WO1998056819A1 - Methods of regulating t cell and macrophage cytokine production, function and pathogenicity - Google Patents

Abstract

Methods and compositions are provided for the modulation of cytokine production by T helper cells and antigen-presenting cells. Compositions are provided that regulate the production of cytokines during the course of an immune response. The cytokine regulatory factor is produced by antigen presenting cells, and can act on uncommitted CD4 + cells to cause them to commit to Th1 cytokine phenotype, on mature, committed Th1 type T cells to decrease the synthesis of pro-inflammatory cytokines, and on macrophages to decrease the production of pro-inflammatory cytokines. The subject compositions can be used in diagnosis or therapy.

Description

METHODS OF REGULATING T CELL AND MACROPHAGE CYTOKINE PRODUCTION,

FUNCTION AND PATHOGENICITY

The development of this invention was supported at least in part under NIH grant R01 AI9512, GM07276, HD07249, and by the Multiple Sclerosis Society grant PP0454 The government may have certain rights in this invention

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U S provisional patent application no 60/049,578, filed June 13, 1997

INTRODUCTION Helper (CD4⁺) 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-γ, TNF-β, GM-CSF and high levels of TNF-α Th2 cells express IL-3, IL-4, IL-5, IL-6, IL-9, IL- 10, IL-13, GM-CSF and low levels of TNF-α These cytokine profiles determine T cell regulatory and effector functions in immune responses The 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 autoimmune and inflammatory diseases, skewing toward Th2 cytokines promotes allergic reactions

Several factors have been shown to influence commitment to Th1 or Th2 profiles The best characterized regulators are cytokines IL-12 and IFN-γ are positive Th1 and negative Th2 regulators IL-12 promotes IFN-γ production, and IFN-γ 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-α by LPS-induced monocytes, in a way similar to IL-4

Signals through the T ceil receptor (TCR) and co-stimulatory molecules have also been shown to influence Th commitment 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 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 However, neither the B7 1/B7 2 effects nor the effects of different APC types in supporting Th1 or Th2 are absolute

The role of T cells in autoimmune disease is of great interest There are a number of mechanisms that cooperate to produce and maintain T-cell tolerance During thymic education, T cells with high affinity for self antigens are clonally deleted However, to ensure that a wide repertoire of T cells is available in the periphery, 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 avidity recognition of self-antigen by T cells permits escape from central tolerance An encephalitogenic epitope of myelin basic protein has been shown to have low affinity for the presenting MHC molecule The avidity of T cell recognition in the thymus may be compromised, enabling autoreactive T cells to escape self-tolerance The specific effects of T cell reactivity toward autoantigens is of great interest for the diagnosis and treatment of autoimmune disease Methods of altering the cytokine profile of autoreactive T cells would be beneficial in the treatment of autoimmune disease

Relevant Literature

The nucleotide and ammo acid sequence of the protein previously identified as cyclophi n C-associated protein (CyCAP) is available at Genbank, accession number L16894 For convenience, 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 muπne 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 For convenience, the polypeptides are referred to herein as cytokine regulatory factor (CYTRF) genencally, and may be further designated as mouse, human, etc

The cloning and characterization of CyCAP is described in Friedman ef al (1993) P N A S 90 6815-6819 The human homolog was independently cloned by a number of researchers, and is described in Rosenberg et al. (1991) J. Biol. Chem. 266:18731-18736; lacobelli et al. (1993) FEBS Lett. 319:59-65; Koths et al. (1993) J. Biol. Chem. 268:14245-14249: Inohara et al. (1994) Biochem Biophvs. Res. Comm. 201 :1366-1375; and Ullrich et al. (1994) _1 Biol. Chem. 269:18401-18407.

Other biological activities of CyCAP (90K protein, Mac-2-binding protein, murine adherent macrophage protein) are described in: Chicheportiche and Vassalli (1994) J. Biol. Chem. 269:5512-5517; Natoli et al. (1993) J. Acquired Imm. Deficiency Syndrome 6:370-375; Brakebusch et al. (1997) J. Biol. Chem. 272:3674-3682; Pearson (1996) Curr. Op. Immunol. 8:20-28; and Ma et al. (1996) J. Exp. Med. 183:147-157.

SUMMARY OF THE INVENTION Methods are provided for the modulation of cytokine production by T helper cells. 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.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 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.

Figures 3A-3C. Down-regulation of B10.BR TCL IFN-γ and TNF-α production by B10.A APC or by 2-hour supernatant from activated B10.A APC.

Figures 4A-4C. Supernatant from B10.A T cells activated with B10.A APC for 2 hours down-regulates IFN-γ and TNF-α production by B10.BR Th1 cells when A^k-expressing L cell fibroblasts are used as APC.

Figures 5A-5C. Down-regulation of IFN-γ and TNF-α levels is not affected when 2-hour B10.A culture supernatant is depleted of known Th1/Th2 cytokine regulators.

Figures 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-γ and TNF-α production by a BIO.BR-derived KLH-specific, E"-restricted T cell clone.

Figure 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.

Figures 10A-10B. Depletion of the B10.A-derived down-regulatory activity with anti- CyCAP antibodies or with the Cyp C-GST fusion protein.

Figures 11A-11D. Purified CyCAP down-regulates TNF-α and IFN-γ production, but not the T cell proliferation. Figures 12A-12B. Proliferative responses to MBP Ac 1-16 and Hb 64-76 peptides.

Figures 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-α and IFN-γ. Figures 15A-15C. Ef ects of CyCAP on Th1 cytokine production in response to strong and to partial agonists.

Figures 16A-16B. IL-12 production by B10.A, B10.BR, (B10.A x B10.BR)F1 , CyCAP

+/+ -/-

, and CyCAP macrophages.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

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. Methods of blocking of CYTRF may also be used, where a pro-inflammatory response is desired.

The subject methods utilize 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-α (TNF-α) and γ-interferon (γ-IFN) The synthesis of interleukin-2 (IL-2), which is involved in all T cell mediated responses, is not affected by the subject methods. 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 γ-IFN and TNF-α 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

Of particular interest are T cell responses to weak antigens, e g the response of post- thymic T cells to autoantigens As used herein, 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) 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-thymιdιne incorporation into the DNA of responding T cells, or production of specific cytokines by responding cells, such as IL-2, IFN-γ, 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 may be arranged according to strength of response (e g proliferation) over 3 logs of arbitrary units The antigen providing for the lowest log of response are negative, the next log are weak antigens and the maximal response are the strongest antigens Generally, a weak antigen will provide a response that may require antigen concentrations of at least about 1 μM, or higher The strong or weak nature of the antigen when bound as a peptide to an MHC protein can also be assessed by the induced changes in the T cell, e g phosphorylation of TCR/CD3-assocιated polypeptides, or down-regulation of TCR expression

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

Methods of Use

Formulations of CYTRF are administered to a host suffering from a pro-inflammatory immune disorder Macrophages are major producers of TNF-α, 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 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 Included are multiple sclerosis (MS), rheumatoid arthritis (RA), reactive arthritis, psoriasis, pemphigus vulgans, Sjogren's disease, thyroid disease, Hashimoto's thyroiditis, myasthenia gravis, insulin dependent diabetes mel tus (IDDM), ulcerative colitis/Crohn's disease, as well as many others 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, muπne, 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

Early treatment is preferable, in order to prevent the loss of function associated with autoimmune tissue damage 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 course of inflammatory arthritis While treatment during early stages is desirable, the adverse symptoms of the disease may be at least partially alleviated by treatment during later stages Clinical indices for the severity of arthritis include pain, swelling, fatigue and morning stiffness, and may be quantitatively monitored by Pannus criteria Disease progression in animal models may be followed by measurement of affected joint inflammation Therapy for inflammatory arthritis may combine the subject treatment with conventional NSAID treatment

A quantitative increase in myelin-autoreactive T cells with the capacity to secrete IFN- gamma is associated with the pathogenesis of MS, suggesting that autoimmune mducer/helper T lymphocytes in the peripheral blood of MS patients may initiate and/or regulate the demye nation process in patients with MS The overt disease is associated with muscle weakness, loss of abdominal reflexes, visual defects and paresthesias During the presymptomatic period there is infiltration of leukocytes into the cerebrosp al fluid, inflammation and demyehnation Family histories and the presence of the HLA haplotype DRB1*1501 , DQA1^*0102, DQB1^*0602 are indicative of a susceptibility to the disease Markers that may be monitored for disease progression are the presence of antibodies in the cerebrospmal fluid, "evoked potentials" seen by electroencephalography in the visual cortex and brainstem, and the presence of spinal cord defects by MRI or computerized tomography Treatment during the early stages of the disease will slow down or arrest the further loss of neural function

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 β-cells The depletion of β-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 In humans a long presymptomatic period precedes the onset of diabetes During this period there is a gradual loss of pancreatic β- 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

(1995) Nature Genetics 9 293-298) 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 After the onset of overt diabetes, patients with residual β-cell function, evidenced by the plasma persistence of insulin C-peptide, may also benefit from administration of the subject polysacchaπdes 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

CYTRF Compounds

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 centπfugation and chromatography, and having the properties of binding to anti-CyCAP antibodies and cyclophilm C When added to Th1 committed T cells, the factor down-regulates expression of the pro-inflammatory cytokines IFN-γ and TNF-α CYTRF also inhibits production of the pro-Th1 cytokine IL-12 by macrophages When the factor is present in stimulated cultures of ThO cells, it shifts the cytokine profile of the stimulated T cells to a Th2 type In mice, 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

For use in the subject methods, 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

The 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

Techniques for in vitro mutagenesis of cloned genes are known Examples of protocols for scanning mutations may be found in Gustm ef al , Biotechmques 14 22 (1993), Barany, Gene 37 111-23 (1985), Colicelli et al , Mol Gen Genet 199 537-9 (1985), and Prentki et al , Gene

29 303-13 (1984) Methods for site specific mutagenesis can be found in Sambrook ef al , Molecular Cloning A Laboratory Manual, CSH Press 1989, pp 15 3-15 108, Werner et al , Gene 126 35-41 (1993), Sayers et al , Biotechmques 13 592-6 (1992), Jones and Winistorfer, Biotechmques 12 528-30 (1992), Barton ef al , Nucleic Acids Res 18 7349-55 (1990), Marotti and Tomich, Gene Anal Tech 6 67-70 (1989), and Zhu Anal Biochem 177 120-4 (1989)

The protein may be joined to a wide variety of other o gopeptides or proteins for a variety of purposes By providing for expression of the subject peptides, various post-expression modifications may be achieved For example, by employing the appropriate coding sequences, one may provide farnesylation or prenylation In this situation, 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 πcin, abnn, diphtheria toxin, or the like, or with specific binding agents that allow targeting to specific moieties on a target cell

Where targeting is desired, 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 multimeπc 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 seπne 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 ) In pharmaceutical dosage forms, 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

For oral preparations, 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, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives The compounds can be utilized in aerosol formulation to be administered via inhalation

The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like

Furthermore, the compounds can be made into suppositories by mixing with a variety of 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 Similarly, 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 For example, 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 The term "unit dosage form," as used herein, 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

The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public Moreover, 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 μg 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

Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects Some of the specific compounds are more potent than others Preferred dosages for a given compound are readily determmable by those of skill in the art by a variety of means A preferred means is to measure the physiological potency of a given compound The use of 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 In one aspect of the invention, 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 lipids, such as phosphatidylcho ne The remaining lipid will normally be neutral lipids, such as cholesterol, phosphatidyl senne, phosphatidyl glycerol, and the like For preparing the liposomes, the procedure described by Kato ef al (1991) J Biol Chem

266 3361 may be used Briefly, 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 After intense agitation for short periods of time, from about 5-60 sec , 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 This method allows for the incorporation into the lumen of high molecular weight molecules As an alternative method of delivery, 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 muπne leukemia virus and modified human immunodeficiency virus, adenovirus vectors, efc that are maintained transiently or stably in mammalian cells A wide variety of vectors can be employed for transfection and/or integration of the gene into the genome of the cells Alternatively, micro- injection may be employed, fusion, or the like for introduction of genes into a suitable host cell See, for example, Dhawan ef a/ (1991) Science 254 1509-1512 and Smith et al (1990) Molecular and Cellular Biology 3268-3271

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 β-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, e g plasmid, retrovirus, e g lentivirus, adenovirus, and the like, where the vectors are able to transiently or stably be maintained in the cells, usually for a period of at least about one day, more usually for a period of at least about several days to several weeks

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. Harlow and Lane eds , Cold Spring Harbor Laboratories, Cold Spring Harbor, New York, 1988 If desired, 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 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 A wide variety of 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

