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WO1998056819A1 - Procedes de regulation de la fonction de production et de pouvoir pathogene de cytokines produites par des macrophages et des lymphocytes t - Google Patents

Procedes de regulation de la fonction de production et de pouvoir pathogene de cytokines produites par des macrophages et des lymphocytes t Download PDF

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Publication number
WO1998056819A1
WO1998056819A1 PCT/US1998/012345 US9812345W WO9856819A1 WO 1998056819 A1 WO1998056819 A1 WO 1998056819A1 US 9812345 W US9812345 W US 9812345W WO 9856819 A1 WO9856819 A1 WO 9856819A1
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leu
cells
ser
ala
pro
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PCT/US1998/012345
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English (en)
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Patricia P. Jones
Irina B. Conboy
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The Board Of Trustees Of The Leland Stanford Junior University
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Priority to AU81431/98A priority Critical patent/AU8143198A/en
Publication of WO1998056819A1 publication Critical patent/WO1998056819A1/fr

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    • 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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • CD4 + T helper T cells orchestrate the immune response of mammals through production of soluble factors that act on other immune system cells, including other T cells
  • Most mature CD4 + T helper cells express one of two cytokine profiles Th1 or Th2 Th1 cells secrete IL-2, IL-3, IFN- ⁇ , TNF- ⁇ , GM-CSF and high levels of TNF- ⁇ Th2 cells express IL-3, IL-4, IL-5, IL-6, IL-9, IL- 10, IL-13, GM-CSF and low levels of TNF- ⁇
  • Th1 subset promotes delayed-type hypersensitivity, cell-mediated immunity, and tmmunoglobulin class switching to lgG2a
  • the Th2 subset induces humoral immunity by activating B cells, promoting antibody production, and inducing class switching to lgG1 and IgE Skewing the T cell responses toward Th1 is thought to result in susceptibility to
  • cytokines IL-12 and IFN- ⁇ are positive Th1 and negative Th2 regulators IL-12 promotes IFN- ⁇ production, and IFN- ⁇ provides positive feedback for IL-12 IL-4 and IL-10 appear to be required for the establishment of the Th2 cytokine profile and to down- regulate Th1 cytokine production, the effects of IL-4 have been demonstrated to be dominant over those of IL-12 IL-13 was shown to inhibit expression of inflammatory cytokines, including IL-12 and TNF- ⁇ by LPS-induced monocytes, in a way similar to IL-4
  • Th1 development was found to be associated with high affinity binding of a peptide antigen to MHC class II and strong signaling through the TCR, whereas lower-affinity antigen-MHC II interactions and weaker signaling through TCR were reported to result in Th2 cytokine responses (Constant ef al (1995) J Exp Med 182 1501-1596)
  • Several cell surface molecules expressed by antigen presenting cells (APC) and T cells have been suggested to influence commitment to Th1 or Th2 response, including CD40-CD40 ligand interactions and B7 1 vs B7 2 signaling
  • APC antigen presenting cells
  • B7 1 vs B7 2 signaling At the cellular level, at least in some cases, macrophages and dendritic cells appear to promote the Th1 response, whereas B cells upregulate Th2 cytokines
  • B7 1/B7 2 effects nor the effects of different APC types in supporting Th1 or Th2 are absolute
  • T cells with high affinity for self antigens are clonally deleted
  • the threshold for clonal deletion may be set low enough so that T cells whose antigen receptors have sub-threshold affinity for self antigens mature and migrate to the periphery T cells that recognize self antigen-derived peptides not expressed or presented in the thymus will also fail to be deleted
  • Self-reactive T cells that are not deleted in the thymus may be tolenzed through other mechanisms, including an alteration of signaling pathways to produce clonal anergy, and lowering the avidity of the TCR for its ligand by downregulating coreceptor and accessory molecules Active suppression of T-cell responses may also be in effect It has been suggested by Liu ef al (1995) Immunity 3 407-415 that low avid
  • cyclophi n C-associated protein is available at Genbank, accession number L16894
  • the complete coding sequence of the mouse CyCAP and its encoded polypeptide are provided as SEQ ID NO 1 and SEQ ID NO 2
  • the protein is also known as 90 K and mu ⁇ ne adherent macrophage protein
  • the human homolog has been previously described as Mac-2- binding protein, which is available at Genbank, accession number 1082577
  • the nucleotide and ammo acid sequences are provided herein as SEQ ID NO 3 and SEQ ID NO 4, respectively
  • the polypeptides are referred to herein as cytokine regulatory factor (CYTRF) genencally, and may be further designated as mouse, human, etc
  • CyCAP 90K protein, Mac-2-binding protein, murine adherent macrophage protein
  • a pharmaceutical composition comprising CYTRF as an active agent is administered in vitro or in vivo, and can act on mature, committed Th1 type T cells to decrease the synthesis of pro- inflammatory cytokines, or to skew the commitment of precursor T helper cells to Th1 or a Th1 cytokine profile. It is particularly effective for T cells having weak response to the stimulating antigen.
  • the inflammatory response of T cells associated with disease conditions is suppressed by the delivery of CYTRF.
  • the administered CYTRF may be a naturally occurring form of the protein, or a synthetic variant derived therefrom. CYTRF signaling and related pathways are also useful for modeling and screening novel pharmacological agents.
  • FIGS 1A-1 F Cytokine levels of MBP-specific T cell lines stimulated overnight with syngeneic APC.
  • Figures 2A-2E Cytokines produced by T cell lines (TCL 3) from B10.A and B10.BR mice are influenced by the source of APC (B10.A, B10.BR, or F1) used for in vitro re-stimulations.
  • FIGS 3A-3C Down-regulation of B10.BR TCL IFN- ⁇ and TNF- ⁇ production by B10.A APC or by 2-hour supernatant from activated B10.A APC.
  • FIGS. 4A-4C Supernatant from B10.A T cells activated with B10.A APC for 2 hours down-regulates IFN- ⁇ and TNF- ⁇ production by B10.BR Th1 cells when A k -expressing L cell fibroblasts are used as APC.
  • FIGS. 5A-5C Down-regulation of IFN- ⁇ and TNF- ⁇ levels is not affected when 2-hour B10.A culture supernatant is depleted of known Th1/Th2 cytokine regulators.
  • FIGS 6A-6B Modulation of down-regulatory activity by addition or neutralization of known cytokine regulators during re-stimulation of B10.BR T cells with either B10.BR or B10.A splenic APC.
  • Figures 7A-7B Down-regulatory effects of B10.A splenic APC on IFN- ⁇ and TNF- ⁇ production by a BIO.BR-derived KLH-specific, E"-restricted T cell clone.
  • FIG. 8 Down-regulation of TNF-a production by purified populations of B10.A splenocytes.
  • Figures 9A and 9B Biochemical properties of the Th1 down-regulatory factor.
  • FIGS. 10A-10B Depletion of the B10.A-derived down-regulatory activity with anti- CyCAP antibodies or with the Cyp C-GST fusion protein.
  • FIGS 11A-11D Purified CyCAP down-regulates TNF- ⁇ and IFN- ⁇ production, but not the T cell proliferation.
