA respectively by post transcriptional modulation.
  • interfering RNA may be used as a method to decrease chemokine RNA levels.
  • Increasing or decreasing the intracellular distribution may occur as a result of the addition of chemokine binding proteins to the intracellular environment.
  • the intracellular distribution may be increased, decreased or altered by the addition or removal of signal sequences and/or leader sequences to the chemokine. Techniques used in such procedures will be familiar to those skilled in the art.
  • Increasing or decreasing the activity of the chemokines can be brought about by bringing the chemokines into contact with inhibitors of chemokines, or activators of chemokines and/or chemokine binding molecules.
  • the term 'contact' in the context of the present invention means does not require a physical contact.
  • Suitable inhibitors and activators include but are not limited to inhibitors of chemokine receptors.
  • the small molecule AMD-3100 inhibits the CXCR4 receptor.
  • co-factors or chemokine binding molecules may affect their activity. Examples include antibodies and fragments thereof (for example FAb, F(Ab') 2 , Fv, disulphide linked Fv, scFv, diabody). It will be appreciated that this list is by no means exhaustive.
  • the functional activity of one or more chemokines is modulated using any one of more of the methods selected from the group consisting of: administering a pharmaceutically effective amount of those one or more chemokine/s to the vertebrate; administering a pharmaceutically effective amount of one or more inhibitor/s of those one or more chemokines to the vertebrate; modulating the transcription of those one or more chemokines in the vertebrate; modulating the translation of those one or more chemokines in the vertebrate; modulating the post-translational modification of those one or more chemokines in the vertebrate and modulating the intracellular or extracellular distribution of those one or more chemokines in the vertebrate.
  • the functional activity of one or more chemokines is modulated by administering a pharmaceutically effective amount of one or more inhibitor/s of those one or more chemokines to the vertebrate.
  • the one or more chemokine inhibitor/s are selected from the group consisting of: chemical chemokine inhibitors, anti -chemokine antibodies and dominant negative mutants of those one or more chemokines described herein.
  • the functional activity of one chemokine may be modulated, or of several chemokines may be modulated.
  • these chemokines may act in isolation or synergistically. In addition there may be functional redundancy in the activity of chemokines.
  • the present invention provides the use of a chemokine which is secreted by antigen presenting cells (APCs) on their activation and which is capable of acting as a chemoattractant for the recruitment of regulatory T-cells to APCs, in the preparation of a medicament for modulating the immune response in a vertebrate.
  • APCs antigen presenting cells
  • the chemokines secreted by the activated APCs are any one or more selected from the group consisting of: CCLl, CCL4, CCL2, CCL3, and XCLl.
  • the chemokine is CCL4 and or CCLl.
  • the chemokine is CCL4.
  • the vertebrate is a mammal.
  • chemokines according to the present invention may act independently of each other, in an additive or subtractive manner or synergistically.
  • there may be functional redundancy that is, one chemokine may act in preference to another chemokine in the recruitment of T cells to APCs.
  • APCs include the following: dendritic cells, macrophages and B cells.
  • dendritic cells include the following: dendritic cells, macrophages and B cells.
  • B cells include the following: dendritic cells, macrophages and B cells.
  • the present inventors have found, using methods described herein, that the chemokines of the present invention modulate the immune response via the recruitment of regulatory T-cells.
  • the present invention provides a method for selecting one or more isolated regulatory T-cells capable of modulating the immune response in a vertebrate, comprising the step/s of: (a) preparing a population of T-cells; and (b) exposing said cells to activated antigen presenting cells (APCs); and/or at least one chemokine which is secreted by APCs on their activation, and
  • step (c) selecting those T-cells which can migrate to those APCs or chemokines defined in step (b).
  • the subset of regulatory T-cells referred to and/or selected are CD25+. In an especially preferred embodiment, they are CD25+ and CD4+. In an alternative embodiment, they are CD25+ and CD8+.
  • a population of regulatory T-cells, and APCs such as B-cells, macrophages and monocytes can be prepared using methods known to those skilled in the art including FACs sorting, MACsorting (sorting with magnetic beads), immunoprecipation and standard chromatographic techniques. All these methods are familiar to one skilled in the art and suitable methods will be described in detail herein. Generally, the ability of a population of T-cells to migrate will be measured by techniques known to those skilled in the art, including, standard cell migration assays. Those skilled in the art will be aware of other suitable methods.
  • the present invention provides the use of one or more isolated regulatory T cell/s which are capable of migrating towards antigen presenting cells (APCs) by chemoattraction mediated by at least one chemokine secreted by APCs on their activation, in the preparation of a medicament for modulating an immune response in a vertebrate.
  • APCs antigen presenting cells
  • the regulatory T-cells are CD25+ positive. In an especially preferred embodiment, they are CD25+ and CD4+ positive. In an alternative embodiment, they are CD25+ and CD8+ positive.
  • the chemokines will be at least one selected from the group consisting of CCLl, CCL4, CCL3, CCL2, and XCLl. More preferably, the chemokines will be CCLl and/or CCL4. In a preferred embodiment of this aspect of the invention, the regulatory T-cells will be capable of migrating towards B-cells.
  • chemokines which are functionally active according to the method of the present invention, or the regulatory T-cells may form therapeutically useful compositions.
  • the present invention provides a composition comprising at least one chemokine which is secreted by antigen presenting cells (APCs) on their activation, and which is capable of acting as a chemoattractant for the recruitment of regulatory T-cells to APCs, or a chemokine binding molecule and a pharmaceutically acceptable carrier, diluent or exipient.
  • APCs antigen presenting cells
  • At least one chemokine is selected from the group consisting of: CCLl, CCL4, CCL2, CCL3, and XCLl.
  • at least one chemokine is CCL4 or CCLl .
  • at least one chemokine is CCL4. It will be appreciated that in any composition comprising many chemokines, they may act synergistically, independently of one another, or there may be functional redundancy in the mixture.
  • the composition comprises a chemokine binding molecule.
  • the present invention provides a composition comprising one or more regulatory T-cells as herein described and a pharmaceutically acceptable carrier, diluent or exipient.
  • the present invention provides the use of at least one chemokine, regulatory T-cells, or a composition according to the present invention in the prophylaxis and/or treatment of disease.
  • the term 'disease' includes amy one or more of the following: cancer, auto-immunity, transplant rejection, infections. It will be appreciated that the list is not exhaustive.
  • FIG. 1 Chemokine expression profile of primary B cells, dendritic cells and macrophages before and after stimulation
  • a-c Radioautographs of arrays probed with labelled cDNA obtained from splenic B cells of MD4 transgenic mice, after the following 24h treatments (a) control, mock incubation (b) 500ng/ml hen egg lysozyme (HEL) (c) 50 ⁇ g/ml LPS.