The term "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 binding molecules include pairs, such as biotm and streptavidm, digoxin and antidigoxm etc For the specific binding members, the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures

A variety of other 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 Depending upon the manner of introduction, 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

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention Efforts have been made to ensure accuracy with respect to the numbers used (e g amounts, temperature, concentrations, etc ) but some experimental errors and deviations should be allowed for Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade, and pressure is at or near atmospheric

Materials and Methods

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 Antι-IL-4 mAb 11 B11 was provided by J O'Hara and W E Paul (NIH) Anti- IFN-γ mAb XMG1 2 was provided by T Mosmann (University of Alberta, Edmonton, Canada) Anti-IFN-γ mAb HB170 was obtained from ATCC Antι-IL-4 mAb BVD4 and BVD6, and antι-IL-10 mAb 2A5 and Sxc 1 were provided by M Howard (DNAX, Palo Alto, CA) Antι-IL-12 p40 mAb C17 8 and C15 6 were provided by G Tπnchieπ (Wistar Institute of Anatomy and Biology,

Philadelphia, PA) Anti-TNF-α mAb was provided by Robert Schreiber (Washington School of Medicine, St Louis, MO) Antι-IL-13 and anti-TGFβ pan-neutralizing antibodies were purchased from R&D Systems (Minneapolis, MN)

Peptides 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 μM 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 μM mMBP Ac1-16 in RPMI 1640 (GIBCO BRL, Gaithersburg, MD) with 2% syngeneic mouse serum Every 10-14 days thereafter 0 5-1x10⁶ T cells were re-stimulated with 30 μM mMBP Ac1-16 and 5x10⁶ 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 μg/ml of Concavalin A was added every other re-stimulation

BR E7 T cell Clone Every 7-10 days 2-5 x 10⁵ T cells per ml were re-stimulated with 2-5 x 10⁶ irradiated (3000 rad) B10 BR splenic APC per ml, 3 μg/ml KLH (Calbiochem, San Diego, CA) and 10% rat ConA supernatant Culture supernatants were harvested 24 hours later and assayed for cytokines

Induction And Scoring OfEAE Three days after the last re-stimulation with 30 μM mMBP Ac1-16, 5x10⁶ irradiated splenic APC, and IL-2, 2-5x10⁷ T cells were washed three times in serum free RPMI and injected i v in the tail veins of non-irradiated recipient mice, 6-9 weeks old Mice were monitored daily, and EAE seventy was measured on the scale 0-5 0, no symptoms, 05 straight tail, 1 , limp tail, 2, paraparesis, 3, paraplegia, 4, quadπplegia, 5, moribund Mice with score 4-5 were euthanized

Induction of Cytokine Responses At the time of re-stimulation aliquots of 0 25-1 x10⁶ T cells were washed and plated in 1 ml of RPMI 1640, 10% FCS 30 μm mMBP Ac1-16, 5x10^s irradiated (3000 rad) splenocytes and 10% rat ConA supernatant (if less than 1x10⁶ T cells were used) were added for 24 hours, after which supernatant fluids were removed and assayed for levels of cytokines For measuring effects of 2 hr supernatants on the levels of IFN-γ and TNF-α, supernatant fluids were obtained from culture of 1x10⁶ B10 A and B10 BR T cells per ml re- stimulated with 30 μm mMBP Ac1-16 and 5x10⁶ splenocytes per ml for 2 hours Alternatively, 1 x 98/56819

10⁷ freshly isolated spleen cells per ml were incubated for 2 hours with 10 μg/ml LPS (E Coli 0127 B8, Difco (Detroit, Ml) The 2 hour supernatants from each strain (fresh or stored frozen at - 80°C) were then used as culture media in which 0 5-1x10⁶B10 BR T cells per ml were re-stimulated with 5x10⁶ irradiated B10 BR splenocytes per ml and 30 μm mMBP for 24 hours In some experiments 5x10⁵ T cells per ml were re-stimulated with 30 μm mMBP Ac1-16 and 5x10⁵ per ml irradiated (10,000 rad) transfected L cell fibroblasts expressing A^k class II proteins (LA^k cells)

Depletion of Splenic T-cells Spleen cells were dissociated in serum free RPMI 1640, resuspended at 4 x 10⁷ per ml and incubated for 10 minutes at 4° with 100 μl/ml (NH4)₂S0₄- concentrated antι-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 antι-CD3 mAb or with isotype-control hamster IgG (Caltag, Burlingame, CA)

Addition or Depletion of Cytokines Cytokines were added as follows mouse recombinant IL-4 (Boehnnger, Indianapolis, IN) at 100 units/ml, mouse recombinant IL-12

(PharMmgen, San Diego, CA) at 10 units/ml, mouse recombinant IFN-γ (Boehnnger, Indianapolis, IN) at 100 ng/ml, mouse recombinant TNF-α (Genentech, South San Francisco, CA) at 2 ng/ml To deplete cytokines from the 2 hour culture supernatants, 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 μg/ml and 20 μg/ml, antι-IL-10 (Sxc 1) at 20 μg/ml and 40 μg/ml, antι-IL-12p40

(C17 8) at 20 μg/ml, antι-IL-13 (goat polyclonal) at 50 μg/mi and 75 μg/ml, anti-IFN-g (XMG1 2) at 10 μg/ml and 20 μg/ml, anti-TNF-α (TN3 19 12) at 50 μg/ml and 100 μg/ml, anti-TGFb (rabbit polyclonal) at 40 μg/ml and 80 μg/ml Following 30 mm incubation at 4°C, 100 μl of protein G- coated agarose beads (Pierce, Rockford, IL) were added to the supernatants After incubation for 30 mm at room temperature, beads were removed from supernatants by centnfugation at 3000 rpm for 3 minutes, and supernatants were then used as a media for the re-stimulation of T cells

ELISA Double-antibody sandwich ELISA assays were used Plates were coated overnight with a monoclonal antibody specific for a particular cytokine, washed and then incubated with culture supernatant or a cytokine standard overnight A second monoclonal antibody, which was biotm-conjugated and which recognizes an independent epitope of the same cytokine, was used for detection Antibodies were biotmylated with D-biotm-N- Hydroxysuccinimide ester (Sigma, St Louis, MO) Biotmylated antibodies on the plate were detected with Streptavidm-HRP (Pierce, Rockford, IL) plus o-Phenylenediamine (Sigma, St Louis, MO) Optical densities were determined at wavelength = 490 nm, using Molecular Devices (Sunnyvale, CA) plate reader Anti- IL-4 mAb BVD4 or 11 B11 was used for coating and BVD6 for detection of IL-4 Mouse recombinant IL-4 from DNAX (Palo Alto, CA) was used as a standard Antι-IL-10 mAb 2A5 (coating) and Sxc 1 (detection) were used for IL-10 Mouse recombinant IL-10 from DNAX (Palo Alto, CA) was used as a standard Antι-IL-12p40 mAb C17 8 (coating) and C15 6 (detection) were used to determine the IL-12 levels Recombinant munne IL-12, provided by S Wolf, Genetics Institute, (Cambridge, MA), was used as a standard Anti-IFN-γ mAb HB170 (coating) and XMG1 2 (detection) were used for the IFN-γ ELISA Recombinant munne IFN-γ (Genentech,

South San Francisco, CA) was used as a standard

IL-2 Bioassay 50 μl of supernatants from T cell lines re-stimulated with serial dilutions of mMBP Ad -16 and irradiated splenic APC for 24 hours were added to 1 x10⁴ HT-2 cells per well in 96 well plates in a final volume of 200 μl IL-4 was neutralized with 11 B11 antι-IL-4 mAb Munne recombinant IL-2 (Sigma, St Louis, MO) was used as a standard 16-18 hours later ³H-thymιdιne

(1 μCi per well in 20 μl) was added to plates After 6 - 7 hours incubation plates were harvested (Ph D Cell Harvester), and incorporation of ³H-thymιdιne was measured by scintillation counting

TNF Bioassay Briefly, 2x10⁴ L929 cells per well were plated in 96 well plates and incubated overnight Serial dilutions of supernatants from T cell lines and 1 μg/ml actinomycm C1 (Boehnnger, Indianapolis, IN) were added to wells in a final volume of 200 μl A standard curve was prepared using recombinant munne TNF-α (Genentech, South San Francisco, CA) After 24 hours incubation 10 μl of 5 mg/ml MTT (Sigma, St Louis, MO) was added to each well 4 hours later 100 μl of solubilization solution containing 50% dimethylformamide and 20% SDS (Sigma, St Louis, MO) were added to each well for 1 hour, and the optical densities were determined using a Molecular Devices (Sunnyvale, CA) plate reader (wavelength = 570 nm)

Results

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) Freshly isolated

B10 A and B10 BR lymph node cells gave very low prohferative responses to MBP compared to those from A^u strain B10 PL, which is susceptible to EAE The differences in susceptibility to EAE and in proliferation of A^u - vs A^k -restricted MBP-pnmed lymph node cells most likely reflects higher affinity binding of MBP N-termmal peptides by A^u compared to A^k class II proteins For adoptive transfer of EAE T cell lines (TCL) specific for mMBP Ac1-16 were generated from B10 PL, B10 A, B10 BR and (B10 AxB10 BR) F1 animals, using splenic APC in the serial re- stimulations After several in vitro re-stimulations T cells were transferred into non-irradiated recipients using the EAE adoptive transfer model In two adoptive transfer experiments two independently-derived sets of TCL specific for mMBP Ac1-16 (TCL 1 and TCL 2) were generated from BIO PL, B10 A, B10 BR and (B10 AxB10 BR) F1 animals Strain B10 PL was used as a positive control for the encephalitogenicity of MBP-specific TCL The first set of TCL (TCL 1 ) was re-stimulated nine times in vitro and the second set of TCL (TCL 2) was re-stimulated five times in vitro prior to transfer into syngeneic recipients TCL from B10 A and B10 BR strains were also transferred into (B10 AxBIO BR) F1 animals The severity of EAE was scored on a standard scale from 0 to 5

The results of these adoptive transfer experiments are presented in Table 1 The positive control B10 PL TCL induced severe EAE Both TCL from B10 BR mice were also highly encephalitogenic in syngeneic recipients and caused disease in F1 recipients In contrast, both B10 A-denved TCL failed to induce significant disease in either B10 A or F1 recipients F1 T cells transferred into F1 mice induced weak EAE, though marginally more severe disease than that induced by B10 A T cells These results show that B10 A and B10 BR strains display a significant phenotypic difference in that TCL from B10 BR induce severe EAE, whereas TCL from B10 A fail to do so Low encephalitogenicity appears to be dominant in the (B10 A x B10 BR) F1 TCL specific for MBP Table 1. MBP-specific TCL from BIO a or (BIO A \ B10 BR) FI Mice Are Less Encephalitogenic than

MBP-specific TCL from BIO BR Strain

Clinical signs of EAE

Strain Maximum score Day of onset

Donor TCL^* No Recipient mice Incidence Average (range) Average (range)

BIO PL TCL 1 BIO PL 5/5 1 9 (1-3) 7 (5-10)

B10 BR TCL 1 B10 BR 5/5 2 7 (2-4) 10 6 (7-14)

TCL 2 B10 BR 5/5 3 4 (3-4) 7 2 (6-10)

B10 BR TCL 1 (A x BR)Fl ^§ 3/5 1 3 (0 5-2) 13 3 (8-17)

TCL 2 (A x BR)F1 5/5 3 8 (3-4) 8 6 (7-10)

B10 A TCL 1 B10 A 4/5^J 0 75 (0 5-1) 7 (4-12)

TCL 2 B10 A 4/5 0 5 (0 5-1) 8 (7-15)

B10 A TCL 1 (A x BR)Fl 3/4 0 66 (0 5-1) 9 (6-1 1)

TCL 2 (A x BR)Fl 3/5 0 5 16 (12-20)

(A x BR)Fl TCL 2 (A x BR)Fl 4/5 1 (0 5-1 5) 14 2 (9-20) 'TCL 1 cells were re-stimulated nine times in vitro before transfer, using syngeneic APC for B10 PL TCL and a random mixture of B10 A and B10 BR APC for B10 A and B10 BR TCL TCL 2 cells were restimulated five times before transfer, using syngeneic splenic APC

*One out of five B10 A mice injected with B10 A T cells died shortly after the experiment, likely due to injury or stress *(A x BR)F 1 is an abbreviation of (B 10 A x B 10 BR)F 1