  • Figures 12A-12B Proliferative responses to MBP Ac 1-16 and Hb 64-76 peptides.
  • FIGS 13A-13B The B10.A-derived Th1 down-regulatory activity depends on the strength of T cell activation.
  • Figures 14A-14B Signaling by a partial agonist is dominant over strong agonist in permitting down-regulation of TNF- ⁇ and IFN- ⁇ .
  • Figures 15A-15C Ef ects of CyCAP on Th1 cytokine production in response to strong and to partial agonists.
  • FIGS 16A-16B IL-12 production by B10.A, B10.BR, (B10.A x B10.BR)F1 , CyCAP
  • Methods and compositions are provided for directing the Th1/Th2 phenotype of T cell subsets, using the protein CYTRF.
  • the protein is effective at suppressing Th1 type responses, particularly in response to weak antigens.
  • CYTRF also inhibits the synthesis of IL-12 by antigen presenting cells, e.g. macrophages, dendritic cells, etc.
  • CYTRF may be administered alone or in combination with other active agents to a patient suffering from pro-inflammatory conditions, e.g.
  • T cell mediated autoimmune diseases T cell mediated autoimmune diseases.
  • Methods of blocking of CYTRF may also be used, where a pro-inflammatory response is desired.
  • CYTRF a novel activity of a previously known polypeptide, herein referred to as CYTRF.
  • the protein sequences are provided in the SEQLIST, and are publically known, as described above. It has been found that CYTRF suppresses the synthesis of pro- inflammatory cytokines, e.g. tumor necrosis factor- ⁇ (TNF- ⁇ ) and ⁇ -interferon ( ⁇ -IFN)
  • TNF- ⁇ tumor necrosis factor- ⁇
  • ⁇ -IFN ⁇ -interferon
  • IL-2 interleukin-2
  • Cytokines produced by Th1 type T cells are associated with inflammation, where capillaries are dilated, fluids accumulate, and migration of phagocytic leukocytes, such as granulocytes and monocytes, to the site of injury or lesion is seen Inflammation is important in defending a host against a variety of infections, but can also have undesirable consequences in inflammatory disorders CYTRF is useful in modulating the T cell response by down-regulating expression of pro-inflammatory cytokines, and in studying the substrates, receptors and pathways of this important biological process
  • Conditions characterized by the undesirable release of pro-inflammatory cytokines include autoimmune diseases, inflammation caused by bacterial, viral and parasitic infection, including response to vaccination, skin sensitivity, e g response to plant toxins or irritants, local inflammation in response to trauma, graft rejection, graft v host disease, and the like
  • Administration of CYTRF is used to decrease the release of ⁇ -IFN and TNF- ⁇ by activated T cells, and to shift the initial response to a Th2 type Administration may be local or systemic
  • Blocking the action of CYTRF by the admmstration of agents that interfere with its biological activity or receptor binding is useful in shifting the initial response to Th1 type This decreases undesirable allergic responses, as may be associated with asthma, etc
  • weak antigens are those peptides that either bind weakly to MHC proteins on antigen-presenting cells, or form MHC peptide complexes that are recognized weakly by T cell receptors (TCR)
  • TCR T cell receptors
  • a weak response may be experimentally characterized Methods are known in the art for measuring the response of a T cell to an antigen, e g by assaying 3H-thym ⁇ d ⁇ ne incorporation into the DNA of responding T cells, or production of specific cytokines by responding cells, such as IL-2, IFN- ⁇ , etc , where the proliferation of a responding cell to an antigen is measured
  • a negative control of no antigen is taken to be a null response
  • the maximal response may be elicited, for example, by stimulating T cells with syngeneic antigen presenting cells and varying concentrations of protein or peptide antigen The spectrum of responses ranging from negative to maximal
  • Characterization of a peptide may also be measured by the affinity of a peptide for the presenting MHC, or by the avidity of the responding T cells for the peptide/MHC complex, using methods known in the art For example, Alam et al (1996) Nature 381 616-620 describe the use of surface plasmon resonance to measure the kinetics of TCR interactions hgands Jorgensen et al (1992) Annu Rev Immunol 10 835-873 review the ternary complex of the T cell receptor (TCR), antigenic peptides, and molecules of the major histocompatibihty complex (MHC) A measure of the affinity of TCR for peptide/MHC complexes comes from competition experiments using soluble MHC complexed with specific peptides
  • Formulations of CYTRF are administered to a host suffering from a pro-inflammatory immune disorder
  • Macrophages are major producers of TNF- ⁇ , and IL-12, which is a major mducer of a Th1 type response
  • Synthesis of these cytokines by macrophages is inhibited by the administration of CYTRF Administration
  • Administration may be topical, localized or systemic, depending on the specific condition Generally the dose will be sufficient to decrease the synthesis of pro- inflammatory cytokines by at least about 50%, and may be higher, at least about 90%, or as much as 99% or higher
  • the compounds of the present invention are administered at a dosage that reduces the cytokine synthesis while minimizing any side-effects It is contemplated that the composition will be obtained and used under the guidance of a physician for in vivo use
  • Autoimmune diseases of interest are associated with T cell-mediated or macrophage- mediated tissue destruction
  • MS multiple sclerosis
  • RA rheumatoid arthritis
  • reactive arthritis psoriasis
  • pemphigus vulgans Sjogren's disease
  • thyroid disease Hashimoto's thyroiditis
  • myasthenia gravis insulin dependent diabetes mel tus
  • IDDM insulin dependent diabetes mel tus
  • Treatment of primates, more particularly humans is of interest, but other mammals may also benefit from treatment, particularly domestic animals such as equine, bovine, ovine, feline, canine, mu ⁇ ne, lagomorpha, and the like
  • the subject therapy will desirably be administered during the presymptomatic or preclmical stage of the disease, and in some cases during the symptomatic stage of the disease
  • the presymptomatic, or preclmical stage will be defined as that period not later than when there is T cell involvement at the site of disease, e g islets of Langerhans, synovial tissue, thyroid gland, etc , but the loss of function is not yet severe enough to produce the clinical symptoms indicative of overt disease T cell involvement may be evidenced by the presence of elevated numbers of T cells at the site of disease, the presence of T cells specific for autoantigens, the release of cytokines, performs and granzymes at the site of disease, response to immunosuppressive therapy, etc
  • Degenerative joint diseases may be inflammatory, as with seronegative spondylarthropathies, e g ankylosmg spondylitis and reactive arthritis, rheumatoid arthritis, gout, and systemic lupus erythematosus
  • the degenerative joint diseases have the common feature in that the cartilage of the joint is eroded, eventually exposing the bone surface Destruction of cartilage begins with the degradation of proteoglycan, mediated by enzymes such as stromelysm and collagenase, resulting in the loss of the ability to resist compressive stress Alterations in the expression of adhesion molecules, such as CD44 (Swissprot P22511), ICAM-1 (Swissprot P05362), and extracellular matrix protein, such as fibronectin and tenascm, follow Eventually fibrous collagens are attacked by metalloproteases, and when the collagenous microskeleton is lost, repair by regeneration is impossible There is significant immunological activity within the synovium during the
  • Human IDDM is a cell-mediated autoimmune disorder leading to destruction of insulm- secretmg b cells and overt hyperglycemia T lymphocytes invade the islets of Langerhans, and specifically destroy insulin-producing ⁇ -cells The depletion of ⁇ -cells results in an inability to regulate levels of glucose in the blood