  • HEL hen egg lysozyme
  • FIG. 1 marks the position of DNA spots of chemokines relevant to the analysis: 1 : CCL22, 2: XCR1, 3: CCL2, 4: CCL3, 5: CCL4, 6:CCL5.
  • Arrow set 2 marks the position of other genes relevant to the analysis: a: CCR6, b:CX 3 CR, c:CRD6, d:PAR3, e:Ig , f: Ig .
  • (d) Summaries of DNA array analysis of primary mouse splenic B cells (Ig HEL transgenic/ MD4), blood B cells (wildtype, C57bl/6xBalb/c), bone marrow derived dendritic cells and peritoneal macrophages (wildtype, C57bl/6xBalb/c). In each case, cDNA preparations from control and activated cells are being compared. The stimulation used in the various cases is denoted on the left side for each particular case.
  • Ig x-linked was by 24h treatment with lOO ⁇ g/ml anti-mouse IgM F(ab)s. Grey scale indicates degree of expression; from light grey for undetectable to black for saturated, (e) Concentration of CCL3 and CCL4 in the supernatants of control B cells (white bars) and B cells activated by Ig cross-linking (grey bars) at 24h and 48h.
  • FIG. 1 Analysis of the migration of CD25 +ve cell from spleens of un-immunised mice
  • (a-f) Representative examples of trans-well migration assays using T-cell column enriched primary splenic cells from Balb/c mice and 500ng/ml of the respective chemokine as chemo-attractant. Shown in red is a FACS histogram of the CD25 stained migrated cells. Each histogram contains the data from four pooled migration wells. Unstained input cells are shown for comparison in black, (a) XCLl, (b) CCL2, (c) CCL3, (d) CCL4, (e) CCL5 and (f) CCL22.
  • a-c Histograms of the CD4 +ve cells migrated towards specific stimuli stained for CD25 in the following trans-well migration assays (FACS gated for CD4 +VC cells only): (a) input cells, (b) cells migrated towards the supernatant of activated B cells and (c) cells migrated towards CCL4.
  • d Summary of three independent migration inhibition experiments using purified T cells (approx. 90% enriched for CD4 +ve cells by depletion) prepared from three independent mice.
  • Frozen spleen sections were prepared from nu-/nu- mice (a) without treatment; (b) 14 days after reconstitution with 5xl0 6 splenic T cells; (c) 14 days after reconstitution with 5xl0 6 splenic T cells depleted of CD25 +ve cells; (d,e) 7 days after i.v. anti-CCL4 treatment on day 0, 1 and 2 (d) without prior reconstitution with T cells and (e) after injection of 10 6 purified splenic T cells from age and gender matched Balb/c mice. Further, frozen spleen sections were prepared from Balb/c mice 7 days after (f ) i.v. injection of anti-CCL4 antibody on day 0, 1 and 2; (g) i.p.
  • FIG. 5 The effect of regulatory T cells on B cell proliferation and blasting.
  • B cells were activated with 50 ⁇ g/ml LPS and incubated with specific T cell populations, which were activated by anti-CD3 cross-linking,
  • Anti-CCL4 treated mice develop IgG switched auto-antibodies. Serial dilutions of the blood taken from 7days after the following treatments (three animals in each group): control (open grey squares); i.p. immunisation with OxCSA at day 0 (filled black squares); i.v. anti-CCL4 treatment on days 0, 1 and 2 (open red circles); i.p. immunisation with OxCSA on day 0 followed by i.v.
  • Figure 7 shows a time course of the relative expression of chemokines upon B cell activation.
  • B cells were activated by cross-linking of surface immunoglobulin with anti-mouse IgM F(ab)2 (15 ⁇ g/ml) to analyse the induction of chemokine expression.
  • Supernatants were harvested at 6 hourly intervals and assayed for the presence of the chemokines CCLl, CCL3, and CCL22 by ELISA. (+) denotes activated supernatant ; (-) supernatant of non-activated controls.
  • Chemokine A chemokine in the context of the present invention includes any cytokine which plays a role in an immune response and/or pro-inflammatory response. Examples include CCL3 (MIP1 alpha), CCL4 (mip 1 beta), MIP2, CCLl 9 (MIP3beta), CCL20 (MIP3 alpha), CXCL8 (IL8). Structurally, they are small disulphide-linked polypeptides of typically 60-70 amino acids (7-lOkDa). They act at a nanomolar concentration. Those skilled in the art should appreciate that the reference model used herein is the mouse. In humans and other mammals the functional homologues to the chemokines herein described may have different names.
  • chemokines with different functions have been given the same name in different mammals. That is, non-functional homologues in different mammals may have same names.
  • the term chemokine includes within its scope functional homologues (as described above) of the chemokines herein described.
  • Antibody An antibody (for example IgG, IgM, IgA, IgD or IgE) or fragment (such as a FAb, F(Ab') 2 , Fv, disulphide linked Fv, scFv, diabody) whether derived from any species naturally producing an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria).
  • antibody for example IgG, IgM, IgA, IgD or IgE
  • fragment such as a FAb, F(Ab') 2 , Fv, disulphide linked Fv, scFv, diabody
  • Antigen Presenting Cell As a first step in mounting an antibody response, foreign antigens are engulfed non-specifically by macrophages or other cells of the reticuloendothelial system. These cells include macrophages and dendritic cells. B cells can also act as antigen presenting cells, either by internalising antigen specifically via their antigen receptor or non-specifically via fluid phase uptake. These cells are collectively known as antigen presenting cells.
  • chemokine in the context of the present invention, refers to the function a chemokine normally performs in its native mammalian environment. These roles include mediators of chemoattraction or repulsion, mediators of cell-cell interactions and effects directly on the target cell.
  • T-cell recruitment In order to elicit an effective immune response in a mammal T- cells must come into contact with APCs.
  • the attraction of T-cells by APCs is known as T-cell recruitment, and it ocurrs on activation of APCs. It is mediated via chemokines of the present invention such as CCL4, CCL3, CCLl and CCL2.
  • APCs Activation of Antigen Presenting Cells Occurs rapidly in an immune response. Once activated, APCs express more MHC class I and II, more Fc receptors and more co- stimulatory adhesion molecules. They also produce more cytokines such as IL-1, IL-6, TNFalpha and other mediators. APC activation is essential for the antigen processing and presentation necessary to elicit an immune response. Activation can be brought about by several methods including treatment with LPS (lipopolysaccharide), or cross- linking the surface with antibodies or F(ab)s, for example cross-linking the surface immunoglobulin (Ig) of B cells with anti-Ig F(ab)s.