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 This suggested that the genetic origin of the APC influenced the encephalitogenicity of TCL To test this hypothesis, 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 After nine re-stimulations with mMBP Ac1- 16 presented by splenic APC of syngeneic or semi-syngeneic F1 genetic origin, the TCL were adoptively transferred into recipients (TCL re-stimulated with the reciprocal parental APC did not expand sufficiently in number to transfer ) As shown in Table 2, neither B10 A TCL caused significant EAE, consistent with the results with TCL1 and TCL2 Strikingly, while B10 BR TCL re- stimulated with B10 BR splenic APC induced severe disease, the B10 BR TCL re-stimulated with F1 APC did not cause EAE These results indicate that B10 BR splenic APC are required for the development of encephalitogenicity of B10 BR TCL and that the non-EAE-promoting phenotype of B10 A is dominant in F1 APC

Table 2. Encephalitogenicity of BIO BR MBP-specific T Cells Is Reduced by Re-stimulation with (BIO A x BIO BRJF1 Splenic APC

Clinical signs of EAE

Strain Maximum score Day of onset

Donor TCL APC Recipient mice Incidence Average (range) Average (range)

(TCL 3)

B10 BR B10 B B10 BR 5/5 3 8 (3-4) 7 5 (5-10)

B10 BR (A x BR)Fl B10 BR 1/4 0 5 9

B10 A B10 A BIO A 4/5 0 5 9 (6-12)

B10 A (A x BR)Fl BIO A 3/5 0 5 12 5 (9-16)

Cytokine Profiles, but not Other Parameters Required for Encephalitogenicity, Differ among BW A B10 BR and (B10 A x B10 BR) F1 T Cell Lines Encephalitogenic B10 BR TCL and non-encephalitogenic B10 A and F1 TCL were characterized for parameters previously shown to be required for the induction of EAE to determine which parameter is affected by the genetic differences between these strains The activation state of TCL following stimulation with mMBP Ac1-16 was assayed by examining proliferation, cell surface levels of Mel-14, LFA-1 , VEA (CD69) CD3, and CD4 antigens and by cell-cycle analysis None of these indices of activation differed between the B10 A and B10 BR TCL, all TCL were virtually 100% CD4⁺ and proliferated vigorously in response to MBP Ac1-16 The proliferative responses of all TCL were strictly mMBP Ac1-16-specιfic and dose-dependent whether re-stimulated with APC of syngeneic, F1, or reciprocal parent strain origin displayed no allogeneic reactivity The potential for homing to the CNS was examined by measuring cell surface levels of α4β1 integnn, VLA-4, no consistent differences were found between B10 A and B10 BR TCL FACS and RT PCR analysis demonstrated that the predominant T cell receptor Vβ regions expressed by B10 A and B10 BR TCL were extremely similar (predominantly Vβ 4, 8, and 13), and somewhat more heterogeneous than that of the B10 PL MBP TCL (50% Vβ 8) The levels and sequences of the A^k class II presenting element for MBP Acl-16 were also examined for strains B10 A and B10 BR By flow cytometry the levels of A^k expressed by splenic B cells, and splenic macrophages, and peritoneal macrophages from B10.A and B10.BR mice were found to be identical. Splenic cDNA for Aα^k and Aβ^k chains were amplified from both strains and sequenced using direct PCR sequencing and were found to be identical over the entire protein coding sequence.

These results indicate that the class II presenting elements, mMBP Ac1-16-specific TCR repertoire, and indices of activation are identical between B10.A and B10.BR.

In contrast, comparison of the cytokine profiles of B10.A and B10.BR TCL revealed differences which correlated with their encephalitogenicity. Data for the TCL 2 lines are shown in Figures 1A-1 F. 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.

Additional cells harvested three days after the same fifth re-stimulation were used for adoptive transfer EAE. Data presented for TNF are means and standard deviations of two bioassays performed on triplicate wells for each sample (n=6). Data presented for IFN-g, IL-12, IL-4 and IL- 10 are means and standard deviations of triplicate wells for each sample (n=3). Data presented for IL-2 are means and standard deviations of two IL-2 bioassays performed in duplicate wells for each sample (n=4).

The B10.A TCL stimulated with peptide and B10.A splenic APC displayed the Th2 phenotype, expressing very low levels of IFN-γ, IL-2 and TNF-α and high levels of IL-4 and IL-10. Levels of Th1 -promoting IL-12 (made by APC) were undetectable. In contrast, the B10.BR TCL displayed the Th1 phenotype, secreting very little IL-4 and IL-10 and high levels of IFN-γ, IL-2 and

TNF-α. IL-12 levels were also significantly higher. TNF-α ELISA results were nearly identical (within 10%) to the TNF bioassay results, and as TNF biological activity was totally blocked with the anti-TNF-α mAb TN3-19.12, all of the TNF activity detected in these TCL supernatants appears to be TNF-α. 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. Thus, TCL from B10.A mice produced a Th2, low TNF-α response, T cells from B10.BR produced a Th1 , high TNF-α response, and the Th2, low TNF-α phenotype was dominant in the (B10.AxB10.BR) F1 T cell cultures. These results indicated that genetic difference between B10.A and B10.BR controls a regulator of Th cytokines.

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. We found that the genetic origin of the splenic APC used for re-stimulations greatly influenced the cytokine profile of the B10.BR TCL. Thus, the Th1 , high TNF-α phenotype of the B10 BR TCL was maintained only by syngeneic B10 BR splenic APC, very little IFN-γ, TNF- α or IL-2 was produced by the B10 BR TCL serially re-stimulated with F1 or B10 A APC (Figure 2A-2E) Figure 2A-D After the third, fourth, fifth, ninth and tenth re-stimulations of T cell lines with mMBP Ac1-16 and irradiated splenic APC of the indicated origin, T cells were re-stimulated for 24 hours with peptide and APC of the same origin Adoptive transfer of EAE was performed after the ninth re-stimulation (See Table 2) Supernatant fluids were analyzed for cytokines In Figure 2 D the points for the B10 BR TCL 3 - B10 A APC are 37 ng/ml of IL-10 after the ninth re-stimulation and 45 ng/ml of IL-10 after the tenth re-stimulation Data presented are average of duplicate wells for each sample Figure 2E At the ninth re-stimulation TCL 3 described for panels A-D were re-stimulated for 24 hours with peptide and APC of the same (indicated) origin Supernatant fluids depleted of IL-4 were analyzed for IL-2 levels, using the growth factor dependent cell line HT-2 Data presented are means and standard deviations of four wells for each sample (n=4)

For IL-4, all three of the B10 A TCL had higher levels than the B10 BR TCL (Figure 2 C), and B10 BR T cells maintained on B10 BR APC produced the lowest IL-4 levels For IL-10, B10 A and F1 APC, but not BIO BR APC supported levels similar to those produced by B10 A TCL,

(Figure 2 D) A Th2, low TNF-α profile was seen for all B10 A TCL irrespective of the source of APC

Expression of the Th1 , high TNF-α phenotype only by B10 BR TCL serially re-stimulated with B10 BR APC correlates well with the encephalitogenicity of this TCL in adoptive transfer of EAE (Table 2) These results indicate that B10 A and F1 APC prevent the development of the

Th1 , high TNF-α encephalitogenic cytokine phenotype in response to MBP

IFN-γ and TNF-α 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 To determine whether a single re-stimulation with B10 A APC can alter the production of IFN-γ and TNF-α by established B10 BR-denved Th1 cells, 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) were re-stimulated overnight with B10 BR or B10 A APC prior to harvesting supernatants for cytokine assays As shown in Figure 3A, B and C (top bars), IFN-γ and TNF-α levels were down-regulated by 40-60% when B10 A instead of B10 BR APC were used for the single re-stimulation Figure 3 A, B and C represent independent experiments with MBP-specific B10 BR TCL 3 (A and B) and TCL 4 (C) TCL were re-stimulated for 24 hours in the presence of B10 BR or B10 A splenic APC, or splenic APC depleted of T cells (panel A, lower bars) In some cultures B10 BR TCL were re-stimulated with B10 BR APC and MBP peptide in the presence of 2-hour supernatant fluids from cultures of B10 BR or B10 A TCL stimulated with syngeneic APC and MBP peptide (Figure 3 B, bottom bars, 3 C, middle bars), or in the presence of 2-hour supernatants from B10 BR or B10 A spleen cells stimulated with LPS (Figure 3 C, bottom bars) Data presented are means and standard deviations of triplicate wells for each sample (n=3) All experiments were repeated at least three times with similar results

Similar down-regulation of IFN-γ and TNF α was observed when B10 A (but not B10 BR) T cell-depleted splenic APC were used to re-stimulate B10 BR TCL (Figure 3 A, bottom bars)

Irrespective of the genetic source of APC IL-2 levels and proliferative responses remained high and IL-4 was not induced

To determine whether the inhibitory activity of B10 A APC on IFN-γ and TNF-α levels was mediated by a soluble factor, the two B10 BR TCL were re-stimulated with mMBP Ac1-16 and B10 BR splenic APC in the presence of supernatant fluids from a culture of B10 A TCL stimulated with B10 A splenic APC and mMBP Ac1-16 for 2 hours The 2-hour time point was chosen because most cytokines induced by activation of T cells and APC are not detectable at this time, except TNF-α and -β The B10 A culture-derived 2-hour supernatant down-regulated IFN-γ and TNF-α production by at least 50% relative to control cultures with 2-hour supernatants from B10 BR T cells and APC plus peptide (Figure 3B, bottom bars and 3C, middle bars), comparable to the effects of B10 A APC themselves Similar results were obtained with B10 BR TCL 4 stimulated with B10 BR splenic APC and MBP peptide in the presence of supernatants from freshly-isolated B10 A and B10 BR spleen cells activated for 2 hours with LPS, (Figure 3C, bottom bars) Thus, down-regulation of IFN-γ and TNF-α production seems to be mediated by a soluble factor secreted by B10 A splenic APC within the first 2 hours after activation by antigen-stimulated

T cells or by LPS However, 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-α producing cells irrespective of the genetic source of APC or 2 hour supernatant

The B10 A-deπved Soluble Cytokine Regulatory Factor Functions when A^k-expressιng L- cell Fibroblasts are Used as APC To begin to clarify the target of action of the B10 A-deπved regulatory factor, B10 BR or B10 A T cells (TCL 4) were re-stimulated twice with mMBP Ac1-16 presented by non-professional APC (A^k-expressιng L cells which do not produce splenic APC- derived cytokines), in the presence of 2-hour supernatant from B10 A cultures (Figures 6A-6B) In either normal medium or in the 2-hour B10 BR supernatant, B10 BR T cells produced high levels of IFN-γ and TNF-α, and B10 A T cells produced high levels of IL-4 with little or no TNF-α and IFN- γ However, the levels of IFN-γ and TNF-α produced by B10 BR TCL were reduced in the presence of 2-hour supernatant from B10 A cultures, similar to the effects seen when professional splenic APC were used (Figures 4A-4C) This result indicates that B10 A-denved factor does not require the presence of splenic APC for its action and probably acts directly on the responding T cells B10 BR (top bars) or B10 A (bottom bars) TCL 4 were activated with mMBP Ac1-16 and A^k- expressmg L cell fibroblasts as APC for two consecutive re-stimulations in either normal medium or in 2-hour supernatants from B10 BR or B10 A T cells activated with syngeneic splenic APC Culture supernatants were harvested 24 hours post re-stimulation and IFN-γ, TNF-α and IL-4 were measured as above Data presented are means and standard deviations of triplicate wells for each sample (n=3) Similar results were obtained in two additional experiments

The B10 A-denved Factor Acts Independently of Known Th1/Th2 Regulatory Cytokines in Down-regulating B10 BR Production of lFN-γand TNF-α To assess whether down-regulation of IFN-γ and TNF-α by B10 A APC is mediated by or depends on any of the known Th1/Th2 regulators, 2-hour culture supernatants derived from B10 BR or B10 A TCL APC cultures were depleted of IL-4, IL-10, IL-12 p40, IL-13, TNF-α, IFN-γ and TGF-β and tested for their effects on IFN-γ and TNF-α production by B10 BR TCL4 re-stimulated with B10 BR APC and mMBP peptide Cytokine depletion did not dimmish the down-regulatory activity of the B10 A culture supernatant was observed (Figure 5 A and B) Serial dilutions of B10 A and B10 BR 2-hour culture supernatants (Figure 5 C) demonstrated that the down-regulatory activity of B10 A-deπved factor is rapidly lost by dilution, indicating that any depletion of the down-regulatory cytokine would have been detected Essentially identical results were obtained when the anti-cytokme antibodies were also used at half the concentrations for which data are shown in Fig 5 Figures A and B B10 BR or B10 A T cells (TCL 4) were re-stimulated with mMBP Ac1-16 and syngeneic splenic APC for 2 hours Neutralizing antibodies specific for mouse IL-4, IL-10, TNF-α, IFN-γ, TGF-β, IL-13 and IL-12p40 were added to these culture supernatants for 30 minutes, and antibody-cytokine complexes were removed using protein G-coated agarose beads Cytokine depleted 2-hour supernatants were then used as media in which B10 BR TCL 4 were re- stimulated with mMBP Ac1-16 and B10 BR splenic APC for 24 hours IFN-γ (panel A) and TNF-α