  • Overt diabetes occurs when the level of glucose in the blood rises above a specific level, usually about 250 mg/dl
  • a long presymptomatic period precedes the onset of diabetes During this period there is a gradual loss of pancreatic ⁇ - cell function
  • the disease progression may be monitored in individuals diagnosed by family history and genetic analysis as being susceptible The most important genetic effect is seen with genes of the major histocompatibiiity locus ⁇ IDDM1), although other loci, including the insulin gene region (IDDM2) also show linkage to the disease (see Davies ef al, supra and Kennedy ef al
  • Markers that may be evaluated during the presymptomatic stage are the presence of msulitis in the pancreas, the level and frequency of islet cell antibodies, islet cell surface antibodies, aberrant expression of Class II MHC molecules on pancreatic b cells, glucose concentration in the blood, and the plasma concentration of insulin
  • An increase in the number of T lymphocytes in the pancreas, islet cell antibodies and blood glucose is indicative of the disease, as is a decrease in insulin concentration
  • patients with residual ⁇ -cell function evidenced by the plasma persistence of insulin C-peptide, may also benefit from administration of the subject polysaccha ⁇ des in order to prevent further loss of function
  • Inflammatory diseases caused by bacterial and viral infection include viral meningitis and bacterial meningitis, herpes encephalitis and viral meningoencephahtis, viral hepatitis, e g Hepatitis A, B, C, D, etc Diseases of interest also include inflammatory response to vaccination, particularly rabies vaccine, varicella zoster vaccine, measles vaccine, efc
  • the cytokine regulatory factor, CYTRF is characterized as an acidic glycoprotem having a molecular weight of approximately 100 Kdal or more, determined by size exclusion cent ⁇ fugation and chromatography, and having the properties of binding to anti-CyCAP antibodies and cyclophilm C
  • the factor down-regulates expression of the pro-inflammatory cytokines IFN- ⁇ and TNF- ⁇ CYTRF also inhibits production of the pro-Th1 cytokine IL-12 by macrophages
  • the factor is present in stimulated cultures of ThO cells, it shifts the cytokine profile of the stimulated T cells to a Th2 type
  • CYTRF activity is genetically upregulated by a non-H2 linked locus derived from A strain, e g B10 A, AWySn, and absent in mice having a C57BL background, e g B10 BR
  • any of the native CYTRF forms, modifications thereof, or a combination of one or more forms may be used.
  • the CYTRF sequence may be from any mammalian or avian species, e g primate sp , particularly humans, rodents, including mice, rats and hamsters, rabbits, equmes, bovines, canines, felines, etc Of particular interest are the human proteins Generally, for in vivo use the CYTRF sequence will have the same species of origin as the animal host For in vitro use, any convenient species having high activity against the microbe being treated may be used.
  • the nucleic acid sequences encoding the above human CYTRF polypeptide may be accessed from public databases as previously cited Identification of non-human CYTRFs is accomplished by conventional screening methods of DNA libraries or biological samples for DNA sequences having a high degree of similarity to known CYTRF sequences
  • sequence of the CYTRF polypeptide may be altered in various ways known in the art to generate targeted changes in sequence
  • the polypeptide will usually be substantially similar to the sequences provided herein, / e will differ by at least one ammo acid, and may differ by at least two but not more than about ten ammo acids
  • the sequence changes may be substitutions, insertions or deletions Scanning mutations that systematically introduce alanme, or other residues, may be used to determine key ammo acids
  • Deletions may further include larger changes, such as deletions of a domain or exon Other modifications of interest include epitope tagging, e g with the FLAG system, HA, efc Such alterations may be used to alter properties of the protein, by affecting the stability, specificity, etc
  • the protein may be joined to a wide variety of other o gopeptides or proteins for a variety of purposes
  • various post-expression modifications may be achieved
  • the subject peptide will be bound to a lipid group at a terminus, so as to be able to be bound to a lipid membrane, such as a posome
  • the subject peptides may be PEGylated, where the polyethyleneoxy group provides for enhanced lifetime in the blood stream
  • the subject peptides may also be combined with other proteins, such as the Fc of an IgG isotype, which may be complement binding, with a toxin, such as ⁇ cin, abnn, diphtheria toxin, or the like, or with specific binding agents that allow targeting to specific moieties on a target cell
  • the active domain of CYTRF may be produced as a fusion protein with an antibody that is specific for a target cell of interest, thereby providing for an antimicrobial antibody composition
  • the antibody may be produced as a single chain, instead of the normal multime ⁇ c structure Single chain antibodies are described in Jost ef al (1994) J B C 269 26267-73, and others DNA sequences encoding the variable region of the heavy chain and the variable region of the light chain are ligated to a spacer encoding at least about 4 ammo acids of small neutral ammo acids, including glycine and/or se ⁇ ne
  • the protein encoded by this fusion allows assembly of a functional variable region that retains the specificity and affinity of the original antibody Formulations
  • the compounds of this invention can be incorporated into a variety of formulations for therapeutic administration More particularly, the compounds of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols As such, administration of the compounds can be achieved in various ways, including oral, buccal, rectal, parenteral, intrapentoneal, intradermal, transdermal, mtracheal, efc , administration
  • the CYTRF may be systemic after administration or may be localized by the use of an implant that acts to retain the active dose at the site of implantation
  • the compounds of the present invention can be administered alone, in combination with each other, or they can be used in combination with other known compounds (e , perform, antibiotics, efc )
  • the compounds may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds
  • the following methods and excipients are merely exemplary and are in no way limiting
  • the compounds can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch, with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins, with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose, with lubricants, such as talc or magnesium stearate, and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents
  • the compounds can be formulated into preparations for injections by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycendes, esters of higher aliphatic acids or propylene glycol, and if desired, with conventional additives such as solubilizers,
  • the compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like
  • the compounds can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases
  • bases such as emulsifying bases or water-soluble bases
  • the compounds of the present invention can be administered rectally via a suppository
  • the suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature
  • Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compounds of the present invention
  • unit dosage forms for injection or intravenous administration may comprise the compound of the present invention in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier
  • Implants for sustained release formulations are well-known in the art implants are formulated as microspheres, slabs, etc with biodegradable or non-biodegradable polymers