  • LPS lipopolysaccharide
  • Ig surface immunoglobulin
  • Modulating an immune response includes either enhancing or diminishing a mammals reaction to foreign or to self-antigen within the mammalian body and/or ex vivo.
  • the term means at least either enhancing or diminishing a mammals reaction to foreign or to self-antigen within the mammalian body.
  • the immune system involves a vast variety of molecules, which are capable of interacting with one another, as well as molecules which themselves are not directly involved in immune responses per se. Thus, certain interactions may be enhanced and others diminished such that apparently a mammals reaction to foreign or self antigen remains unaltered. Changes in immune responses of this sort are also contemplated according to the present invention.
  • modulating the immune response according to the present invention includes within its scope the substantial prevention or purposeful induction of autoimmunity.
  • a systematic analysis of the changes in the chemokine and related receptor expression before and after activation by physiological stimuli is performed in order to identify those chemokines involved in the recruitment of regulatory T cells to APCs.
  • LPS is used for activation.
  • cross linking of surface Ig or antigen is used for the activation of B cells.
  • the use of DNA array technology is employed for this purpose.
  • the chemokines are detected in the supernatant of the cells by chemokine specific ELISA.
  • the samples (for example, members of a library, or PCR products or oligonucleotides) are generally fixed or immobilised onto a solid phase, preferably a solid substrate, to limit diffusion and admixing of the samples.
  • the samples may be immobilised to a substantially planar solid phase, including membranes and non- porous substrates such as plastic and glass.
  • the samples are preferably arranged in such a way that indexing (i.e., reference or access to a particular sample) is facilitated.
  • the samples are applied as spots in a grid formation.
  • Common assay systems may be adapted for this purpose.
  • an array may be immobilised on the surface of a microplate, either with multiple samples in a well or with a single sample in each well, or fibre optic bundles.
  • the solid substrate may be a membrane, such as a nitrocellulose or nylon or polyvinylidene membrane (for example, membranes used in blotting experiments).
  • Alternative substrates include gold, glass, plastic, or silica based substrates.
  • the substrates may be coated with a reagent(s) for example poly-L-lysine or polyacrylamide.
  • the samples are immobilised by any suitable method known in the art, for example, by charge interactions, or by chemical coupling to the walls or bottom of the wells, or the surface of the membrane.
  • Other means of arranging and immobilising may be used, for example, pipetting, drop-touch, piezoelectric means, ink-jet and bubblejet technology or electrostatic application.
  • photolithography may be utilised to arrange and fix the samples on the chip.
  • Nomenclature has developed in the art, and is used herein, whereby the samples (for example, members of a library, or PCR products or oligonucleotides) immobilised on the array are termed probes and the sample (for example, libraries, or PCR products, or DNA, or cDNA, or RNA, or oligonucleotides) for hybridisation with the array is termed target.
  • the probes may be arranged by being "spotted” onto the solid substrate; this may be done by hand or by making use of robotics to deposit the sample.
  • Array formats may be described as, for example, macroarrays, microarrays, high-density oligonucleotide arrays and microelectronic arrays.
  • targets and/or probes may be labelled with any readily detectable reporter, for example, a fluorescent, bioluminescent, phosphorescent, or radioactive reporter. Labelling of probes and targets is disclosed in Shalon et al., 1996, Genome Res 6:639-45. Arrays are based on the same steps. An array of the probe sample is constructed. The target sample is labelled. The labelled target sample is hybridised to the arrayed probe samples and the relative signals are measured.
  • any readily detectable reporter for example, a fluorescent, bioluminescent, phosphorescent, or radioactive reporter.
  • the raw data from a microarray experiment typically are images, which need to be transformed into gene expression matrices (tables where rows represent for example genes, columns represent for example various samples such as tissues or experimental conditions, and numbers in each cell for example characterise the expression level of the particular gene in the particular sample). These matrices have to be analysed further, if any knowledge about the underlying biological processes is to be extracted.
  • Methods of data analysis including supervised and unsupervised data analysis as well as bioinformatic approaches) are disclosed in Brazma and Nilo J (2000) FEBS Lett 480:17-24.
  • a custom made DNA array may be used.
  • a custom made DNA array containing probes for 29 chemokine genes as well as two additional putative chemokine genes was generated using the methods described above. Also included were probes for 61 non-chemokine related genes implicated in regulating cell migration.
  • probes for 61 non-chemokine related genes implicated in regulating cell migration.
  • Suitable methods for the preparation of a uniform population of APCs are generally known to those skilled in the art and include FACs sorting, MACsorting and standard chromatographic techniques as well as the use of isolated cells from trangenic animals.
  • the FACS allows cells (lymphocytes) to be separated and isolated through the use of tagged antibodies or anti-immunoglobulins.
  • the sample mixture is funneled through a nozzle which isolates cells and charges them.
  • At the base of the FACS there are 2 variably charged deflection plates and cell collectors.
  • the individual cells in a resting lymphocyte pool must be isolated. This is accomplished by suspending the cells in a fluid (eg., saline). This suspension of cells is then forced through a fine, high-pressure nozzle or fluidic diluting system which distributes the cells into a single-file line or flow cell (Kidd and Nicholson 229).
  • Light is an integral part of the FACS technique. Light striking each cell is scattered.
  • FACSs have two types of data collecting hardware: light scatter sensors and photomultiplier tubes (PMTs).
  • the light scatter sensors measure the light that is scattered by each cell from two different angles.
  • the forward angle light scatter sensor (FALS) gathers light scattered in the forward direction. This type of scattered light gives a clue as to a cell's size.
  • Right angle, orthogonal, or side light scatter (SS) sensors gather light scattered at 90° from the original direction of the light source. This light reveals cell granularity, refractiveness, and the presence of intracellular structures that reflect light (Darzynkiewicz, et al. 335).
  • Scatter sensors are useful in distinguishing cells based on the cells' different structures.
  • Neutrophiles for example, display more SS than lymphocytes (Kidd and Nicholson 229).
  • different cell lineages or cells at different stages of development i.e., pre B cells versus plasma B cells
  • PMTs detect fluorescent emissions from the fluorescent dyes on antibodies bound to cells or from auto-fluorescence of the cells.
  • the cells are passed through an electric field generated by oppositely- charged plates. By changing the direction of the electric field between the plates, selected cells can be directed into precise collection areas.
  • the use of APCs isolated from transgenic animals may be employed.