(panel B) levels were measured as above Lower levels of TNF-α following treatment with anti- TNF-α antibody and protein G beads were due to the presence of residual anti-TNF-α antibody, which has low affinity for protein G in medium with serum Neutralizing antibodies were used at concentrations equal to and twice recommended by the manufacturers for complete neutralization of cytokine levels produced by high expressing cells, giving similar results Data presented, for the higher antibody concentration, are means and standard deviations of triplicate wells for each sample (n=3) Figure 5C B10 BR TCL-4 cells were re-stimulated with B10 BR splenic APC and mMBP Ac1-16 in 2-hour culture supernatants from B10 A or B10 BR TCL-4, used either undiluted or at serial 1 2 dilutions in RPMI, 10% FCS TNF-α levels are means and standard deviations of triplicate wells for each sample (n=3) As summarized in Figures 6A-6B, blocking or adding IL-4 and IL-12 (alone or in combinations) did not reduce the down-regulatory effects of B10 A APC on TNF-α levels, although there were effects on the absolute levels of TNF-α production B10 BR T cells (TCL 4) were re- stimulated with mMBP Ac1-16 and either B10 BR or B10 A splenic APC for 24 hours in the absence or presence of mouse recombinant IL-4, IFN-γ, TNF-α and IL-12 alone or combination with neutralizing antibody specific for mouse IL-12, or mouse IL-4 Cytokine levels resulting from each treatment are expressed as % of control (B10 BR TCL, B10 BR APC and peptide with no cytokine or antibody additions) Data presented are means and standard deviations of triplicate wells for each sample (n=3) At least two independent experiments were performed for each treatment, yielding similar results In contrast, adding IFN-γ significantly reduced the down- regulation by B10 A APC For IFN-γ production, adding TNF-α did not prevent the down- regulation of IFN-γ by B10 A APC, and adding IL-4 did not synergize with this down-regulation (Figure 6 B) As expected because of their pro-Th1 effects, addition of IL-12 alone, or in combination with antι-IL-4, did increase the overall IFN-γ levels and reduced the down-regulation of IFN-γ by B10 A APC

In the experiments shown in Figures 5 and 6, none of the cytokines or anti-cytokme antibodies induced detectable levels of IL-4 production by the B10 BR TCL Overall the results suggest that the B10 A-denved factor acts independently of known cytokine regulators, and that strong pro-Th1 conditions can overcome its down-regulatory effect on TNF-α and IFN-γ

B10 A APC Down-regulate IFN-γ and TNF-α 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-restπcted 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-γ and TNF-α levels produced by this Th1 clone Thus, the B10 A APC-denved factor acts independently of the specificity of the Th1 cell and is able to down-regulate IFN-γ and TNF-α 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-stimulations Culture supernatants were harvested and assayed for TNF-α (Figure 7A) and IFN-γ (Figure 7B) 24 hours post stimulation Data presented are means and standard deviations of triplicate wells for each sample (n=3) Similar results were observed in at least two additional experiments

A genetic dιfference(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-specιfιc 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-γ, and TNF-α production, and in the production of IFN-γ, and TNF-α 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 down-regulation of IFN-γ, and TNF-α production by a B10 BR- denved KLH-specific, E^k-restrιcted T cell clone when re-stimulated by KLH presented by B10 A APC demonstrates that the regulatory effects of B10 A-denved factor 1) are not limited to responses of MBP-specific, A^k-restrιcted Th1 cells, and 2) can act by affecting individual clonal populations of mature Th1 cells rather than by expanding uncommitted precursors or ThO cells The induction of comparable proliferative and IL-2 responses in Th1 B10 BR TCL by MBP peptide presented by either B10 A or B10 BR APC indicates that B10 A APC do not inhibit the activation of these T cells but down-regulate production of IFN-γ and TNF-α The target of the factor appears to be the responding T cells, as the down-regulatory effects of the B10 A supernatants are observed when MBP peptide is presented to B10 BR T cells by non-professional APC, A^k - expressing L cell fibroblasts B10 A TCL maintain a Th2, low TNF-α cytokine profile irrespective of the genetic source of the APC or 2-hour supernatant, suggesting that they had become committed in vivo, perhaps due to the pro-Th2 effects of their own APC The Th2-ιnducιng phenotype of B10 A is dominant in (B10 A x B10 BR) F1 cells

The identity of the APC cell type(s) within the irradiated B10 A splenocyte population that is responsible for the down-regulation of IFN-γ and TNF-α production has not yet been determined Splenic T cells seem not to be required for the down-regulation Preliminary results indicate that B10 A-denved splenic macrophages have down-regulatory activity, while splenic B cells do not The results to date indicate that the down-regulation of IFN-γ and TNF-α production by B10 BR Th1 cells is mediated by B10 A APC, but it is possible that activated B10 A T cells also produce the down-regulatory factor

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-γ and TNF-α The B10 A-denved factor also acts independently of prostaglandm E2, an APC-denved molecule known to inhibit production of IFN-γ by activated CD4+ T cells, indicated by the failure of mdomethacin to block down-regulation of IFN-γ and TNF-α production by B10 A APC The activity of this factor in down-regulating IFN-γ production also differs from that of IL-4 in that IL-4, but not the factor, blocks IFN-γ induction by IL- 12 through its inhibition of signalling through the IL-12 receptor The finding that the down-regulatory effects of the B10 A-denved factor on Th1 IFN-γ production is overcome by exogenous IFN-γ and by IL-12, especially in combination with antι-IL-4, suggests that the in vivo effects of the B10 A-denved factor should be most evident in the absence of strong pro-Th1 conditions This may occur during initial antigen priming, especially when T cell responses are weak - for example, with poor antigens such as the MBP Ac1-16 peptide, and perhaps more generally with self antigens The apparent direct effect of B10 A APC and of the secreted cytokine regulatory factor on IFN-γ production by mature Th1 cells, even when IL-4 has been depleted, suggests that inhibition of IFN-γ production may be the primary mechanism by which the B10 A-denved factor skews initial Th1/Th2 subset determination When T cells are strongly activated, or where significant levels of IL-12 and/or IFN-γ are already present, the B10 A-denved factor may not down-regulate IFN-γ production Unlike IFN-γ, down-regulation of TNF-α is maintained in the presence of exogenous IL-12, implying IFN-γ and TNF-α are regulated independently

The basis for the difference between 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-lιnked, 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 telomeπc to Ea) was derived from strain A/WySn However, preliminary tests of (B10 A x B10 BR) F2 progeny and (F1 x B10 BR) backcross progeny, in which their splenic APC were assayed for the ability to down-regulate TNF-α production by MBP-specific B10 BR TCL, suggest that the polymorphic gene(s) controlling factor production is/are not linked to H-2 If confirmed, this would indicate that strains B10 A and B10 BR also differ by a non-H-2 locus or loci Splenic APC from A strain sublme A/WySn and from the A strain background H-2congenιc A TL down- regulate IFN-γ and TNF-α production by B10 BR TCL in response to MBP Ad -16 Thus the production of the down-regulatory factor by B10 A appears to be due to a contaminating non-H-2 gene(s) derived from the A/WySn donor of the H-2* haplotype, despite the ten backcrosses to the C57BIJ10 inbred partner during the initial derivation of B10 A The genetic and phenotypic properties of the B10 A-denved factor clearly distinguish it from another major polymorphic gene previously shown to regulate Th1/Th2 cytokine production The Leishmania resistance gene acts in T cells to regulate their Th1/Th2 cytokine phenotypes, whereas the results reported here show that the B10 A-denved factor acts in the APC used for T- cell activation (Howard ef al (1980) Parasite Immunol 2 303-314) Also, for Leishmania, most inbred strains, including B10 A and A strains, have the resistant (Th1) phenotype Finally, in the

Leishmania system the resistant (Th1) phenotype is largely dominant in heterozygotes, whereas for the B10 A-denved factor discussed here the Th2-promotιng 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 Identification of the soluble cytokine regulatory factor controlled by the B10 A-denved gene, its cellular origin, and its mode of action, contribute to deciphering the mechanisms regulating Th1 and Th2 cells and their pathogenicity in autoimmune, inflammatory, and allergic disorders

Example 2 Characterization of the Cytokine Regulatory Factor and its Activities

B10 A APC produce a soluble factor that down-regulates production of TNF-α and IFN-γ 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-α and IFN-γ 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 T cell stimulation is very strong, B10 A and B10 BR APC support similar levels of TNF-α and IFN-γ

To test this hypothesis directly, two experimental systems have been used, in which 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) It has been previously established that an MBP analogue with lysine at position 4 substituted by glutamic acid, MBP Ac 1-16(4E), binds at least 100X stronger to A^k than the wild type peptide (Tate ef al (1995) Intl Immunol l,141-l<ξ>\ , Lee et al (1998. J Exp Med 187(9) 1505-1516) Such differential affinity reflects the nature of the polymorphic ammo acid residue at position 9 in the class II Aβ chain As expected, glutamic acid instead of lysine in peptide position 4 was also shown to result in a significantly lower dose of peptide required for activation of T cells specific for the N-termmal MBP peptides

The second experimental system utilizes a panel of peptide analogues derived from the Hb-64-76 peptide (Hb-β^dallele), 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 CD3ζ chains (Kersh ef al (1996) J

Exp Med 184, 1259-1268) Similar studies using altered peptide ligands were also performed by other research groups, with the general conclusion that a higher state of CD3ζ phosphorylation and of ZAP-70 association with CD3ζ chain stimulated by strong agonist peptides results in more pronounced Th1 response (Constant and Bottomly (1997) Annu Rev Immunol 15, 297-332) CYTRF was also analyzed for its effects on cytokine production by macrophages

Consistent with its activity in inhibiting production of Th1 cytokines IFN-γ and TNF-α, CYTRF inhibits production of the pro-Th1 cytokine IL-12 by macrophages

Materials and Methods Origin, maintenance, and stimulation of T cells The B10 BR-deπved MBP-specific T cell line TCL-4 was generated as described above 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-specιfic 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 μM wild type Hb presented by B10 BR lethally-irradiated splenic APC, in the presence of 0 2 U/ml mouse recombinant IL-2 (Sigma)

Peptides and antibodies used for functional assays of T cell responses Mouse MBP Ac1 -

16 and mouse MBP Ac1-16(4E) were synthesized by the Stanford Protein and Nucleic Acid facility (Stanford, CA), wild type Hb 64-76 and Hb with substitutions of threonme, Hb(T), and isoleucme, Hb(l) at position 72 were either obtained from P Allen's laboratory or were synthesized by the Stanford Protein and Nucleic Acid facility Antι-CD4 antibody (L3/T4 rat anti-mouse lgG2b) was purchased from PharMingen

Purification of splenic macrophages and B cells 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 μg/ml macrophage-specific F4/80-bιotιnylated 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.

Induction of lL-12 and TNF-α production by macrophages. Resident peritoneal macrophages were harvested by peritoneal lavage, plated at 2.5 -5 x 10⁵ cells/ml, and purified by adherence for 1 hour in complete RPMI. Then macrophages were treated with 20 μg/ml LPS (E.coli, 0127:B8, Sigma) and 100 U/ml mouse recombinant IFN-γ (Sigma). Culture supernatants were collected at 2 hours and at 24 hours, and the levels of IL-12 and TNF-α were measured as described above.

Biochemical characterization of CYTRF.

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-α 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-digested, concentrated 2 hour culture supernatants were treated with 0.1 mM PMSF (Sigma) and their ability to down-regulate TNF- α_was tested on the B10.BR TCL-4 Th1 line, as described. Untreated 2 hour culture supernatants processed similarly were used as a positive control.