  • biodegradable or non-biodegradable polymers polymers of lactic acid and/or glycolic acid form an erodible polymer that is well-tolerated by the host
  • the implant containing CYTRF is placed in proximity to the site of infection or inflammation, so that the local concentration of active agent is increased relative to the rest of the body
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle
  • the specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host
  • pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public
  • Typical dosages for systemic administration range from 0 1 ⁇ g to 100 milligrams per kg weight of subject per administration
  • a typical dosage may be one tablet taken from two to six times daily, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient
  • the time-release effect may be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release
  • liposomes as a delivery vehicle is one method of interest
  • the posomes fuse with the cells of the target site and deliver the contents of the lumen intracellularly
  • the liposomes are maintained in contact with the cells for sufficient time for fusion, using various means to maintain contact, such as isolation, binding agents, and the like
  • liposomes are designed to be aerosolized for pulmonary administration
  • Liposomes may be prepared with purified proteins or peptides that mediate fusion of membranes, such as Sendai virus or influenza virus, efc
  • the lipids may be any useful combination of known liposome forming lipids, including cationic lipid
  • the lipids and lumen composition containing the nucleic acids are combined in an appropriate aqueous medium, conveniently a saline medium where the total solids will be in the range of about 1 -10 weight percent
  • an appropriate aqueous medium conveniently a saline medium where the total solids will be in the range of about 1 -10 weight percent
  • the tube is placed in a warm water bath, from about 25-40° C and this cycle repeated from about 5-10 times
  • the composition is then sonicated for a convenient period of time, generally from about 1-10 sec and may be further agitated by vortexing
  • the volume is then expanded by adding aqueous medium, generally increasing the volume by about from 1-2 fold, followed by shaking and cooling
  • the CYTRF genes are used for modulating expression in vitro and in vivo
  • Vectors useful for introduction of the gene include plasmids and viral vectors Of particular interest are retroviral-based vectors, e g Moloney mu ⁇ ne leuk
  • the expression vector will have a transcnptional initiation region oriented to produce functional mRNA
  • the native transcnptional initiation region or an exogenous transcnptional initiation region may be employed
  • the promoter may be introduced by recombmant methods in vitro, or as the result of homologous integration of the sequence into a chromosome
  • Many strong promoters are known in the art, including the ⁇ -actin promoter, SV40 early and late promoters, human cytomegalovirus promoter, retroviral LTRs, methallothionem responsive element (MRE), tetracycline-mducible promoter constructs, etc
  • Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences
  • Transcription cassettes may be prepared comprising a transcription initiation region, the target gene or fragment thereof, and a transcnptional termination region
  • the transcription cassettes may be introduced into a variety of vectors,
  • CYTRF Blocking Agents The CYTRF polypeptides are used for the production of antibodies, where short fragments provide for antibodies specific for the particular polypeptide, and larger fragments or the entire protein allow for the production of antibodies over the surface of the polypeptide
  • Antibodies may be raised to the wild-type or variant forms of CYTRF Antibodies may be raised to isolated peptides corresponding to these domains, or to the native protein, e g by immunization with cells expressing CYTRF, immunization with liposomes having CYTRF inserted in the membrane, etc
  • Antibodies are prepared in accordance with conventional ways, where the expressed polypeptide or protein is used as an immunogen, by itself or conjugated to known immunogenic carriers, e g KLH, pre-S HBsAg, other viral or eukaryotic proteins, or the like Various adjuvants may be employed, with a series of injections, as appropriate For monoclonal antibodies, after one or more booster injections, the spleen is isolated, the lymphocytes immortalized by cell fusion, and then screened for high affinity antibody binding The immortalized cells, / e hybndomas, producing the desired antibodies may then be expanded For further description, see Monoclonal Antibodies A Laboratory Manual.
  • the mRNA encoding the heavy and light chains may be isolated and mutagenized by cloning in E coli, and the heavy and light chains mixed to further enhance the affinity of the antibody
  • Antisense molecules are used to down-regulate expression of CYTRF in cells
  • the anti- sense reagent may be antisense o gonucleotides (ODN), particularly synthetic ODN having chemical modifications from native nucleic acids, or nucleic acid constructs that express such anti- sense molecules as RNA
  • ODN antisense o gonucleotides
  • the antisense sequence is complementary to the mRNA of the targeted gene, and inhibits expression of the targeted gene products
  • Antisense molecules inhibit gene expression through various mechanisms, e g by reducing the amount of mRNA available for translation, through activation of RNAse H, or stenc hindrance
  • One or a combination of antisense molecules may be administered, where a combination may comprise multiple different sequences Drug Screening
  • the CYTRF polypeptide may be used for binding assays, for elucidation of signaling pathways involved in the determination of cytokine synthesis by T cells and macrophages, and for identifying ligands or substrates that bind to, modulate or mimic the action of CYTRF, that block CYTRF binding to its receptor, or that mimic CYTRF triggering of its receptor
  • Drug screening identifies agents that provide a replacement or enhancement for CYTRF function Of particular interest are screening assays for agents that have a low toxicity for human cells
  • assays may be used for this purpose, including labeled in vitro protein-protein binding assays, protem-DNA binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, and the like
  • the purified protein may also be used for determination of three-dimensional crystal structure, which can be used for modeling intermolecular interactions, transcnptional regulation, efc
  • agent as used herein describes any molecule, e g protein or pharmaceutical, with the capability of mimicking or modulating the activity of CYTRF Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations Typically, one of these concentrations serves as a negative control, / e at zero concentration or below the level of detection
  • Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups
  • Candidate agents are also found among biomolecules including peptides, sacchandes, fatty acids, steroids, punnes, pynmidines, derivatives, structural analogs or combinations thereof
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and o gopeptides Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, efc to produce structural analogs Where the screening assay is a binding assay, one or more of the molecules may be joined to a label, where the label can directly or indirectly provide a detectable signal Various labels include radioisotopes, fluorescers, chemilummescers, enzymes, specific binding molecules, particles, e g magnetic particles, and the like Specific
  • reagents may be included in the screening assay These include reagents like salts, neutral proteins, e g albumin, detergents, etc that are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc may be used.