  • Ig HEL (MD4) trangenic mice all express the same anti-hen egg lysozyme (HEL) immunoglobulin transgene and can be activated by HEL.
  • HEL anti-hen egg lysozyme
  • APCs Methods for the activation of APCs are known to those skilled in the art (Antibodies, Harlow and Lane, Cold Spring Harbour, 1998). Purified APCs can be activated by
  • Ig (MD4) B cells can be activated by anti-hen egg lysozyme (HEL) immunoglobulin.
  • HEL anti-hen egg lysozyme
  • purified B cells from the blood of wild type mice can be activated by exposure to anti-mouse Ig F(Ab)s or LPS. It will be appreciated that the choice of ligand used to activate the purified APCs depends upon the APC sample used.
  • transwell migration assays can be performed using methods known to one skilled in the art, and detailed in (Antibodies, Harlow and Lane, Cold Spring Harbour, 1998). Those skilled in the art will be aware of other suitable methods.
  • a typical protocol is detailed below: Migration assays are carried out in 6.5mm diameter, 5.0 ⁇ m pore size polycarbonate membrane filter trans-well plates (Costar Corning). 0.6 ml RPMI medium containing the chemokine (500ng/ml) and/or antibody (2 ⁇ g/ml) or supernatant of in vitro activated B cells was placed in the lower chamber. 0.1 ml of T lymphocyte suspension (lxlO 7 cells/ml) was added to the upper filters. Following incubation at 37°C 10% CO 2 for 3 hours the cells migrated into the lower chamber were stained with anti-CD4 PE (Pharmingen) and anti-CD25 FITC (Pharmingen) and analysed by FACS and quantified using beads.
  • chemokines which are secreted by APCs on their activation, and which are capable of acting as a chemo-attractant for the recruitment of regulatory T-cells are identified.
  • the chemokines are any one or more selected from the group consisting of: CCLl, CCL4, CCL3, CCL2, and XCLl.
  • the chemokine/s are CCL4 and/or CCLl.
  • at least one chemokine which is secreted by APCs on their activation, and which is capable of acting as a chemo-attractant for the recruitment of regulatory T cells is CCL4.
  • the chemokines of the present invention comprise a narrow range of those chemokines already known in the art. These chemokines have been found, surprisingly, to modulate immune responses in a mammal, and this effect is brought about by the action of these chemokines in recruiting regulatory T-cells to APCs.
  • chemokines as described herein may be effective in regulatory T cell recruitment in isolation, or when acting in conjunction with one or more further chemokines. That is, a given subset of chemokines may show regulatory T-cell recruitment effects whereas each chemokine in isolation may show negligible effects. Furthermore, one skilled in the art will appreciate that two or more chemokines may act synergistically in their effects. Those skilled in the art will appreciate that within a mixture of chemokines, one or several chemokines may influence the recruitment effects as herein described of one or more further chemokines within that mixture, be it by synergistic, additive, subtractive or other modulating effects.
  • modulating the immune response involves either enhancing or diminishing a mammals reaction to foreign or to self-antigen within the body.
  • the immune system involves a vast variety of molecules, which are capable of interacting with one another as well as molecules which are not involved in immune responses. Thus, certain interactions may be enhanced and others diminshed such that apparently a mammals reaction to foreign or self antigen remains unaltered. Changes in immune responses of this sort are also crossmplated according to the present invention.
  • modulating the immune response includes within its scope the substantial prevention or purposeful induction of autoimmunity.
  • the present invention provides a method for modulating the immune response in a mammal comprising the step of increasing or decreasing, or otherwise altering the functional activity of at least one chemokine secreted by antigen presenting cells (APCs) on their activation, and which is capable of acting as a chemoattractant for the recruitment of regulatory T-cells to APC.
  • APCs antigen presenting cells
  • chemokine in the context of the present invention refers to the function the chemokine normally performs in its native mammalian environment. These may include roles such as chemoattractants, mediators of cell-cell interactions and so on.
  • chemokine includes within its scope functional homologues (as described above) of the chemokines herein described.
  • the term 'increasing or decreasing the functional activity of at least one chemokine' in the context of the present invention includes within its scope increasing or decreasing the expression and/or activity of at least one chemokine as described herein. Otherwise altering' the functional activity may result from adjusting the intracellular and/or extracellular distribution of the chemokine/s, or the nucleic acid encoding them.
  • Increasing or decreasing the expression may occur as a result of increasing or decreasing mRNA expression respectively, or by increasing or decreasing gene transcription respectively using methods known to those skilled in the art.
  • Increasing or decreasing the expression and/or functional activity of a chemokine may also be brought about via posttranslational modications using methods known to those skilled in the art.
  • intracellular distribution or extracellular distribution may occur as a result of the addition of nucleic acid binding molecules, or chemokine binding molecules, to the inta or extracellular environment, using techniques known to one skilled in the art.
  • Increasing or decreasing the activity of the chemokines can be brought about by bringing the chemokines into contact with inhibitors of the chemokines, or activators of chemokines and/or chemokine binding molecules.
  • contact in the context of the present invention means does not require a physical contact.
  • a functional contact, that is where the presence of the inhibitor or activator or chemokine binding protein affects the activity of the chemokine is sufficient.
  • Suitable chemokine binding molecules include antibodies as herein defined and chemokine dominant negative mutants.
  • Antibodies may be prepared using standard laboratory techniques. Either recombinant proteins or those derived from natural sources can be used to generate antibodies. For example, the protein (or "immunogen") is administered to challenge a mammal such as a monkey, goat, rabbit or mouse. The resulting antibodies can be collected as polyclonal sera, or antibody- producing cells from the challenged animal can be immortalized (e.g. by fusion with an immortalizing fusion partner to produce a hybridoma), which cells then produce monoclonal antibodies.
  • the antigen protein is either used alone or conjugated to a conventional carrier in order to increases its immunogenicity, and an antiserum to the peptide-carrier conjugate is raised in an animal, as described above.
  • Coupling of a peptide to a carrier protein and immunizations may be performed as described (Dymecki et al. (1992) J. Biol. Chem., 267: 4815).
  • the serum is titered against protein antigen by ELISA or alternatively by dot or spot blotting (Boersma and Van Leeuwen (1994) J. Neurosci. Methods, 51: 317).
  • the serum is shown to react strongly with the appropriate peptides by ELISA, for example, following the procedures of Green et al. (1982) Cell, 28: 477.
  • monoclonal antibodies may be prepared using any candidate antigen, preferably bound to a carrier, as described by Arnheiter et al. (1981) Nature, 294, 278.