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 α-glucose After concentration and dialysis into RPMI 1640, flow through and bound fractions were tested for their ability to down-regulate TNF-α production by B10 BR TCL-4 Th1 cells

CyCAP depletion, purification and addition, and metabolic labeling

Depletion B10 A TCL-1s and B10 BR TCL-1s 2 hour culture supernatants concentrated with Centriplus 100 were depleted of CyCAP by adding 1 250 final dilution of rat anti-CyCAP antiserum, with subsequent removal of the CyCAP-antibody complexes by a 1 5 hour incubation with protein A beads (Pierce) at 4°C In a separate experiment Centriplus 100-concentrated 2 hour culture supernatants were incubated for 3 hours at 4°C with the glutathione beads (Sigma) coated with Cyp C-GST fusion protein

Purification and Addition 3 x 10⁷ thioglycollate-elicited peritoneal lavage cells from 3 mice were plated at 1 x 10⁶/ml in complete RPMI and purified by adherence Macrophage culture supernatants harvested after one hour adherence and peritoneal fluids from which the macrophages were obtained were concentrated with Centriplus 100, and CyCAP was purified by binding to the Cyp C-GST-coated glutathione beads and subsequent elution with 30 μg/ml CsA, as previously described (Friedman ef al (1993) Proc Natl Acad Sci CSA 90, 6815-6819) Eluted CyCAP (and 30 μg/ml CsA used as a negative control) were then dialyzed in PBS overnight and further concentrated with Centπcon 100 spin filters

Metabolic labeling Thioglycollate-elicited peritoneal lavage cells were cultured at 3 x 10⁶ cell/ml in methionme-free, cysteme-free RPMI 1640 (Sigma), 2 5% FCS for 1 hour, after which non-adherent cells were removed and 0 5 mCi/ml of S³⁵-methιonιne (Amersham) was added to the macrophages After four hours of the metabolic labeling, culture supernatants were harvested, concentrated with Centncon 100 columns, and CyCAP was purified with the Cyp C-GST-coated glutathione beads and eluted with CsA, as above CyCAP was then resolved on a 10% SDS- PAGE protein gel

CyCAP-specific RT PCR Total RNA was extracted with RNeasy kit (Qiagen) from 1 x 10^s thioglycollate-elicited peritoneal macrophages at 0, 1 , 2 and 24 hours post isolation, according to the Qiagen protocol 100 μl cDNA was synthesized from 1 μg of RNA, using o go dT primers and MMLV reverse transcπptase (GIBCO), as recommended by GIBCO Then RT PCR was performed with Taq polymerase (GIBCO) and CyCAP-specific primers [SEQ ID NO 5] 5' AT G GCT CTC CTG TGG CTC CTC TC, 3' [SEQ ID NO 6] CAC CAT GTC AGT GGA GTT AGT GAG The following conditions were used for the amplification 2 mm at 95°C, 35 amplification cycles of 1 mm at 94°C, 1 mm at 58°C, 3 mm at 72°C, then 10 mm at 72°C Amplified cDNA was resolved on 1% agarose gel containing 0 5 μg/ml ethidium bromide, and visualized using digital camera

Results 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-α produced by the TCL-4 were reduced by about 70% when purified APC were derived from the B10 A strain In contrast, B10 A-denved B cells showed significantly diminished capacity to down- regulate TNF-α production, which was inhibited only by 20% compared to the B10 BR APC (Figure 8) 2 x 10⁵ TCL-4 cells/ml were stimulated with 30 μM mMBP Ac1-16 presented by B10 A or B10 BR APC as follows 1) 2 x 10⁶ unsorted splenic APC, 2) 2 x 10⁵ macrophages, 3) 8 x 10⁶ B cells Culture supernatants were harvested 24 hours later and assayed for TNF-α Presented are the means and standard deviations of triplicate wells of TNF-α bioassay

These results indicated that the cytokine regulatory factor mediating the genetic difference between the B10 congenics is produced by macrophages Therefore, subsequent experiments based on the comparison of Th1 down-regulatory activity in B10 A and B10 BR strains were performed mostly with pure peritoneal macrophages These experiments confirmed that B10 A macrophages are responsible for the Th1 inhibitory activity

CYTRF is a glycosylated protein of about 100 kD To characterize the nature of CYTRF, several biochemical characterizations were performed with 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-α_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 10⁵ TCL-4 cells were stimulated with 30 μM mMBP Ac1-16 and 5 x 10⁶ B10 BR splenic APC in either B10 A or B10 BR two-hour culture supernatants (unmampulated, treated with trypsin, absorbed by or eluted from the DE52 or Concanavalin A beads, as indicated) Shown are means and standard deviations of triplicate wells of the TNF-α bioassay, similar data were obtained in an additional experiment

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 These results demonstrate that CYTRF is a glycosylated acidic soluble protein

Comparison between 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 Following this result, 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-α by Th1 cells As shown in Figure 9B, the down-regulatory activity was present in several fractions ranging from 100 kD to 470 kD, with the most prominent inhibition of the TNF-α production mediated by the 100 kD fraction 5 x 10⁵ BR E7 cells were stimulated with 10 μM KLH and 5 x 10⁶ B10 BR splenic APC in the indicated FPLC fractions derived from two-hour B10 A culture supernatant The RPMI line corresponds to the levels of TNF-α produced in complete RPMI medium Addition of the FPLC buffer to the stimulated BR E7 cells did not reduce levels of TNF-α Presented are the means of duplicate wells of TNF-α bioassay with variance within 15% of the mean Thus, the Th1 inhibitory protein has a molecular weight of approximately 100 kD or higher

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

To assay whether CyCAP participates in the observed down-regulation of the Th1 cytokine production, two approaches were used CyCAP-depleted two-hour B10 A or B10 BR culture supernatants were tested for CyCAP activity Alternatively, purified B10 A-denved CyCAP was added during the activation of mMBP-specific Th1 cells by B10 BR APC As shown in Figures 10A-10B, 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 10⁵ TCL-4 cells/ml were stimulated with 30 μM mMBP Ac 1-16 and 2 5 - 5 x 10⁵ 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 protein A beads (A), or by using Cyp C-

GST-glutathione beads (B) or in supernatant incubated with controls Normal Rat Serum for the antibody-based depletion and PBS for the Cyp C-GST-based depletion Results represent means and variances of duplicate wells of TNF-α_bιoassay Two total experiments each with the addition of antibody and Cyp C-GST depletion methods produced similar results The levels of TNF-α produced by MBP-specific TCL-4, stimulated with mMBP Ac 1-16 and B10 BR APC, increased and became similar to those produced in the presence of the B10 BR two hour culture supernatant The residual down-regulatory activity seen in panel B may represent residual CyCAP, as the amount of Cyp C-GST necessary for the maximal depletion was not determined

To confirm the role of CyCAP in CYTRF activity, purified native B10 A CyCAP was added to the Th1 cells during the antigenic stimulation with syngeneic B10 BR peritoneal macrophages

Figures 11A-11D show that B10 A-denved CyCAP down-regulated TNF-α (A) and IFN-γ (B) levels produced by mMBP-specific TCL 4 5 x 10⁵TCL-4 were stimulated with 30 μM mMBP Ad -16 and 5 x 10⁵ 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-α (A) and IFN-γ (B) levels in the supernatants were

3 determined after 24 hours, and the incorporation of H-thymidine (C) was assayed after 48 hours, as above Similar results were obtained in an additional experiment

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

SDS-PAGE gel of the ³⁵S-metabolιcally labeled, macrophage secreted proteins, purified with the Cyp C-GST affinity beads and eluted with CsA 1 x 10^s thioglycollate-elicited B10 A peritoneal macrophages/ml were metabolically labeled with ³⁵S-methιonιne, and CyCAP purified with Cyp C- GST-coated glutathione beads and eluted with CsA was resolved on a 10% SDS-PAGE gel Shown CyCAP purified from duplicate wells, as imaged by digital camera The migration position of 97 4 kD marker protein is indicated at the right Similarly, highly enriched CyCAP protein was identified by silver-stained SDS-PAGE gel These results suggest that CYTRF activity is associated with CyCAP

Effectiveness of CYTRF in down-regulating IFN- and TNF-α depends on the nature of signaling via TCR The effects of CYTRF on IFN-γ and TNF-α production were tested in the MBP and Hb systems Th1 cells were differentially stimulated by the wild type peptide or peptide analogues known to elicit different T cell responses Figures 12A-12B show proliferative responses of TCL 4 (Panel A) and 3L2 (Panel B) to different antigens, reflecting the effects of the altered peptide ligands on the level of T cell stimulation 2 x 10⁴ T cells/100 μl were stimulated with serial dilutions of peptides presented by 1 x 10⁵ B10 BR lethally-irradiated splenic APC in one well of 96 well plate 48 hours later 1 μCi ³H-thymιdιne was added to each well, and incorporation of the ³H-thymιdιne was measured as described above Similar results were obtained in at least two independent experiments The strong agonist peptide mMBP Ac 1-16(4E) induced proliferation of T cells at about 1 -

2 log lower concentration compared to the partial agonist mMBP Ac 1-16 Addition of the inhibitory anti-CD4 antibody shifted the mMBP Ac 1-16(4E) dose-response toward that of the mMBP Ac 1- 16. Similarly, the wt Hb peptide induced proliferative responses at significantly lower doses, compared to partial agonist Hb(T). The antagonist peptide Hb(l) did not induce proliferative response even at 100 μM. 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 EC₄₀ 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 EC₄₀ was established to be 0.0004 μM for wt Hb, 0.023 μM for Hb(T), and 7 μM for Hb(l). Figure 13A shows that when TCL 4 cells were stimulated with the wild type mMBP Ad -

16, CYTRF down-regulated TNF-α and IFN-γ_production; however, stimulation of Th1 cells with a stronger agonist, mMBP Ac1-16(4E), did not permit such down-regulation. 3 x 10⁵TCL-4 cells/ml were stimulated with 30 μM mMBP Ad-16, 0.3 μM mMBP Ac1-16(4E), or 3 μM mMBP Ac1- 16(4E) + 1 μg/ml anti-CD4 presented by 3 x 10⁵ B10.A or B10.BR peritoneal macrophages. Figure 13B: 2x 10⁵ 3L2 cells/ml were stimulated with 10 μM wild type (wt) Hb 64-76 or 100 μM

Hb(T), or 100 μM Hb(l) presented by 3 x 10⁵ B10.A or B10.BR peritoneal macrophages. Culture supernatants were assayed for TNF-α and IFN-γ 24 hours later. Presented are means and standard deviations of triplicate wells of the TNF-α bioassay and IFN-γ ELISA; except for TNF-α levels in 6A, which represent means and variances of duplicate wells of the TNF-α bioassay. Similar results were obtained in at least three experiments.

Decreasing T cell stimulation by blocking CD4 signaling with anti-CD4 antibody again restored the inhibitory activity of the B10.A macrophages. Similar results were observed with B10.BR APC in the presence of B10.A vs. B10.BR two-hour culture supernatants. Adding the blocking anti-CD4 antibodies to TCL 4 stimulated by the wild type mMBP Ad -16 resulted in a very low cytokine response, and did not increase, but at times even diminished differences in responses elicited by the B10.A and B10.BR APC.

Similarly, B10.A-derived down-regulatory effects were significantly lower when the strong agonist, the wild type 64-76 Hb peptide, was used in the stimulation of the 3L2 cells compared to the partial agonist Hb(T) (Figure 13B). 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-α was not observed; although IFN-γ production was inhibited, very low levels of IFN-γ were induced by the stimulation with APC from either strain. Stimulation of T cells with a combination of wt Hb and Hb(T), potentially allowing signaling by both peptide ligands, resulted in the diminished production of TNF-α and IFN-γ compared to that observed in the presence of wt Hb alone and again restored the down-regulatory activity of B10.A-derived CYTRF (Figures 14A-14B). 3 x 10⁵ B10.A or B10.BR peritoneal macrophages/ml were cultured with 10 μM wt Hb during adherence purification After 1 hour macrophages were washed with cRPMI, and 3 x 10⁵ 3L2 T cells were added to these APC simultaneously with 100 μM Hb(T) As controls, 3L2 cells were stimulated by 10 μM wt Hb and 100 μM Hb(T) alone 24 hours later culture supernatants were collected and assayed for TNF-α and IFN-γ levels

These results suggest that 1) Hb(T) displays antagonistic properties, and 2) 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-α and IFN-γ by B10 A-denved CYTRF Similar results were observed when 100 μM mMBP Ac 1-16 was used in combination with 0 1 - 5 μM mMBP Ad- 16(4E) At lower than 100 μM concentrations mMBP Ac 1-16 could not restore the ability of

B10 A-denved CYTRF to down-regulate Th1 cytokine production, probably because it could not productively compete with the mMBP Ad-16(4E) for the binding to the IA

The Th1 down-regulatory effects of purified CyCAP were also tested in Hb system 10 nM CyCAP significantly inhibited the production of TNF-α and IFN-γ by the Hb-specific 3L2 cells (Figures 15A and 15B) 3 x 10⁵ 3L2 cells/ml were stimulated with 2 x 10⁵ B10 BR peritoneal macrophages and either 10 μM wt Hb or 100 μM Hb (T) in the presence or in the absence of 10 nM CyCAP TNF-α (A) and IFN-γ (B) were assayed 24 hours after stimulation Serial dilutions of B10 A-denved CyCAP (C) were added to the similarly stimulated 3L2 cells, but 1 μM wt Hb and 10 μM Hb (T) were used Shown are means and standard deviations or variances of triplicate wells for TNF-α bioassay and duplicate wells for the IFN-γ ELISA Similar results were obtained in an additional experiment