  • the mixture of components are added in any order that provides for the requisite binding Incubations are performed at any suitable temperature, typically between 4 and 40°C Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening Typically between 0 1 and 1 hours will be sufficient
  • the compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host for treatment of infection
  • the compounds may be formulated in a variety of ways
  • the concentration of therapeutically active compound in the formulation may vary from about 0 1-100 wt %
  • the following examples are offered by way of illustration and not by way of limitation
  • mice Animals B10 A, B10 BR, and B10 PL mice, six to nine weeks old, were obtained from Jackson Laboratory (Bar Harbor, ME), (B10 AxB10 BR) F1 , F2 and (B10 BRxF1) backcross mice were bred in our facility 98/56819
  • Antibodies Ant ⁇ -IL-4 mAb 11 B11 was provided by J O'Hara and W E Paul (NIH)
  • Anti- IFN- ⁇ mAb XMG1 2 was provided by T Mosmann (University of Alberta, Edmonton, Canada)
  • Anti-IFN- ⁇ mAb HB170 was obtained from ATCC Ant ⁇ -IL-4 mAb BVD4 and BVD6, and ant ⁇ -IL-10 mAb 2A5 and Sxc 1 were provided by M Howard (DNAX, Palo Alto, CA)
  • Ant ⁇ -IL-12 p40 mAb C17 8 and C15 6 were provided by G T ⁇ nchie ⁇ (Wistar Institute of Anatomy and Biology,
  • Anti-TNF- ⁇ mAb was provided by Robert Schreiber (Washington School of Medicine, St Louis, MO) Ant ⁇ -IL-13 and anti-TGF ⁇ pan-neutralizing antibodies were purchased from R&D Systems (Minneapolis, MN)
  • Mouse MBP Ac1-16 Ac-ASQKRPSQRSKYLATA was purchased from the Protein and Nucleic Acids Facility (Stanford U , Stanford, CA)
  • T Cell Lines Mice were immunized in the base of the tail with 100 ⁇ M mouse MBP Ac1 - 16 in CFA H37 Ra (Difco, Detroit, Ml) Eight days later superficial inguinal and sacral lymph nodes were removed, and lymph node cells were stimulated with 30 ⁇ M mMBP Ac1-16 in RPMI 1640 (GIBCO BRL, Gaithersburg, MD) with 2% syngeneic mouse serum every 10-14 days thereafter 0 5-1x10 6 T cells were re-stimulated with 30 ⁇ M mMBP Ac1-16 and 5x10 6 irradiated (3000 rad) splenocytes per ml Starting from the third re-stimulation T cell lines were cultured in RPMI 1640 with 10% FCS (GIBCO BRL, Gaithersburg, MD), and 10% supernatant from rat splenocytes activated overnight by 2 ⁇ g/ml of Concavalin A was added every other re-stimulation
  • Spleen cells were dissociated in serum free RPMI 1640, resuspended at 4 x 10 7 per ml and incubated for 10 minutes at 4° with 100 ⁇ l/ml (NH4) 2 S0 4 - concentrated ant ⁇ -Thy-1 mAb (Tib 99 from ATCC, Rockville, MD) Following incubation for 30 minutes at 37° with 1 8 diluted Low-Tox Baby Rabbit Complement (Cedarlme, Westbury, NY), surviving cells were washed three times in RPMI 1640 with 10% FCS and analyzed by FACS with ant ⁇ -CD3 mAb or with isotype-control hamster IgG (Caltag, Burlingame, CA)
  • Cytokines were added as follows mouse recombinant IL-4 (Boehnnger, Indianapolis, IN) at 100 units/ml, mouse recombinant IL-12
  • anti-cytokme antibodies were added to 1 ml of 2-hour supernatants from B10 BR and B10 A cultures at the following concentrations anti- IL-4 (11B11) at 10 ⁇ g/ml and 20 ⁇ g/ml, ant ⁇ -IL-10 (Sxc 1) at 20 ⁇ g/ml and 40 ⁇ g/ml, ant ⁇ -IL-12p40
  • T Cells from B10 BR Mice are Significantly More Encephalitogemc in Adoptive Transfer of EAE than B10 A-denved T Cells
  • Initial comparison of susceptibility of strains B10 A and B10 BR to EAE induced in vivo by immunization with MBP N-termmal peptides were uninformative, as both strains demonstrated comparably low susceptibility to EAE (average score 0 5)
  • BIO PL TCL 1 BIO PL 5/5 1 9 (1-3) 7 (5-10)
  • the two B10 BR TCL differed in the severity of EAE induced in F1 recipients B10 BR TCL 1 which had been re-stimulated with a random mixture of B10 BR and B10 A splenocytes, induced less severe EAE in (B10 A x B10 BR) F1 mice than did B10 BR TCL 2, which had been re-stimulated with B10 BR APC only
  • B10 BR TCL 2 additional TCL (TCL 3) were generated, in which lymph node T cells from B10 A and B10 BR mice were re-stimulated as separate lines with B10 A, B10 BR, or (B10 AxBIO BR) F1 splenic APC
  • TCL were adoptively transferred into recipients
  • TCL 2 lines were established lymph node T cell lines (TCL 2) from mMBP Ac1-16- immunized B10.A, B10.BR and (B10.AxB10.BR) F1 mice were re-stimulated for the fifth time with mMBP Ac1-16 and irradiated splenic APC. After 24 hours, culture supernatants were harvested.
  • TCL 2 lymph node T cell lines
  • TNF TNF-derived neurotrophic factor
  • TNF- ⁇ TNF- ⁇ .
  • IL-12 levels were also significantly higher.
  • TNF- ⁇ ELISA results were nearly identical (within 10%) to the TNF bioassay results, and as TNF biological activity was totally blocked with the anti-TNF- ⁇ mAb TN3-19.12, all of the TNF activity detected in these TCL supernatants appears to be TNF- ⁇ .
  • Supernatants from the (B10.AxB10.BR) F1 TCL stimulated with peptide and F1 APC contained intermediate amounts of the tested cytokines, but the levels were closer to those from B10.A than B10.BR TCL.
  • TCL from B10.A mice produced a Th2, low TNF- ⁇ response
  • T cells from B10.BR produced a Th1 , high TNF- ⁇ response
  • the Th2 low TNF- ⁇ phenotype was dominant in the (B10.AxB10.BR) F1 T cell cultures.
  • the Cytokine Profile of the B10.BR TCL is Influenced by the Genetic Origin of the APC Used for in vitro Re-stimulation.
  • the cytokine profiles of the third set (TCL 3) of MBP-specific T cell lines serially re-stimulated with splenic APC of varying genetic origin were also examined.
  • the cytokine responses of the six TCL were determined after the third, fourth, fifth, ninth, and tenth re-stimulations.