  • Monoclonal antibodies are typically obtained from hybridoma tissue cultures or from ascites fluid obtained from animals into which the hybridoma tissue was introduced. Nevertheless, monoclonal antibodies may be described as being “raised against” or “induced by” a protein.
  • monoclonal antibodies are tested for function and specificity by any of a number of means. Similar procedures can also be used to test recombinant antibodies produced by phage display or other in vitro selection technologies. Monoclonal antibody-producing hybridomas (or polyclonal sera) can be screened for antibody binding to the immunogen, as well. Particularly preferred immunological tests include enzyme-linked immunoassays (ELISA), immunoblotting and immunoprecipitation (see Voller, (1978) Diagnostic Horizons, 2: 1, Microbiological Associates Quarterly Publication, Walkersville, MD; Voller et al. (1978) J. Clin. Pathol., 31: 507; U.S. Reissue Pat. No.
  • ELISA enzyme-linked immunoassays
  • Suitable activators or inhibitors of chemokines which may be of use in the method of the present invention include but are not limited to inhibitors of chemokine receptors.
  • the small molecule AMD-3100 inhibits the CXCR4 receptor.
  • One skilled in the art will be aware of other suitable inhibitors or activators.
  • Increasing or decreasing the functional activity of the chemokines of the present invention leads to a modulation of the recruitment of regulatory T-cells to APCs, and therefore a concomitant modulation of the immune response.
  • a further aspect of the present invention provides the use of one or more isolated regulatory T cell/s which are capable of migrating towards antigen presenting cell/s (APCs) by chemoattraction mediated by at least one chemokine secreted by APCs on their activation, in the preparation of a medicament for modulating an immune response in a vertebrate.
  • APCs antigen presenting cell/s
  • the vertebrate is a mammal.
  • T cells can be isolated and purified using methods known to those skilled in the art. These include standard chromatographic methods including immunoprecipitation, and FACs sorting.
  • a suitable source of T cells is spleens from un-immunised mice.
  • One skilled in the art will be aware of other suitable sources of T cells.
  • An example of a protocol for the isolation of T-cells is described below:
  • T lymphocytes were prepared from Balb/c mice using Lympholyte M followed by a T cell column (Cedarlane Laboratories). CD4 +ve T lymphocytes enriched by negative selection using MACS reagents (Miltenyi biotech) and antibodies to CD 19, CD l ib, CD8a, CD80, CD86 and CD 16/32 (Pharmingen).
  • the present invention provides a method for selecting one or more regulatory T cells capable of modulating the immune response in a vertebrate comprising the steps of:- (a) preparing a population of T cells, and
  • step (c) Selecting those T cells which can migrate towards those APCs or chemokines defined in step (b).
  • a population of T-cells is prepared as herein described.
  • the selection step (c) also preferably involves a cell-migration assay, followed by the physical selection of those cells which have migrated towards the chemokines or APCs as described above.
  • step (b), (that is exposing the cells to activated APCs) also includes exposing the cells to supematants from in vitro activated APCs.
  • at least one chemokine used is selected from the group consisting of CCLl, CCL4, CCL3 and CCL2, and XCLl.
  • Suitable APCs for use in the method of this aspect of the present invention include macrophages, dendritic cells and B cells, which can be isolated using standard laboratory techniques as herein described.
  • the vertebrate is a mammal.
  • Those regulatory T-cells selected according to the method of the present invention are preferably CD25+ positive. Even more preferably they are CD25+ and CD4+ positive. In an alternative embodiment of this aspect of the invention, they are CD25+ and CD8+.
  • the present invention provides a composition comprising at least one chemokine, or chemokine binding protein, according to the present invention, and a pharmaceutically acceptable carrier, diluent or exipient.
  • the present invention provides a composition comprising at least one or more regulatory T cells as herein described, and a pharmaceutically acceptable carrier, diluent or exipient.
  • Chemokines, chemokine binding proteins, T-cells and compositions according to the present invention may be employed in in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assay and reagent applications, and the like.
  • chemokines chemokine binding proteins
  • T-cells T-cells and compositions prepared according to the invention involve the administration of the above to a recipient mammal, such as a human.
  • chemokines, binding proteins thereof of at least 90 to 95% homogeneity are preferred for administration to a mammal, and 98 to 99% or more homogeneity is most preferred for pharmaceutical uses, especially when the mammal is a human.
  • the chemokines, binding proteins and T-cells may be used diagnostically or therapeutically (including extracorporeally) or in developing and performing assay procedures using methods known to those skilled in the art.
  • the selected chemokines, or binding proteins thereof, or T-cells of the present invention will typically find use in preventing, suppressing or treating inflammatory states, allergic hypersensitivity, cancer, bacterial or viral infection, and autoimmune disorders (which include, but are not limited to, Type I diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease and myasthenia gravis), and in preventing transplant rejection.
  • autoimmune disorders which include, but are not limited to, Type I diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease and myasthenia gravis
  • depletion of the regulatory T cells or interference with their recruitment may result in an enhanced immune response which may be of particular use in the treatment of infections which otherwise escape a normal immune response.
  • the additional administration of regulatory T cells of the present invention may result in a dampened immune response or even complete suppression of an immune response.
  • chemokines of the present invention may lead to the generation of an autoimmune response, which may be of use in the destruction of certain cells types. This may be of particular use in the treatment of cancer.
  • chemokines or regulatory T cells as herein described may be useful for modulating a immune response in regions of a vertebrate where they are not normally located.
  • one or more chemokines used as herein described may be perfused, injected, or the nucleic acid encoding them expressed within a tissue of a vertebrate, using techniques known to those skilled in the art.
  • the presence of a chemokine and/or a regulatory T-cell as described herein, in such an ectopic environment may be useful in the modulation of an immune response during for example, transplant rejection and the like.
  • prevention involves administration of the protective composition prior to the induction of the disease.
  • suppression refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease.
  • Treatment involves administration of the protective composition after disease symptoms become manifest.
  • EAE in mouse and rat serves as a model for MS in human.
  • the demyelinating disease is induced by administration of myelin basic protein (see Paterson (1986) Textbook of Immunopathology, Mischer et al., eds., Grune and Stratton, New York, pp. 179-213; McFarlin et al. (1973) Science, 179: 478: and Satoh et al. (1987) J. Immunol., 138: 179).
  • the selected chemokines, or binding proteins thereof, or T-cells of the present invention will be utilised in purified form together with pharmacologically appropriate carriers.
  • these carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, any including saline and/or buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's.
  • Suitable physiologically-acceptable adjuvants, if necessary to keep a polypeptide complex in suspension may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.
  • Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).
  • the selected chemokines, or binding proteins thereof, or T-cells of the present invention may be used as separately administered compositions or in conjunction with other agents. These can include various immunotherapeutic drugs, such as cylcosporine, methotrexate, adriamycin or cisplatinum, and immunotoxins. Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents in conjunction with the chemokines, or binding proteins thereof, or T-cells of the present invention or even combinations of selected chemokines, or binding proteins thereof, according to the present invention.
  • immunotherapeutic drugs such as cylcosporine, methotrexate, adriamycin or cisplatinum
  • Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents in conjunction with the chemokines, or binding proteins thereof, or T-cells of the present invention or even combinations of selected chemokines, or binding proteins thereof, according to the present invention.
  • the route of administration of pharmaceutical compositions according to the invention may be any of those commonly known to those of ordinary skill in the art.
  • the selected antibodies, receptors or binding proteins thereof of the invention can be administered to any patient in accordance with standard techniques.
  • the administration can be by any appropriate mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or also, appropriately, by direct infusion with a catheter.
  • the dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counterindications and other parameters to be taken into account by the clinician.
  • the selected chemokines, or binding proteins, of this invention can be lyophilised for storage and reconstituted in a suitable carrier prior to use.
  • Known lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of functional activity loss and that use levels may have to be adjusted upward to compensate.
  • compositions containing the present selected chemokines, or binding proteins thereof, or T-cells of the present invention or a cocktail thereof can be administered for prophylactic and/or therapeutic treatments.
  • an adequate amount to accomplish at least partial inhibition, suppression, modulation, killing, or some other measurable parameter, of a population of selected cells is defined as a "therapeutically-effective dose”. Amounts needed to achieve this dosage will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from 0.00005 to 5.0 mg of selected chemokine or chemokine binding-protein per kilogram of body weight, with doses of 0.0005 to 2.0 mg/kg/dose being more commonly used.
  • compositions containing the present selected polypeptides or cocktails thereof may also be administered in similar or slightly lower dosages.
  • a composition containing one or more selected chemokines, or binding proteins thereof, or T-cells according to the present invention may be utilised in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or removal of a select target cell population in a mammal.
  • the selected repertoires of polypeptides described herein may be used extracorporeally or in vitro selectively to kill, deplete or otherwise effectively remove a target cell population from a heterogeneous collection of cells.
  • Blood from a mammal may be combined extracorporeally with the selected antibodies, cell-surface receptors or binding proteins thereof whereby the undesired cells are killed or otherwise removed from the blood for return to the mammal in accordance with standard techniques.
  • Regulatory T cells isolated from a patient may be enriched by migration.
  • these cells may be treated such that their responsiveness to one or more chemokines is modulated.
  • Such cells may subsequently introduced into the same or a different patient.
  • adoptive transfer of ex vivo enriched regulatory T cells from the patient him/herself or a donor is also contemplated herein. The invention is further described, for the purposes of illustration only, in the following examples which are in no way limiting of the invention.
  • chemokines changes. Whilst some of these changes can be deduced from studies performed with the affimetrix oligonucleotide expression array (R Glynne et al, Nature 403, 672-6 (2000)), a systematic analysis of changes in the chemokine and related receptor expression during B cell differentiation, after B cell activation and during cell-cell interaction had not until now been performed. For this purpose the present inventors developed a custom made DNA array containing probes for 29 chemokine genes as well as two additional putative chemokine genes. Also included were probes for 61 non-chemokine related genes implicated in regulating cell migration.
  • splenic B cells from Ig HEL transgenic mice were stimulated for 24-48h with HEL.
  • splenic B cells from wild-type Balb/c mice were stimulated with either anti mouse Ig F(ab)s or LPS.
  • purified B cells from the blood of C57/B16 mice were stimulated for 24-48h with either lOO ⁇ g/ml anti mouse Ig F(ab)s or LPS.
  • B lymphocytes were prepared from the spleen of Ig HEL transgenic (MD4) mice or blood of C57B16xBalb/c mice using Lympholyte M or Mammal, followed by a B cell column (Cedarlane Laboratories).
  • the cells were activated for 24 hours using either 500ng/ml of hen egg lysozyme (Sigma) or 50 ⁇ g of anti-mouse IgM F(ab) 2 (Jackson labs). Macrophages were harvested by peritoneal lavage and further enriched by adhesion to tissue culture plates. Bone-marrow-derived dendritic cells obtained as described [K. Inaba, et al. J. Exp. Med. 76, 1693 (1992)]. Both APC types were activated with 250ng/ml LPS (Sigma). The cDNA preparations obtained from these samples were hybridised to the DNA arrays.
  • RNA from 10 cells was reverse transcribed using ( ⁇ 32 P) dATP (Amersham) and a mix of gene specific primers. After removal of unincorporated nucleotides by G50 sephadex column centrifugation, the probe was denatured and then added directly to the 'Expresshyb' solution (ClontechyiOO ⁇ g herring sperm DNA, in which the blot was pre-hybridised for lhr.
  • CCL5 is the only chemokine detected in un-stimulated splenic B cells (Fig. la, d), while un-stimulated blood B cells also express CCLl 8 and CXCL4, suggesting a possible functional difference (Fig. Id).
  • Stimulation with antigen Fig. lb, d
  • cross- linking of surface Ig Fig. Id
  • LPS Fig.lc, d
  • Antigen activation and Ig cross-linking but not LPS activation leads to a marked induction of CCL3 and CCL4 in both cell populations (Fig.lc, d).
  • CCL2 In splenic B cells CCL2, CCL22 and XCLl are also up-regulated. All the other 26 chemokines remain unaffected by both specific and unspecific stimulation.
  • ELISA assays for CCL3 and CCL4 using supernatant derived from either non-activated or surface Ig cross- linked B cells. High levels of both chemokines can be detected upon B cell activation (Fig.le).
  • chemokines likely to play a recruitment role early in an immune response we then enquired whether other antigen presenting cell types show a similar activation dependent chemokine/chemokine receptor profile.
  • CCL2, CCL3 and CCL4 three of the five chemokines up-regulated in activated B cells (CCL2, CCL3 and CCL4) are also induced upon activation of dendritic cells and macrophages (Fig. Id).
  • CCL22 which is only slightly induced upon activation in B cells and could not be detected in macrophages, appears to be constitutively present in dendritic cells with a further increase in expression upon activation.
  • both macrophages and dendritic cells appear to constitutively express CCL9/10 and the putative chemokine nap212.
  • CCLl 7 can only be detected in dendritic cells whilst CCL20 can only be detected in macrophages.