In surprising contrast with the results seen with either B10 A macrophages or with B10 A two-hour culture supernatant, the magnitude of inhibition by 10 nM highly purified CyCAP was at least as pronounced for the strong antigen, the wild type Hb, as for the Hb(T) However, the inhibitory effects of the B10 A-denved CyCAP were sustained at lower CyCAP concentrations when T cells were stimulated by the partial agonist Hb(T) (Figure 15C), suggesting that at higher than physiological levels CyCAP can down-regulate Th1 cytokine production induced by strong agonists The effectiveness of the B10 A-denved down-regulatory activity depends on the strength or nature of T cell stimulation The most prominent down-regulation is observed during sub-optimal levels of T cell activation These data also support the involvement of CyCAP in the

CYTRF-mduced down-regulation of Th1 cytokines

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 These results imply that the genetic difference between the B10 A and B10 BR in CYTRF activity does not reflect differences in the levels of CyCAP mRNA, and that CyCAP mRNA levels are either positively regulated in vivo or are negatively affected by some in vitro culture conditions Multiple mRNA species could represent genes with regions homologous to the PCR primers, or alternatively spliced forms of

CyCAP mRNA

Production of lL-12 by B10 A, B10 BR, (B10 A x B10 BR)F1, CyCAP^+/+, and CyCAP ' macrophages As described above, CYTRF down-regulatory activity for IFN-γ and TNF-α was shown to be produced by macrophages Serum levels of IFN-γ and TNF-α are elevated in

CyCAP ' mice following injections of LPS As macrophages are known to produce IL-12, a major positive regulator of IFN-γ, 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-γ IFN-γ 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 10⁵/ml and stimulated with 20 μg/ml LPS and 100 U/ml IFN-γ 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 10⁵/ml and stimulated with 10 μg/ml LPS and 100 U/ml IFN-γ In experiment #2 adherence-purified thioglycollate-elicited macrophages were cultured at 4 x 10⁵/ml and similarly stimulated 9 hours later supernatants were harvested and tested for levels of

IL-12 using IL-12 ELISA Results represent means of duplicate wells with variances within 22%

The lower IL-12 production was dominant in the (B10 A x B10 BR)F1 macrophages Levels of TNF-α measured either at 2 hours or at 24 hours after the activation were not consistently different between the two B10 strains, with macrophages from each strain variably producing higher, lower, or similar levels of this cytokine Variation in TNF-α production may reflect differential delivery of thioglycollate injections and/or differences in macrophage isolation/viability Therefore, only when TNF-α levels were similar between the macrophages from two B10 strains were IL-12 levels compared

Similar experiments were performed with macrophages from CyCAP ^{+ +} and CyCAP ' mice As shown in Figure 16B, CyCAP ' macrophages produced higher levels of IL-12 compared to macrophages from CyCAP ^+/+ strain This general conclusion, however, was accompanied by a large variation between experiments in total levels of IL-12 being produced and in the magnitude of differences between CyCAP ^+/+ and CyCAP ' mice Macrophages used in experiment #1 were from mice derived from three backcrosses onto the C57/BL background and were not elicited with thioglycollate However, IL-12 production by these resident cells was at least three fold higher compared to macrophages used in the experiment #2, derived from F3 mice of crosses onto the

129 background and elicited with thioglycollate, which usually results in higher macrophage activation In a third experiment, normal resident macrophages were derived from CyCAP ^+/+ and CyCAP ' F3 mice on the 129 background These cells produced only low levels of IL-12 and did not show a significant difference in IL-12 levels between CyCAP ^{+ +} and CyCAP ' macrophages These results suggest that genetic difference between B10 A and B10 BR strains may control IL-12 production in response to LPS Similarly to the effects of B10 A-denved CYTRF, CyCAP appears to be involved in the down-regulation of the IL-12 response, indicated by the enhanced IL-12 production when this gene is not functional

Characterization of CYTRF activity has identified several physiologically important points First, the inhibitory activity is made by macrophages As these cells are efficient APC which, unlike B cells, generally promote Th1 cytokine responses, the differential genetic control of TNF-α and IFN-γ production by macrophages is very relevant The capability of B10 A macrophages to inhibit the effector functions of Th1 cells, while not affecting IL-2 and IL-4 production, would limit the induction of DTH The physiological significance of CYTRF is suggested by the studies connecting the effectiveness of the B10 A-denved inhibitory activity with the degree of T cell activation Strong T cell activation signals induced by true agonists appear to make the cells resistant to the down-regulation of TNF-α and IFN-γ by CYTRF However, inhibition of CD4 signaling or signaling by partial agonists/antagonists (alone or in combination with the strong agonist) permitted the down-regulation of these Th1 cytokines by the B10 A-denved factor Thus, the genetically-determined difference between B10 A and B10 BR strains in Th1 cytokine production is manifested mainly when T cells are activated sub-optimally, reflecting the strength and/or nature of the activating stimuli

This conclusion is relevant to the regulation of potential self-reactivity, as T cells strongly responding to self are eliminated and/or inactivated by several layers of negative control, but weakly self-reactive T cells may escape such negative regulation Th1 cytokine production in response to weak self-antigens may suffice to induce an autoimmune disease The activity of CYTRF allows inhibition of the Th1 effector responses to partial agonists, either self-peptides or pathogenic peptides, while allowing T cell proliferation and help for anti-pathogenic antibody production The present results suggest that susceptibility to CYTRF-mediated inhibition of the TNF-α and IFN-γ cytokine production is controlled by qualitatively and not by quantitatively different signals through the TCR The dosage of an antigen is not as important as its agonistic character, as TNF-α and IFN-γ were successfully down-regulated when a 10-fold range of concentrations of partial agonists were used, and similarly, resistance to the Th1 cytokine inhibition by CYTRF was observed in a 10-fold range of strong agonists As signaling by agonists, partial agonists and antagonists corresponds to qualitatively-different patterns of the CD3 and ZAP-70 tyrosme phosphorylation, one can identify "permissive" vs "inhibitory" signal transduction pathways downstream of the TCR, leading to the characterization of the critical kinetic and/or molecular events in T cell activation determining whether TNF-α and IFN-γ will be produced at high levels or down- regulated The B10 A-denved down-regulatory activity is dependent on CyCAP Effects of depletion and addition of CyCAP on TNF-α and IFN-γ production, as well as the biochemical parameters support this conclusion The addition of CyCAP does not affect T cell proliferation, which is also similar to the phenotypes of the B10 A-denved CYTRF Finally, IL-12 levels produced by CyCAP knockout macrophages in response to LPS + IFN-γ were often higher than those produced by the CyCAP wild type cells, suggesting that CyCAP similarly to CYTRF negatively regulates production of IL-12

Highly-purified B10 A-denved CyCAP down-regulated TNF-α and IFN-γ production by mMBP-specific and Hb-specific mature Th1 cells The same preparation of CyCAP also significantly down-regulated IFN-γ levels produced by naive HEL-specific TCR transgenic T cells As predicted, 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

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: Conboy, Irina

Jones, Patricia

(ii) TITLE OF THE INVENTION: METHODS OF REGULATING T CELL AND MACROPHAGE CYTOKINE PRODUCTION, FUNCTION AND PATHOGENICITY

(iii) NUMBER OF SEQUENCES: 6

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Bozicevic & Reed, LLP

(B) STREET: 285 Hamilton Avenue, Suite 200

(C) CITY: Palo Alto

(D) STATE: CA

(E) COUNTRY: USA

(F) ZIP: 94301

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ for Windows Version 2.0

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 60/049,578

(B) FILING DATE: 13-JUN-1997

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Sherwood, Pamela J

(B) REGISTRATION NUMBER: 36,677 (C) REFERENCE/DOCKET NUMBER:

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 650-327-3400

(B) TELEFAX: 650 327-3231

(C) TELEX:

(2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2176 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE:

(A) NAME/KEY: Coding Sequence

(B) LOCATION: 176...1897

(D) OTHER INFORMATION: Mouse CYTRF cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

CAGCCTCGTC CCAGGAAAGC CACATGGTGG AGCTTTTCTC TGCTGAACAG TCTACAGAAG 60

GCTTCCAGTT GCAGCCAGGA AGGAGGCTCC AGGACTGCCT TCAGCCACTG CCTCGCTGCC 120

TGAGGGACAG TTGAGTCCCT CTCTTGCTCC CAGGGTTGGG CTTCTAGGCC AGGCA ATG 178

Met

1

GCT CTC CTG TGG CTC CTC TCT GTG TTC TTG CTG GTT CCA GGG ACT CAA 226 Ala Leu Leu Trp Leu Leu Ser Val Phe Leu Leu Val Pro Gly Thr Gin 5 10 15

GGT ACA GAA GAT GGA GAC ATG GGC TTG GTT AAC GGG GCC TCA GCC AAT 274 Gly Thr Glu Asp Gly Asp Met Gly Leu Val Asn Gly Ala Ser Ala Asn 20 25 30

GAG GGC CGC GTG GAG ATC TTC TAC AGA GGC CGG TGG GGG ACA GTG TGT 322 Glu Gly Arg Val Glu lie Phe Tyr Arg Gly Arg Trp Gly Thr Val Cys 35 40 45 GAC AAC CTC TGG AAC CTT TTG GAT GCC CAC GTC GTC TGC CGG GCC CTG 370 Asp Asn Leu Trp Asn Leu Leu Asp Ala His Val Val Cys Arg Ala Leu

50 55 60 65

GGC TAT GAG AAC GCC ACC CAA GCA CTG GGC AGA GCT GCC TTC GGG CCA 418

Gly Tyr Glu Asn Ala Thr Gin Ala Leu Gly Arg Ala Ala Phe Gly Pro 70 75 80

GGA AAG GGA CCG ATC ATG CTG GAT GAG GTG GAA TGT ACA GGG ACC GAG 466

Gly Lys Gly Pro lie Met Leu Asp Glu Val Glu Cys Thr Gly Thr Glu 85 90 95

TCC TCA CTG GCC AGT TGC AGA TCC CTG GGT TGG ATG GTG AGC CGC TGT 514

Ser Ser Leu Ala Ser Cys Arg Ser Leu Gly Trp Met Val Ser Arg Cys 100 105 110

GGG CAC GAG AAG GAC GCA GGC GTG GTC TGC TCC AAC GAT ACC ACG GGG 562

Gly His Glu Lys Asp Ala Gly Val Val Cys Ser Asn Asp Thr Thr Gly 115 120 125

CTT CAC ATC CTG GAC CTC TCT GGA GAG CTC TCA GAT GCA CTG GGC CAG 610

Leu His lie Leu Asp Leu Ser Gly Glu Leu Ser Asp Ala Leu Gly Gin

130 135 140 145

ATC TTT GAC AGC CAG CAG GGC TGC GAC CTA TTC ATC CAG GTG ACA GGG 658 lie Phe Asp Ser Gin Gin Gly Cys Asp Leu Phe lie Gin Val Thr Gly 150 155 160

CAG GGG TAT GAG GAC CTG AGC CTC TGT GCC CAC ACG CTG ATC CTG CGC 706

Gin Gly Tyr Glu Asp Leu Ser Leu Cys Ala His Thr Leu lie Leu Arg 165 170 175

ACC AAC CCC GAG GCC CAG GCC CTG TGG CAA GTG GTG GGC AGC AGC GTC 754

Thr Asn Pro Glu Ala Gin Ala Leu Trp Gin Val Val Gly Ser Ser Val 180 185 190

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

AGG TAC TTT TAC TCC CGA AGA ATC GAG GTC AGC ATG TCT TCT GTT AAG 850

Arg Tyr Phe Tyr Ser Arg Arg lie Glu Val Ser Met Ser Ser Val Lys

210 215 220 225 TGC TTG CCA AAG CTA GCC TCT GCC TAT GGG GCC ACA GAG CTT CAG GAC 898 Cys Leu Pro Lys Leu Ala Ser Ala Tyr Gly Ala Thr Glu Leu Gin Asp 230 235 240

TAC TGT GGA CGG CTT TTT GCC ACC CTC CTC CCC CAA GAC CCC ACT TTC 946 Tyr Cys Gly Arg Leu Phe Ala Thr Leu Leu Pro Gin Asp Pro Thr Phe 245 250 255

CAT ACT CCC TTG GAC CTT TAT GCG TAC GCA CGG GCC ACC GGG GAC TCT 994 His Thr Pro Leu Asp Leu Tyr Ala Tyr Ala Arg Ala Thr Gly Asp Ser 260 265 270