  • Th1 high TNF- ⁇ encephalitogenic cytokine phenotype in response to MBP
  • IFN- ⁇ and TNF- ⁇ Production by Established Th1 B10 BR TCL can be Down-regulated by a Single Re-stimulation with BIO A APC or in the Presence of 2-hour Supernatants from Activated B10 A Cultures
  • two independently- derived Th1 MBP-specific B10 BR TCL TCL 3 and TCL 4, both of which had been maintained with syngeneic B10 BR APC
  • TCL 3 and TCL 4 both of which had been maintained with syngeneic B10 BR APC
  • IFN- ⁇ and TNF- ⁇ levels were down-regulated by 40-60% when B10 A instead of B10 BR APC were used for the single re-stimulation
  • B and C represent independent experiments with MBP-specific B
  • B10 BR TCL T cells or by LPS
  • a single re-stimulation of B10 BR TCL with B10 A splenic APC or in the presence of 2-hour supernatant from B10 A cultures did not induce any IL-4 expression in B10 BR T cells
  • B10 A TCL remained Th2, low TNF- ⁇ producing cells irrespective of the genetic source of APC or 2 hour supernatant
  • B10 BR or B10 A T cells were re-stimulated twice with mMBP Ac1-16 presented by non-professional APC (A k -express ⁇ ng L cells which do not produce splenic APC- derived cytokines), in the presence of 2-hour supernatant from B10 A cultures ( Figures 6A-6B)
  • B10 BR T cells produced high levels of IFN- ⁇ and TNF- ⁇
  • B10 A T cells produced high levels of IL-4 with little or no TNF- ⁇ and IFN- ⁇
  • the levels of IFN- ⁇ and TNF- ⁇ produced by B10 BR TCL were reduced in the presence of 2-hour supernatant
  • B10 A APC Down-regulate IFN- ⁇ and TNF- ⁇ Production by a Thl T Cell Clone with Different Antigen and MHC Restriction Specificities
  • the cytokines produced by the B10 BR- denved KLH-specific, E k -rest ⁇ cted Th1 clone BR E7 were assayed following two consecutive re- stimulations with B10 BR or B10 A splenic APC As shown in Figure 7, presentation of KLH by B10 A APC results in the progressive reduction of IFN- ⁇ and TNF- ⁇ levels produced by this Th1 clone
  • the B10 A APC-denved factor acts independently of the specificity of the Th1 cell and is able to down-regulate IFN- ⁇ and TNF- ⁇ production by a fully-committed clonal Th1 cell population
  • the BR E7 T cell clone was activated with KLH and either B10 BR or B10 A splenic APC for two consecutive re-
  • a genetic d ⁇ fference(s) between inbred strains B10 A and B10 BR act(s) in APC to control Th 1 Th2 cytokine profiles and the encephalogemcity of MBP-Ac1- 16-spec ⁇ f ⁇ c T cells
  • the regulatory effects are manifested both at the time of commitment to Th1/Th2 phenotypes, as B10 A APC block the development of IL-2, IFN- ⁇ , and TNF- ⁇ production, and in the production of IFN- ⁇ , and TNF- ⁇ by committed Th1 cells, as a single re-stimulation with B10 A-denved APC or in the presence of B10 A-denved supernatant down-regulates the production of these cytokines by established Th1 B10 BR cells
  • the B10 A-denved soluble factor that modulates Th cytokine production is novel
  • the results of adding or blocking cytokines implicated in regulating Th1/Th2 cytokine production indicate that the down-regulatory effects of this factor probably do not act through or require IL-4, IL-10, IL-13, IL-12p40, TGF-b, IFN- ⁇ and TNF- ⁇
  • the B10 A-denved factor also acts independently of prostaglandm E2, an APC-denved molecule known to inhibit production of IFN- ⁇ by activated CD4+ T cells, indicated by the failure of mdomethacin to block down-regulation of IFN- ⁇ and TNF- ⁇ production by B10 A APC
  • the activity of this factor in down-regulating IFN- ⁇ production also differs from that of IL-4 in that IL-4, but not the factor, blocks IFN- ⁇ induction by IL- 12 through its inhibition of signalling through the IL-12 receptor
  • B10 A and B10 BR APC in down-regulatory factor production remains to be determined
  • the gene for the factor itself could be polymorphic, resulting in differences in the levels or activity of the factor produced Alternatively, the polymorphism could be in gene(s) that indirectly affect factor activity, by controlling its synthesis, modification, or secretion
  • Initial analysis of the genetics down-regulatory activity have yielded surprising results It was anticipated that the gene would be /-/-2-l ⁇ nked, as B10 A and B10 BR are H-2 congenic strains on the C57BL/10 background the H-2 k haplotype of B10 BR was derived from C57BR and the H-2 recombinant haplotype of B10 A (k haplotype in the H-2K-Ea region, d haplotype telome ⁇ c to Ea) was derived from strain A/WySn However, preliminary tests of (B10 A x B10 BR) F2 progeny and (F1 x B10 BR) back
  • Th1 Leishmania system the resistant (Th1) phenotype is largely dominant in heterozygotes, whereas for the B10 A-denved factor discussed here the Th2-promot ⁇ ng phenotype is dominant or co- dominant in (B10 A x B10 BR)F1 mice (Seder ef al (1992 J E M 176 1091-1098.
  • This gene represents a new member of a growing number of polymorphic genes that influence susceptibility to autoimmune diseases
  • B10 A APC produce a soluble factor that down-regulates production of TNF- ⁇ and IFN- ⁇ by Th1 cells
  • B10 BR APC either make no or lower levels of this factor, or at reduced activity
  • This soluble molecule, cytokine regulatory factor (CYTRF) is herein characterized in terms of both its biological activity and its biochemical properties
  • the screening assay used in the characterization of CYTRF was based on the fact that in a single stimulation of Th1 cell lines or clones B10 A APC or B10 A-denved culture supernatant reduces production of TNF- ⁇ and IFN- ⁇ compared to APC or culture supernatant from strain B10 BR
  • the results suggest that CYTRF reduces the magnitude of the Th1 response to weak and/or self antigens
  • Studies with MBP and Hb peptide analogues provided herein demonstrate that the down-regulatory effects of CYTRF are inversely related to the strength of signaling via TCR, and that when
  • Th1 cells are stimulated by antigenic peptides that bind 1) with different affinity to MHC class II (MBP system), or 2) with different affinities to the TCR when complexed to MHC class II (hemoglobin (Hb) system)
  • MBP system MHC class II
  • Hb hemoglobin
  • the second experimental system utilizes a panel of peptide analogues derived from the Hb-64-76 peptide (Hb- ⁇ d allele), which is presented by l-E k , and has been tested for their antigenic properties using a specific T cell clone, 3L2 (Sloan-Lancaster ef al (1994) Cell 79, 913-922) It has been shown that position 72 (N in the wild type Hb) is a TCR contact site and is important for T cell activation, substitutions at this site do not affect binding affinity to class II E k Am o acid substitutions at position 72 produced a set of altered peptide ligands with partially agonistic and antagonistic properties For some, but not all Hb analogs, the differences in the T cell responses were linked to qualitatively-different signal transduction downstream of TCR, manifested by distinct patterns of tyrosine phosphorylation of ZAP-70 and CD3 ⁇ chains (Kersh ef al (1996) J
  • CYTRF inhibits production of the pro-Th1 cytokine IL-12 by macrophages
  • TCL-4 Materials and Methods Origin, maintenance, and stimulation of T cells
  • TCL-1s lines were established from B10 A and B10 BR mice by two consecutive immunizations with mMBP Ac1-16, following by isolation and serial re-stimulations of spleen cells from the primed animals After 3 - 4 in vitro serial re- stimulations both B10 A and B10 BR strains generated mMBP Ac1-16-spec ⁇ fic T cell lines producing Th1 cytokines All mMBP-specific TCL were maintained by antigenic re-stimulations every 10 - 14 days The 3L2 T cell clone was maintained by 14 day re-stimulations with 1 ⁇ M wild type Hb presented by B10 BR lethally-irradiated splenic APC, in the presence of 0 2 U/ml mouse recombinant IL-2 (Sigma)
  • Spleens were freshly dissociated in cold complete RPMI (RPMI 1640, 10% FCS), and erythrocytes were removed by a Ficoll gradient (Histopaq, Sigma) Cells from the Ficoll interface were washed two times in PBS, 2% FCS and reacted with 1 ⁇ g/ml macrophage-specific F4/80-b ⁇ ot ⁇ nylated antibody (monoclonal rat anti mouse lgG2b, Caltag) in the same buffer for 20 minutes at 4°C. Then macrophages were purified with streptavidin-coated magnetic beads (Miltenyi, Auburn, CA) according to the Miltenyi protocol.
  • the flow through F4/80 negative cells were then purified according to the Miltenyi protocol using magnetic beads coated with goat-anti-mouse IgG antibodies, selecting B cells. Macrophages were purified by the overnight adherence in complete RPMI. The purity of macrophage and B cell populations was assayed by FACS, using Mac-1 and B220 antibodies (both monoclonal rat anti- mouse lgG2b and lgG2a, respectively, from Caltag), and was found to be 98% for both cell types.