  • Migration assays were carried out in 6.5mm diameter, 5.0 ⁇ m pore size polycarbonate membrane filter trans-well plates (Costar Corning). 0.6 ml RPMI medium containing the chemokine (500ng/ml) and/or antibody (2 ⁇ g/ml) or supernatant of in vitro activated B cells was placed in the lower chamber. 0.1 ml of T lymphocyte suspension (1x10 cells/ml) was added to the upper filters.
  • MCP-1 has been demonstrated to play a major role in the polarisation of the immune response (L Gu et al, Nature 404, 407-8 (2000)).
  • XCLl was found to be an inhibitor of T-cell proliferation (C Cerdan et al, Blood 96 (420-8) (2000)).
  • the present inventors were interested in the signals that promote cell-cell interaction at the initiation of an immune response. Therefore a more detailed analysis of cell migration induced by chemokines that show activation dependent expression in B cells and professional APC (Fig.2b-g) was performed. Using T cells purified from spleens of un-immunised mice it was observed that CCL4 induced the migration of a substantial population of CD25 +ve cells (Fig.2e). This was somewhat surprising since the number of CD25 +ve activated T cells in an un-immunised mouse spleen would be expected to be low. Furthermore, it was found that the other chemokines whose expression is activation dependent in B cells also can recruit CD25 +ve cells albeit with markedly reduced efficiency (Fig2b-g). CCL22 and XCLl are comparably poor at attracting T cells in general (Fig.2a,b,g).
  • CD4 +ve CD25 +ve could not be stimulated by CD3 cross- linking confirming that they are indeed anergic.
  • CD4 +ve CD25 +ve cells migrating to CCL4 are anergic. This coupled with the fact that depletion of CD25 +ve cells from the input population led to a loss of CD4 +ve migrated cells makes it unlikely that CCL4 non-selectively attracts T cells and subsequently activates them, leading to expression of CD25 and CTLA4.
  • regulatory T cells can influence a normal B cell immune response by reconstituting nu-/nu- mice with either CD25 depleted T cells or all T cells.
  • PALS peri-arteriolar lymphoid sheets
  • Germinal centres can be detected by day 6-7 when they are characterised by displacement of small, non-dividing IgD +ve , PNA l0 follicular B cells by the rapidly proliferating IgD "ve , PNA hl blasts.
  • the T cell compartment of nu-/nu- mice was reconstituted with either 'total' T cells or with T cells depleted of their CD25 expressing sub-populations.
  • the transferred cells were obtained from age, gender and background matched Balb/c mice.
  • T lymphocytes from the spleen of Balb/c mice were prepared using Lympholyte M followed by a T cell column (Cedarlane Laboratories).
  • CD25+ cells were depleted by FACsorting.
  • nu- /nu- mice were injected i.v with 5xl0 6 -10 7 'total' T cells or CD25 depleted T cells. For the temporal depletion of CCL4, mice were injected i.v.
  • mice When mice are reconstituted with CD25 depleted T cells found an abundance of germinal centres all of which contained a substantial number of IgGl switched cells were found (Fig.4c). In addition, a dramatic increase in IgGl switched cells was found in extra follicular areas (Fig. 4c). In contrast to this, few germinal centres were seen in mice reconstituted with total T cells and these never contained any IgG switched cells (Fig.4b). However, it is clear that reconstitution with total T cells alone can cause inconsistent but usually low level switching of B cells to IgGl in the spleen (Fig. 4b). This effect is transient with a return to normal levels after 3 weeks.
  • Example 7 Since our studies implicated CCL4 as the most effective chemo-attractant for regulatory T cells, which can clearly effect the humoral immune response, the role of CCL4 in vivo was studied.
  • the chemokine was temporarily depleted by injection of an anti-serum, which neutralises the biological activity of CCL4 in migration assays using primary T cells (Fig.3f). It was not possible to detect any change in the spleen of any of the analysed nu-/nu- mice after injection of anti-CCL4 serum (Fig. 4d).
  • mice were reconstituted with 'total' T cells prior to anti- CCL4 serum injection, a dramatic effect on the kinetics of the humoral response and the architecture of the spleen was observed (Fig. 4e).
  • the same effect was observed in Balb/c mice treated with the neutralising anti-serum.
  • Fig. 4f By day 7 nearly all follicles developed large germinal centres (Fig. 4f). This is in marked contrast to a normal immunisation (Fig. 4g), in which a minority of follicles contain nascent germinal centres.
  • the presence of isotype switched cells in the germinal centres reflects their mature nature (Fig. 4f,h).
  • Combinations of cells were incubated for 60-84h in RPMI (10% FCS): 5xl0 5 B cells, 4xl0 5 FACsorted CD25 + or CD25 " CD4+ cells, 2xl0 5 splenic 'input' T cells and cells migrated to CCL4.
  • Wells were pre-coated with 2 ⁇ g/ml anti-CD3e (Pharmingen) and LPS was used at 25 ⁇ g/ml. Cells were counted by FACS using beads for normalisation. Two independent sources of regulatory T cells, FACS sorted small CD4 +ve CD25 +ve cells and T cells that had actually migrated to CCL4 in vitro.
  • Activation of B cells leads to the production of a chemokine that recruits regulatory T cells, which can directly inhibit B cell activation. Interuption of this pathway by either the removal of the regulatory cells themselves or the chemokine that recruits them leads to the generation of auto-antibodies. Plates were coated with either 50 ⁇ l of 5 ⁇ g/ml linear dsDNA in TE pH7.4 following pre-treatment with 5 ⁇ g/ml Poly-L-Lysine in water, or lOO ⁇ g/ml cardiolipin in absolute ethanol, or 250ng/ml myeloperoxidase in bicarbonate buffer pH9.3.
  • IgGl level measurement plates were incubated overnight at 4°C with 8 ⁇ g/ml of IgGl capture antibody (Pharmingen) in sterile 0.1M NaHCO 3 . Plates were blocked with 10% FCS in PBS. 3 fold sera dilution steps were incubated, washed and bound antibody was detected using 2 ⁇ g/ml biotinylated anti-mouse IgG, followed by 0.5 ⁇ g/ml Streptavidin-HRP or Avidin-AP (myeloperoxidase plates). Colorimetric detection was by ABTS or pNPP.
  • Example 9 The inventors have shown that upon activation B cells and professional APC up-regulate CCLl and/or CCL2 and/or CCL3 and/or CCL4.