ATG CTG GAA GAT CTG TGT GTA CAG TTT CTG GCC TGG AAC TTC GAG CCT 1042 Met Leu Glu Asp Leu Cys Val Gin Phe Leu Ala Trp Asn Phe Glu Pro 275 280 285

CTG ACA CAG TCT GAG TCC TGG TCG GCT GTT CCC ACC ACC TTG ATC CAG 1090 Leu Thr Gin Ser Glu Ser Trp Ser Ala Val Pro Thr Thr Leu lie Gin 290 295 300 305

GCT CTC CTC CCC AAG AGT GAG CTG GCT GTG TCT AGT GAG CTG GAT CTG 1138 Ala Leu Leu Pro Lys Ser Glu Leu Ala Val Ser Ser Glu Leu Asp Leu 310 315 320

CTG AAG GCA GTG GAC CAG TGG AGC ACA GAA ACC ATT GCC TCA CAC GAG 1186 Leu Lys Ala Val Asp Gin Trp Ser Thr Glu Thr lie Ala Ser His Glu 325 330 335

GAT ATA GAG CGC CTG GTG GAA CAG GTC CGC TTC CCC ATG ATG CTG CCC 1234 Asp lie Glu Arg Leu Val Glu Gin Val Arg Phe Pro Met Met Leu Pro 340 345 350

CAG GAG CTG TTC GAG CTG CAG TTC AAC CTG TCC TTG TAC CAA GAT CAC 1282 Gin Glu Leu Phe Glu Leu Gin Phe Asn Leu Ser Leu Tyr Gin Asp His 355 360 365

CAG GCA CTG TTC CAG AGG AAG ACC ATG CAG GCC TTG GAG TTC CAC ACA 1330 Gin Ala Leu Phe Gin Arg Lys Thr Met Gin Ala Leu Glu Phe His Thr 370 375 380 385

GTG CCT GTC GAA GTG CTG GCC AAG TAC AAA GGC CTG AAC CTC ACG GAG 1378 Val Pro Val Glu Val Leu Ala Lys Tyr Lys Gly Leu Asn Leu Thr Glu 390 395 400 GAC ACC TAC AAG CCC CGC CTT TAC ACC TCT TCC ACC TGG AGT AGC TTG 1426 Asp Thr Tyr Lys Pro Arg Leu Tyr Thr Ser Ser Thr Trp Ser Ser Leu 405 410 415

GTG ATG GCC TCC ACC TGG AGG GCA CAA AGA TAT GAA TAC AAT CGA TAC 1474 Val Met Ala Ser Thr Trp Arg Ala Gin Arg Tyr Glu Tyr Asn Arg Tyr 420 425 430

AAT CAG CTC TAT ACA TAT GGC TAT GGC TCA GTA GCC CGG TAC AAT AGC 1522 Asn Gin Leu Tyr Thr Tyr Gly Tyr Gly Ser Val Ala Arg Tyr Asn Ser 435 440 445

TAC CAG TCC TTC CAA ACC CCA CAA CAC CCC AGC TTC CTC TTC AAG GAC 1570 Tyr Gin Ser Phe Gin Thr Pro Gin His Pro Ser Phe Leu Phe Lys Asp 450 455 460 465

AAC GAG ATC TCC TGG TCA GCC ACC TAC CTC CCC ACC ATG CAG AGC TGC 1618 Asn Glu lie Ser Trp Ser Ala Thr Tyr Leu Pro Thr Met Gin Ser Cys 470 475 480

TGG AAC TAT GGC TTC TCG TGT ACC TCT AAC GAG CTC CCT GTA CTG GGC 1666 Trp Asn Tyr Gly Phe Ser Cys Thr Ser Asn Glu Leu Pro Val Leu Gly 485 490 495

CTC ACC ACA TCC AGC TAC TCC AAT CCG ACA ATT GGC TAT GAG AAC AGA 1714 Leu Thr Thr Ser Ser Tyr Ser Asn Pro Thr lie Gly Tyr Glu Asn Arg 500 505 510

GTA CTG ATC CTC TGC GGA GGC TAC AGT GTG GTG GAT GTC ACC AGC TTT 1762 Val Leu lie Leu Cys Gly Gly Tyr Ser Val Val Asp Val Thr Ser Phe 515 520 525

GAA GGC TCC AAG GCC CCT ATT CCC ACT GCC CTG GAC ACC AAT AGT TCC 1810 Glu Gly Ser Lys Ala Pro lie Pro Thr Ala Leu Asp Thr Asn Ser Ser 530 535 540 545

AAG ACT CCC TCC CTC TTT CCC TGT GCC TCA GGG GCC TTT AGC AGC TTC 1858 Lys Thr Pro Ser Leu Phe Pro Cys Ala Ser Gly Ala Phe Ser Ser Phe 550 555 560

CGT GTG GTC ATA CGC CCC TTC TAC CTC ACT AAC CTC CAC TGACATGGTG TA 1909 Arg Val Val lie Arg Pro Phe Tyr Leu Thr Asn Leu His 565 570 AATGGTACAT CTCAGTGGTG GGGACGCAGA CATTCCTGTG TCCCCTCCTT CGCCTCCAGC 1969

TGCTTTGTAA GCATAAACTG ATTGTATTCA AATGAAAATT TACTAGAAGG TTTCAGCCAG 2029

CACTCACTCC AGGACTGAGA GTCCCAGGGC CCTCACTGCA GGTACAAGCA GGTTCCATGA 2089

GGTCCTGTGG GATTTCCTGT GCCTACTGCA GTAGCCCCAT CTGTCACAGT CACTCATCAA 2149

AAATCATTAA AGTCTCACGT GCTTCTC 2176

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 574 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Ala Leu Leu Trp Leu Leu Ser Val Phe Leu Leu Val Pro Gly Thr

1 5 10 15

Gin Gly Thr Glu Asp Gly Asp Met Gly Leu Val Asn Gly Ala Ser Ala

20 25 30

Asn Glu Gly Arg Val Glu lie Phe Tyr Arg Gly Arg Trp Gly Thr Val

35 40 45

Cys Asp Asn Leu Trp Asn Leu Leu Asp Ala His Val Val Cys Arg Ala

50 55 60

Leu Gly Tyr Glu Asn Ala Thr Gin Ala Leu Gly Arg Ala Ala Phe Gly 65 70 75 80

Pro Gly Lys Gly Pro lie Met Leu Asp Glu Val Glu Cys Thr Gly Thr

85 90 95

Glu Ser Ser Leu Ala Ser Cys Arg Ser Leu Gly Trp Met Val Ser Arg

100 105 110

Cys Gly His Glu Lys Asp Ala Gly Val Val Cys Ser Asn Asp Thr Thr

115 120 125

Gly Leu His lie Leu Asp Leu Ser Gly Glu Leu Ser Asp Ala Leu Gly

130 135 140

Gin He Phe Asp Ser Gin Gin Gly Cys Asp Leu Phe He Gin Val Thr 145 150 155 160

Gly Gin Gly Tyr Glu Asp Leu Ser Leu Cys Ala His Thr Leu He Leu 165 170 175 Arg Thr Asn Pro Glu Ala Gin Ala Leu Trp Gin Val Val Gly Ser Ser

180 185 190

Val He Met Arg Val Asp Ala Glu Cys Met Pro Val Val Arg Asp Phe

195 200 205

Leu Arg Tyr Phe Tyr Ser Arg Arg He Glu Val Ser Met Ser Ser Val

210 215 220

Lys Cys Leu Pro Lys Leu Ala Ser Ala Tyr Gly Ala Thr Glu Leu Gin 225 230 235 240

Asp Tyr Cys Gly Arg Leu Phe Ala Thr Leu Leu Pro Gin Asp Pro Thr

245 250 255

Phe His Thr Pro Leu Asp Leu Tyr Ala Tyr Ala Arg Ala Thr Gly Asp

260 265 270

Ser Met Leu Glu Asp Leu Cys Val Gin Phe Leu Ala Trp Asn Phe Glu

275 280 285

Pro Leu Thr Gin Ser Glu Ser Trp Ser Ala Val Pro Thr Thr Leu He

290 295 300

Gin Ala Leu Leu Pro Lys Ser Glu Leu Ala Val Ser Ser Glu Leu Asp 305 310 315 320

Leu Leu Lys Ala Val Asp Gin Trp Ser Thr Glu Thr He Ala Ser His

325 330 335

Glu Asp He Glu Arg Leu Val Glu Gin Val Arg Phe Pro Met Met Leu

340 345 350

Pro Gin Glu Leu Phe Glu Leu Gin Phe Asn Leu Ser Leu Tyr Gin Asp

355 360 365

His Gin Ala Leu Phe Gin Arg Lys Thr Met Gin Ala Leu Glu Phe His

370 375 380

Thr Val Pro Val Glu Val Leu Ala Lys Tyr Lys Gly Leu Asn Leu Thr 385 390 395 400

Glu Asp Thr Tyr Lys Pro Arg Leu Tyr Thr Ser Ser Thr Trp Ser Ser

405 410 415

Leu Val Met Ala Ser Thr Trp Arg Ala Gin Arg Tyr Glu Tyr Asn Arg

420 425 430

Tyr Asn Gin Leu Tyr Thr Tyr Gly Tyr Gly Ser Val Ala Arg Tyr Asn

435 440 445

Ser Tyr Gin Ser Phe Gin Thr Pro Gin His Pro Ser Phe Leu Phe Lys

450 455 460

Asp Asn Glu He Ser Trp Ser Ala Thr Tyr Leu Pro Thr Met Gin Ser 465 470 475 480

Cys Trp Asn Tyr Gly Phe Ser Cys Thr Ser Asn Glu Leu Pro Val Leu

485 490 495

Gly Leu Thr Thr Ser Ser Tyr Ser Asn Pro Thr He Gly Tyr Glu Asn

500 505 510

Arg Val Leu He Leu Cys Gly Gly Tyr Ser Val Val Asp Val Thr Ser 515 520 525 Phe Glu Gly Ser Lys Ala Pro He Pro Thr Ala Leu Asp Thr Asn Ser

530 535 540

Ser Lys Thr Pro Ser Leu Phe Pro Cys Ala Ser Gly Ala Phe Ser Ser

545 550 555 560

Phe Arg Val Val He Arg Pro Phe Tyr Leu Thr Asn Leu His 565 570

(2) INFORMATION FOR SEQ ID NO : 3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2257 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE:

(A) NAME/KEY: Coding Sequence

(B) LOCATION: 180...1934

(D) OTHER INFORMATION: Human CYTRF cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

AATCGAAAGT AGACTCTTTT CTGAAGCATT TCCTGGGATC AGCCTGACCA CGCTCCATAC 60

TGGGAGAGGC TTCTGGGTCA AAGGACCAGT CTGCAGAGGG ATCCTGTGGC TGGAAGCGAG 120

GAGGCTCCAC ACGGCCGTTG CAGCTACCGC AGCCAGGATC TGGGCATCCA GGCACGGCC A 180

Me

TGA CCC CTC CGA GGC TCT TCT GGG TGT GGC TGC TGG TTG CAG GAA CCC 228 t Thr Pro Pro Arg Leu Phe Trp Val Trp Leu Leu Val Ala Gly Thr Gl 1 5 10 15

AAG GCG TGA ATG ATG GTG ACA TGC GGC TGG CCG ATG GGG GCG CCA CCA 276 n Gly Val Asn Asp Gly Asp Met Arg Leu Ala Asp Gly Gly Ala Thr As 20 25 30

ACC AGG GCC GCG TGG AGA TCT TCT ACA GAG GCC AGT GGG GCA CTG TGT 324 n Gin Gly Arg Val Glu He Phe Tyr Arg Gly Gin Trp Gly Thr Val Cy 35 40 45 GTG ACA ACC TGT GGG ACC TGA CTG ATG CCA GCG TCG TCT GCC GGG CCC 372 s Asp Asn Leu Trp Asp Leu Thr Asp Ala Ser Val Val Cys Arg Ala Le 50 55 60 6

TGG GCT TCG AGA ACG CCA CCC AGG CTC TGG GCA GAG CTG CCT TCG GGC 420 u Gly Phe Glu Asn Ala Thr Gin Ala Leu Gly Arg Ala Ala Phe Gly Gl 70 75 80

AAG GAT CAG GCC CCA TCA TGC TGG ACG AGG TCC AGT GCA CGG GAA CCG 468 n Gly Ser Gly Pro He Met Leu Asp Glu Val Gin Cys Thr Gly Thr Gl 85 90 95

AGG CCT CAC TGG CCG ACT GCA AGT CCC TGG GCT GGC TGA AGA GCA ACT 516 u Ala Ser Leu Ala Asp Cys Lys Ser Leu Gly Trp Leu Lys Ser Asn Cy 100 105 110