  • peritoneal macrophages were harvested by peritoneal lavage, plated at 2.5 -5 x 10 5 cells/ml, and purified by adherence for 1 hour in complete RPMI. Then macrophages were treated with 20 ⁇ g/ml LPS (E.coli, 0127:B8, Sigma) and 100 U/ml mouse recombinant IFN- ⁇ (Sigma). Culture supernatants were collected at 2 hours and at 24 hours, and the levels of IL-12 and TNF- ⁇ were measured as described above.
  • LPS E.coli, 0127:B8, Sigma
  • FPLC fractionation B10.A 2 hour culture supernatant was prepared, as described above, but was derived from Th1 B10.A TCL-1s. After 10X concentration with Centriplus 100 column (molecular weight cut off of 97 kD, Amicon, Beverly, MA), retained proteins were separated on an FPLC Superose 6 column (Pharmacia FPLC, Pinscataway, NJ). Molecular weight calibration of
  • FPLC column was determined with chymotrypsin, ferritin, thyroglobin, and blue dextran standards. FPLC fractions were then separately tested for their ability to down-regulate TNF- ⁇ production by BR E7 Th1 clone, as described above. Complete RPMI and FPLC buffer were used as negative controls. Trypsin digest: B10.A TCL-1s and B10.BR TCL-1s 2 hour culture supernatants were treated with trypsin/EDTA (GIBCO) for 2 hours at 37°C. Then supernatants were concentrated with Centriplus 100 columns and complete RPMI was added to block trypsin, after which another Centriplus 100 concentration was performed.
  • Trypsin digest B10.A TCL-1s and B10.BR TCL-1s 2 hour culture supernatants were treated with trypsin/EDTA (GIBCO) for 2 hours at 37°C. Then supernatants were concentrated with Centriplus 100 columns and complete RPMI was added to block tryp
  • DE52 and Concanavalin A separation B10.A TCL-1s and B10.BR TCL-1s 2 hour culture supernatants concentrated with Centriplus 100 were absorbed with DE52 beads (Sigma) in low pH, low salt buffer (20 mM TrisHCI, 50 mM NaCl), or with concanavalin A beads (type IV, Sigma) in PBS modified according to Sigma instructions.
  • Proteins bound to the DE52 beads were eluted with 0 5 M NaCl, 20 mM Tns-HCI, proteins bound to the concanavalin A beads were eluted with 100 mg/ml ⁇ -glucose After concentration and dialysis into RPMI 1640, flow through and bound fractions were tested for their ability to down-regulate TNF- ⁇ production by B10 BR TCL-4 Th1 cells
  • the B10 A-denved cytokine regulatory activity is produced by macrophages B 10 B R mMBP-specific Th1 TCL-4 was stimulated with mMBP Ac1-16 presented by pure populations of splenic mature macrophages or B cells or by unfractionated splenic APC Unfractionated splenic APC and macrophages displayed the B10 A-denved down-regulatory activity, as levels of TNF- ⁇ produced by the TCL-4 were reduced by about 70% when purified APC were derived from the B10 A strain
  • B10 A-denved B cells showed significantly diminished capacity to down- regulate TNF- ⁇ production, which was inhibited only by 20% compared to the B10 BR APC ( Figure 8) 2 x 10 5 TCL-4 cells/ml were stimulated with 30 ⁇ M mMBP Ac1-16 presented by B10 A or B10 BR APC as follows 1) 2 x 10 6 unsorted splenic APC, 2) 2 x 10 5 macrophages, 3)
  • CYTRF is a glycosylated protein of about 100 kD
  • two-hour culture supernatants from B10 A or B10 BR TCL-1s (both producing Th1 cytokines but differing in CYTRF activity) stimulated by syngeneic APC
  • These 2 hour culture supernatants were digested with trypsin, or incubated with anion-exchange resin DE52 or with concanavalin A-coated beads, followed by elution of bound proteins
  • Figure 9A shows levels of TNF- ⁇ _produced by B10 BR Th1 cells stimulated with mMBP Ac1-16 and B10 BR APC in the presence of control or treated B10 A or B10 BR two hour culture supernatants 5 x 10 5 TCL-4 cells were stimulated with 30 ⁇ M mMBP Ac1-16 and 5 x 10 6 B10 BR splenic APC in either B10 A or B10 BR two-hour culture super
  • the B10 A-denved down-regulatory activity was destroyed by trypsin and was absorbed either by DE52 in the low salt buffer, or by the concanavalin A-coated beads
  • the Th1 down- regulatory activity of the B10 A two-hour culture supernatant was present in the DE52 0 5 M NaCl eluent or in the concanavalin A eluent
  • B10 A and B10 BR two-hour culture supernatants demonstrated that B10 A-denved down-regulatory activity was present in the top, and not in the bottom fraction of the Centncon 100 protein concentrator, which has the molecular weight cut off of about 97 kD
  • B10 A two-hour culture supernatant was fractionated on FPLC, and the CYTRF activity of fractions with different molecular weight was tested as above for their ability to down-regulate production of TNF- ⁇ by Th1 cells
  • the down-regulatory activity was present in several fractions ranging from 100 kD to 470 kD, with the most prominent inhibition of the TNF- ⁇ production mediated by the 100 kD fraction 5 x 10 5 BR E7 cells were stimulated with 10 ⁇ M KLH and 5 x 10 6 B10 BR splenic APC in the indicated FPLC fractions derived from two-hour B10 A culture supernatant
  • the RPMI line corresponds to
  • the B10 A-denved CYTRF activity is associated with CyCAP CyCAP was considered a candidate for the activity of CYTRF because of its similarity in size and secretion by macrophages
  • CyCAP-depleted two-hour B10 A or B10 BR culture supernatants were tested for CyCAP activity
  • purified B10 A-denved CyCAP was added during the activation of mMBP-specific Th1 cells by B10 BR APC
  • depletion of CyCAP using either specific antiserum followed by protein A beads (A) or Cyp C-GST fusion protein bound to glutathione-agarose (B) significantly diminished the down-regulatory activity of the B10 A two-hour culture supernatant 2.5 - 5 x 10 5 TCL-4 cells/ml were stimulated with 30 ⁇ M mMBP Ac 1-16 and 2 5 - 5 x 10 5 B10 BR peritoneal macrophages in either B10 A or B10 BR two-hour culture supernatants depleted of CyCAP by using anti-CyCAP specific antibody followed by
  • FIGS 11A-11D show that B10 A-denved CyCAP down-regulated TNF- ⁇ (A) and IFN- ⁇ (B) levels produced by mMBP-specific TCL 4 5 x 10 5 TCL-4 were stimulated with 30 ⁇ M mMBP Ad -16 and 5 x 10 5 B10 BR macrophages with addition of serial dilutions of the B10 A-denved CyCAP purified from either peritoneal fluid or from culture supernatants from peritoneal macrophages cultured for 1 hour in vitro with no added stimuli TNF- ⁇ (A) and IFN- ⁇ (B) levels in the supernatants were
  • CyCAP did not dimmish the proliferative response to mMBP peptide (Figure 11C), which is consistent with previous observed finding of similar proliferative responses of B10 BR TCL stimulated with either B10 A or B10 BR APC The purity of the CyCAP was confirmed by the
  • Hb-specific 3L2 cells The proliferative responses of Hb-specific 3L2 cells correlated very well with the previously-determined induction of IL-2 production and of cytotoxic activity on a B cell line used as APC, the assay previously used to characterize agonistic/anagonistic properties and EC 40 of the Hb analogues.