  • CCL4 is the most efficient chemo-attractant for regulatory T cells in vitro. It is possible that in vivo synergistic or additive effects amongst these chemokines may come into play. However, the profound effect of CCL4 depletion suggests that this chemokine plays a central role in vivo. Regulatory T cells have been implicated in modulating T cell function and results show regulatory T cell recruitment occurs at the level of APC activation. Furthermore, these cells are not only recruited efficiently by B cells, but directly affect the humoral response.
  • regulatory T cells can clearly influence T helper cells our findings suggest an additional more direct effect on B cells. Indeed, our data indicates that regulatory T cells can affect B cell function in vitro in the absence of helper T cells (Fig. 5).
  • the functional interaction between regulatory T cells and APC is further supported by a number of gene deletion models involving genes implicated in this network. Whilst having diverse phenotypes resulting from pleiotropic effects, all lead to exaggerated lympho-proliferation and in many cases to auto-immunity. These include CD25, CTLA-4 and TGF ⁇ all of which are markers for regulatory T cells.

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Abstract

L'invention concerne des chimiokines, des compositions ainsi que des procédés efficaces permettant de moduler la réponse immune chez un mammifère, via recrutement de lymphocytes T régulateurs contre des cellules de présentation d'antigènes. Elle concerne, en particulier, la modulation d'une réponse immune par augmentation ou diminution, ou par altération, de l'activité fonctionnelle d'une quelconque ou de plusieurs des chimiokines CCL1, CCL4, CCL3, CCL2, et XCL1. Elle concerne aussi des utilisations de chimiokines, des compositions et des procédés.
PCT/GB2002/004083 2001-09-06 2002-09-06 Chimiokines WO2003022885A1 (fr)

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GBGB0121579.7A GB0121579D0 (en) 2001-09-06 2001-09-06 Chemokines
GB0121579.7 2001-09-06

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004050706A2 (fr) * 2002-12-03 2004-06-17 Medical Research Council Lymphocytes t regulateurs
WO2006083289A2 (fr) * 2004-06-04 2006-08-10 Duke University Methodes et compositions ameliorant l'immunite par depletion in vivo de l'activite cellulaire immunosuppressive
WO2007085464A1 (fr) * 2006-01-26 2007-08-02 Universität Duisburg-Essen Cellules cd88 t régulatrices et cytotoxiques
WO2018049120A1 (fr) * 2016-09-09 2018-03-15 The General Hospital Corporation Cellules présentatrices d'antigène ex vivo ou cellules t cd positives activées pour le traitement du cancer
EP3447066A1 (fr) * 2017-08-21 2019-02-27 Herlev Hospital Nouveaux composés ccl2 pour utilisation dans le traitement anti-cancéreux

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113893331A (zh) * 2020-07-06 2022-01-07 中国农业科学院特产研究所 一种诱导母鹿生茸的产品及其制备方法和诱导母鹿生茸的方法和应用

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
ANDRIAN VON U H ET AL: "T-CELL FUNCTION AND MIGRATION TWO SIDES OF THE SAME COIN", NEW ENGLAND JOURNAL OF MEDICINE, THE, MASSACHUSETTS MEDICAL SOCIETY, WALTHAM, MA, US, vol. 343, no. 14, 5 October 2000 (2000-10-05), pages 1020 - 1034, XP001029001, ISSN: 0028-4793 *
BYSTRY R S ET AL: "B cells and professional APCs recruit regulatory T cells via CCL4.", NATURE IMMUNOLOGY. UNITED STATES DEC 2001, vol. 2, no. 12, December 2001 (2001-12-01), pages 1126 - 1132, XP001120877, ISSN: 1529-2908 *
IELLEM ANDREA ET AL: "Unique chemotactic response profile and specific expression of chemokine receptors CCR4 and CCR8 by CD4+CD25+ regulatory T cells.", JOURNAL OF EXPERIMENTAL MEDICINE, vol. 194, no. 6, 17 September 2001 (2001-09-17), pages 847 - 853, XP002223042, ISSN: 0022-1007 *
JONULEIT H ET AL: "Dendritic cells as a tool to induce anergic and regulatory T cells", TRENDS IN IMMUNOLOGY, ELSEVIER, CAMBRIDGE, GB, vol. 22, no. 7, 1 July 2001 (2001-07-01), pages 394 - 400, XP004247295, ISSN: 1471-4906 *
READ S ET AL: "CD4regulatory T cells", CURRENT OPINION IN IMMUNOLOGY, CURRENT BIOLOGY LTD,, vol. 13, no. 6, 1 December 2001 (2001-12-01), pages 644 - 649, XP004311242, ISSN: 0952-7915 *
READ S ET AL: "Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation.", THE JOURNAL OF EXPERIMENTAL MEDICINE. UNITED STATES 17 JUL 2000, vol. 192, no. 2, 17 July 2000 (2000-07-17), pages 295 - 302, XP002223043, ISSN: 0022-1007 *
TAKAHASHI T ET AL: "Immunologic self-tolerance maintained by CD4+CD25+ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4", JOURNAL OF EXPERIMENTAL MEDICINE, TOKYO, JP, vol. 192, no. 2, 2000, pages 303 - 309, XP002174070, ISSN: 0022-1007 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004050706A2 (fr) * 2002-12-03 2004-06-17 Medical Research Council Lymphocytes t regulateurs
WO2004050706A3 (fr) * 2002-12-03 2004-09-16 Medical Res Council Lymphocytes t regulateurs
WO2006083289A2 (fr) * 2004-06-04 2006-08-10 Duke University Methodes et compositions ameliorant l'immunite par depletion in vivo de l'activite cellulaire immunosuppressive
WO2006083289A3 (fr) * 2004-06-04 2006-12-21 Univ Duke Methodes et compositions ameliorant l'immunite par depletion in vivo de l'activite cellulaire immunosuppressive
WO2007085464A1 (fr) * 2006-01-26 2007-08-02 Universität Duisburg-Essen Cellules cd88 t régulatrices et cytotoxiques
WO2018049120A1 (fr) * 2016-09-09 2018-03-15 The General Hospital Corporation Cellules présentatrices d'antigène ex vivo ou cellules t cd positives activées pour le traitement du cancer
US11364264B2 (en) 2016-09-09 2022-06-21 The General Hospital Corporation Ex vivo antigen-presenting cells or activated CD-positive T cells for treatment of cancer
EP3447066A1 (fr) * 2017-08-21 2019-02-27 Herlev Hospital Nouveaux composés ccl2 pour utilisation dans le traitement anti-cancéreux
WO2019038235A1 (fr) * 2017-08-21 2019-02-28 Herlev Hospital Nouveaux peptides ccl2 destinés à être utilisés en thérapie anticancéreuse

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