  • the relative activities of T cell stimulation were determined as 100% for the wt Hb, 2% for Hb(T) and 0.006% for Hb(l); the EC 40 was established to be 0.0004 ⁇ M for wt Hb, 0.023 ⁇ M for Hb(T), and 7 ⁇ M for Hb(l).
  • Figure 13A shows that when TCL 4 cells were stimulated with the wild type mMBP Ad -
  • Hb(T) allowed B10.A macrophages to robustly inhibit Th1 cytokine production. Under very low levels of T cell stimulation (Hb(l)) the difference between the
  • B10.A and B10.BR macrophages in down-regulation of TNF- ⁇ was not observed; although IFN- ⁇ production was inhibited, very low levels of IFN- ⁇ were induced by the stimulation with APC from either strain.
  • 3 x 10 5 B10.A or B10.BR peritoneal macrophages/ml were cultured with 10 ⁇ M wt Hb during adherence purification After 1 hour macrophages were washed with cRPMI, and 3 x 10 5 3L2 T cells were added to these APC simultaneously with 100 ⁇ M Hb(T) As controls, 3L2 cells were stimulated by 10 ⁇ M wt Hb and 100 ⁇ M Hb(T) alone 24 hours later culture supernatants were collected and assayed for TNF- ⁇ and IFN- ⁇ levels
  • Hb(T) displays antagonistic properties
  • signaling by a partial agonist/antagonist is dominant over that of a strong agonist in the induction of intracellular events that allow down-regulation of TNF- ⁇ and IFN- ⁇ by B10 A-denved CYTRF
  • 100 ⁇ M mMBP Ac 1-16 was used in combination with 0 1 - 5 ⁇ M mMBP Ad- 16(4E)
  • 100 ⁇ M concentrations mMBP Ac 1-16 could not restore the ability of
  • CyCAP mRNA levels in B10 A and B10 BR macrophages RT PCR analysis of the CyCAP mRNA demonstrated that 1) there is no major difference in the levels of CyCAP mRNA between B10 A and B10 BR macrophages, 2) levels of CyCAP mRNA decline by 24 hours of culture, and 3) in addition to the CyCAP mRNA of the predicted 1902 bp size (12) other mRNA(s) of lower molecular weight are consistently amplified
  • CyCAP ' mice following injections of LPS As macrophages are known to produce IL-12, a major positive regulator of IFN- ⁇ , I L-12 production by activated B10 A, B10 BR and (B10 A x B10 BR)F1 macrophages, and by macrophages from CyCAP +/+ and CyCAP ' mice has been compared
  • B10 A, B10 BR, and (B10 A x B10 BR)F1 peritoneal macrophages elicited with thioglycollate were activated in vitro by LPS and IFN- ⁇ IFN- ⁇ is known to augment LPS-mduced
  • IL-12 production in macrophages Levels of IL-12 in the supernatants were tested 24 hours later As shown in Figure 16A, IL-12 levels produced by B10 A macrophages were down-regulated by 60% compared to those produced by B10 BR macrophages
  • Figure 16A adherence-purified thioglycollate-elicited peritoneal macrophages were cultured at 2 5-5 x 10 5 /ml and stimulated with 20 ⁇ g/ml LPS and 100 U/ml IFN- ⁇ 24 hours later supernatants were harvested and tested for levels of IL-12 using IL-12 ELISA Results represent means and standard errors of four independent experiments Figure 16B In experiment #1 , adherence-purified resident peritoneal macrophages were cultured at 2 5 x 10 5 /ml and stimulated with 10 ⁇ g/ml LPS and 100 U/ml IFN- ⁇ In experiment #2 adherence-purified thioglycollate-elicited macrophages were cultured
  • CyCAP down-regulated TNF- ⁇ and IFN- ⁇ production by mMBP-specific and Hb-specific mature Th1 cells
  • CyCAP also significantly down-regulated IFN- ⁇ levels produced by naive HEL-specific TCR transgenic T cells
  • exogenous CyCAP did not induce IL-4 by the HEL TCR transgenic T cells
  • CYTRF exhibits a novel mechanism of genetic and molecular regulation of cytokine production and of the control of susceptibility/resistance to autoimmune diseases in mice and in humans
  • Gly Lys Gly Pro lie Met Leu Asp Glu Val Glu Cys Thr Gly Thr Glu 85 90 95
  • Leu His lie Leu Asp Leu Ser Gly Glu Leu Ser Asp Ala Leu Gly Gin
  • ATC ATG AGA GTG GAT GCT GAG TGC ATG CCT GTC GTC AGA GAC TTC CTC 802 lie Met Arg Val Asp Ala Glu Cys Met Pro Val Val Arg Asp Phe Leu 195 200 205
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • Gly Leu His lie Leu Asp Leu Ser Gly Glu Leu Ser Asp Ala Leu Gly
  • GCA GGC ACG AGA GAG ACG CTG GTG TGG TCT GCA CCA ATG AAA CCA GGA 564 s Arg His Glu Arg Asp Ala Gly Val Val Cys Thr Asn Glu Thr Arg Se 115 120 125
  • CAATGACCTT CAACAACCGG CCACCAGATG TCGCCCTACT CACCTGAGCG CTCAGCTTCA 2082
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal

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Abstract

La présente invention concerne des procédés et des compositions destinés à moduler la production de cytokines par des lymphocytes T auxiliaires et des cellules présentrices de l'antigène. Les compositions obtenues régulent la production de cytokines pendant la réponse immunitaire. Le facteur régulateur des cytokines est produit par des cellules présentrices de l'antigène et peut agir sur des cellules CD4 + non sensibilisées de façon à les sensibiliser à un phénotype de cytokine de type Th1, sur des lymphocytes T de type Th1 sensibilisés, mûrs, de façon à réduire la synthèse des cytokines pro-inflammatoires, et sur des macrophages de façon à réduire la production de cytokines pro-inflammatoires. Les compositions peuvent également être utilisées pour un diagnostic ou une thérapie.
PCT/US1998/012345 1997-06-13 1998-06-12 Procedes de regulation de la fonction de production et de pouvoir pathogene de cytokines produites par des macrophages et des lymphocytes t WO1998056819A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7541184B2 (en) 2000-02-24 2009-06-02 Invitrogen Corporation Activation and expansion of cells
US7572631B2 (en) 2000-02-24 2009-08-11 Invitrogen Corporation Activation and expansion of T cells
US9528088B2 (en) 2002-06-28 2016-12-27 Life Technologies Corporation Methods for eliminating at least a substantial portion of a clonal antigen-specific memory T cell subpopulation

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