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

GCA CCC ACA CCC TGG ACC TCT CCA GGG AGC TCT CGG AGG CCC TTG GCC 612 r Thr His Thr Leu Asp Leu Ser Arg Glu Leu Ser Glu Ala Leu Gly Gl 130 135 140 1

AGA TCT TTG ACA GCC AGC GGG GCT GCG ACC TGT CCA TCA GCG TGA ATG 660 n He Phe Asp Ser Gin Arg Gly Cys Asp Leu Ser He Ser Val Asn Va 5 150 155 160

TGC AGG GCG AGG ACG CCC TGG GCT TCT GTG GCC ACA CGG TCA TCC TGA 708 1 Gin Gly Glu Asp Ala Leu Gly Phe Cys Gly His Thr Val He Leu Th 165 170 175

CTG CCA ACC TGG AGG CCC AGG CCC TGT GGA AGG AGC CGG GCA GCA ATG 756 r Ala Asn Leu Glu Ala Gin Ala Leu Trp Lys Glu Pro Gly Ser Asn Va 180 185 190

TCA CCA TGA GTG TGG ATG CTG AGT GTG TGC CCA TGG TCA GGG ACC TTC 804 1 Thr Met Ser Val Asp Ala Glu Cys Val Pro Met Val Arg Asp Leu Le 195 200 205

TCA GGT ACT TCT ACT CCC GAA GGA TTG ACA TCA CCC TGT CGT CAG TCA 852 u Arg Tyr Phe Tyr Ser Arg Arg He Asp He Thr Leu Ser Ser Val Ly 210 215 220 2 AGT GCT TCC ACA AGC TGG CCT CTG CCT ATG GGG CCA GGC AGC TGC AGG 900 s Cys Phe His Lys Leu Ala Ser Ala Tyr Gly Ala Arg Gin Leu Gin Gl 5 230 235 240

GCT ACT GCG CAA GCC TCT TTG CCA TCC TCC TCC CCC AGG ACC CCT CGT 948 y Tyr Cys Ala Ser Leu Phe Ala He Leu Leu Pro Gin Asp Pro Ser Ph 245 250 255

TCC AGA TGC CCC TGG ACC TGT ATG CCT ATG CAG TGG CCA CAG GGG ACG 996 e Gin Met Pro Leu Asp Leu Tyr Ala Tyr Ala Val Ala Thr Gly Asp Al 260 265 270

CCC TGC TGG AGA AGC TCT GCC TAC AGT TCC TGG CCT GGA ACT TCG AGG 1044 a Leu Leu Glu Lys Leu Cys Leu Gin Phe Leu Ala Trp Asn Phe Glu Al 275 280 285

CCT TGA CGC AGG CCG AGG CCT GGC CCA GTG TCC CCA CAG ACC TGC TCC 1092 a Leu Thr Gin Ala Glu Ala Trp Pro Ser Val Pro Thr Asp Leu Leu Gl 290 295 300 3

AAC TGC TGC TGC CCA GGA GCG ACC TGG CGG TGC CCA GCG AGC TGG CCC 1140 n Leu Leu Leu Pro Arg Ser Asp Leu Ala Val Pro Ser Glu Leu Ala Le 5 310 315 320

TAC TGA AGG CCG TGG ACA CCT GGA GCT GGG GGG AGC GTG CCT CCC ATG 1188 u Leu Lys Ala Val Asp Thr Trp Ser Trp Gly Glu Arg Ala Ser His Gl 325 330 335

AGG AGG TGG AGG GCT TGG TGG AGA AGA TCC GCT TCC CCA TGA TGC TCC 1236 u Glu Val Glu Gly Leu Val Glu Lys He Arg Phe Pro Met Met Leu Pr 340 345 350

CTG AGG AGC TCT TTG AGC TGC AGT TCA ACC TGT CCC TGT ACT GGA GCC 1284 o Glu Glu Leu Phe Glu Leu Gin Phe Asn Leu Ser Leu Tyr Trp Ser Hi 355 360 365

ACG AGG CCC TGT TCC AGA AGA AGA CTC TGC AGG CCC TGG AAT TCC ACA 1332 s Glu Ala Leu Phe Gin Lys Lys Thr Leu Gin Ala Leu Glu Phe His Th 370 375 380 3

CTG TGC CCT TCC AGT TGC TGG CCC GGT ACA AAG GCC TGA ACC TCA CCG 1380 r Val Pro Phe Gin Leu Leu Ala Arg Tyr Lys Gly Leu Asn Leu Thr Gl 5 390 395 400 AGG ATA CCT ACA AGC CCC GGA TTT ACA CCT CGC CCA CCT GGA GTG CCT 1428 u Asp Thr Tyr Lys Pro Arg He Tyr Thr Ser Pro Thr Trp Ser Ala Ph 405 410 415

TTG TGA CAG ACA GTT CCT GGA GTG CAC GGA AGT CAC AAC TGG TCT ATC 1476 e Val Thr Asp Ser Ser Trp Ser Ala Arg Lys Ser Gin Leu Val Tyr Gl 420 425 430

AGT CCA GAC GGG GGC CTT TGG TCA AAT ATT CTT CTG ATT ACT TCC AAG 1524 n Ser Arg Arg Gly Pro Leu Val Lys Tyr Ser Ser Asp Tyr Phe Gin Al 435 440 445

CCC CCT CTG ACT ACA GAT ACT ACC CCT ACC AGT CCT TCC AGA CTC CAC 1572 a Pro Ser Asp Tyr Arg Tyr Tyr Pro Tyr Gin Ser Phe Gin Thr Pro Gl 450 455 460 4

AAC ACC CCA GCT TCC TCT TCC AGG ACA AGA GGG TGT CCT GGT CCC TGG 1620 n His Pro Ser Phe Leu Phe Gin Asp Lys Arg Val Ser Trp Ser Leu Va 5 470 475 480

TCT ACC TCC CCA CCA TCC AGA GCT GCT GGA ACT ACG GCT TCT CCT GCT 1668 1 Tyr Leu Pro Thr He Gin Ser Cys Trp Asn Tyr Gly Phe Ser Cys Se 485 490 495

CCT CGG ACG AGC TCC CTG TCC TGG GCC TCA CCA AGT CTG GCG GCT CAG 1716 r Ser Asp Glu Leu Pro Val Leu Gly Leu Thr Lys Ser Gly Gly Ser As 500 505 510

ATC GCA CCA TTG CCT ACG AAA ACA AAG CCC TGA TGC TCT GCG AAG GGC 1764 p Arg Thr He Ala Tyr Glu Asn Lys Ala Leu Met Leu Cys Glu Gly Le 515 520 525

TCT TCG TGG CAG ACG TCA CCG ATT TCG AGG GCT GGA AGG CTG CGA TTC 1812 u Phe Val Ala Asp Val Thr Asp Phe Glu Gly Trp Lys Ala Ala He Pr 530 535 540 5

CCA GTG CCC TGG ACA CCA ACA GCT CGA AGA GCA CCT CCT CCT TCC CCT 1860 o Ser Ala Leu Asp Thr Asn Ser Ser Lys Ser Thr Ser Ser Phe Pro Cy 5 550 555 560

GCC CGG CAG GGC ACT TCA ACG GCT TCC GCA CGG TCA TCC GCC CCT TCT 1908 s Pro Ala Gly His Phe Asn Gly Phe Arg Thr Val He Arg Pro Phe Ty 565 570 575 ACC TGA CCA ACT CCT CAG GTG TGG AC TAGACGGCGT GGCCCAAGGG TGGTGAGA 1962 r Leu Thr Asn Ser Ser Gly Val Asp 580 585

ACCGGAGAAC CCCAGGACGC CCTCACTGCA GGCTCCCCTC CTCGGCTTCC TTCCTCTCTG 2022

CAATGACCTT CAACAACCGG CCACCAGATG TCGCCCTACT CACCTGAGCG CTCAGCTTCA 2082

AGAAATTACT GGAAGGCTTC CACTAGGGTC CACCAGGAGT TCTCCCACCA CCTCACCAGT 2142

TTCCAGGTGG TAAGCACCAG GACGCCCTCG AGGTTGCTCT GGGATCCCCC CACAGCCCCT 2202

GGTCAGTCTG CCCTTGTCAC TGGTCTGAGG TCATTAAAAT TACATTGAGG TTCCT 2257

(2) INFORMATION FOR SEQ ID NO : 4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 585 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :

Met Thr Pro Pro Arg Leu Phe Trp Val Trp Leu Leu Val Ala Gly Thr

1 5 10 15

Gin Gly Val Asn Asp Gly Asp Met Arg Leu Ala Asp Gly Gly Ala Thr

20 25 30

Asn Gin Gly Arg Val Glu He Phe Tyr Arg Gly Gin Trp Gly Thr Val

35 40 45

Cys Asp Asn Leu Trp Asp Leu Thr Asp Ala Ser Val Val Cys Arg Ala

50 55 60

Leu Gly Phe Glu Asn Ala Thr Gin Ala Leu Gly Arg Ala Ala Phe Gly 65 70 75 80

Gin Gly Ser Gly Pro He Met Leu Asp Glu Val Gin Cys Thr Gly Thr

85 90 95

Glu Ala Ser Leu Ala Asp Cys Lys Ser Leu Gly Trp Leu Lys Ser Asn

100 105 110

Cys Arg His Glu Arg Asp Ala Gly Val Val Cys Thr Asn Glu Thr Arg

115 120 125

Ser Thr His Thr Leu Asp Leu Ser Arg Glu Leu Ser Glu Ala Leu Gly 130 135 140 Gin He Phe Asp Ser Gin Arg Gly Cys Asp Leu Ser He Ser Val Asn 145 150 155 160

Val Gin Gly Glu Asp Ala Leu Gly Phe Cys Gly His Thr Val He Leu

165 170 175

Thr Ala Asn Leu Glu Ala Gin Ala Leu Trp Lys Glu Pro Gly Ser Asn

180 185 190

Val Thr Met Ser Val Asp Ala Glu Cys Val Pro Met Val Arg Asp Leu

195 200 205

Leu Arg Tyr Phe Tyr Ser Arg Arg He Asp He Thr Leu Ser Ser Val

210 215 220

Lys Cys Phe His Lys Leu Ala Ser Ala Tyr Gly Ala Arg Gin Leu Gin 225 230 235 240

Gly Tyr Cys Ala Ser Leu Phe Ala He Leu Leu Pro Gin Asp Pro Ser

245 250 255

Phe Gin Met Pro Leu Asp Leu Tyr Ala Tyr Ala Val Ala Thr Gly Asp

260 265 270

Ala Leu Leu Glu Lys Leu Cys Leu Gin Phe Leu Ala Trp Asn Phe Glu

275 280 285

Ala Leu Thr Gin Ala Glu Ala Trp Pro Ser Val Pro Thr Asp Leu Leu

290 295 300

Gin Leu Leu Leu Pro Arg Ser Asp Leu Ala Val Pro Ser Glu Leu Ala 305 310 315 320

Leu Leu Lys Ala Val Asp Thr Trp Ser Trp Gly Glu Arg Ala Ser His

325 330 335

Glu Glu Val Glu Gly Leu Val Glu Lys He Arg Phe Pro Met Met Leu

340 345 350

Pro Glu Glu Leu Phe Glu Leu Gin Phe Asn Leu Ser Leu Tyr Trp Ser

355 360 365

His Glu Ala Leu Phe Gin Lys Lys Thr Leu Gin Ala Leu Glu Phe His

370 375 380

Thr Val Pro Phe Gin Leu Leu Ala Arg Tyr Lys Gly Leu Asn Leu Thr 385 390 395 400

Glu Asp Thr Tyr Lys Pro Arg He Tyr Thr Ser Pro Thr Trp Ser Ala

405 410 415

Phe Val Thr Asp Ser Ser Trp Ser Ala Arg Lys Ser Gin Leu Val Tyr

420 425 430

Gin Ser Arg Arg Gly Pro Leu Val Lys Tyr Ser Ser Asp Tyr Phe Gin

' 435 440 445

Ala Pro Ser Asp Tyr Arg Tyr Tyr Pro Tyr Gin Ser Phe Gin Thr Pro

450 455 460

Gin His Pro Ser Phe Leu Phe Gin Asp Lys Arg Val Ser Trp Ser Leu 465 470 475 480

Val Tyr Leu Pro Thr He Gin Ser Cys Trp Asn Tyr Gly Phe Ser Cys 485 490 495 Ser Ser Asp Glu Leu Pro Val Leu Gly Leu Thr Lys Ser Gly Gly Ser

500 505 510

Asp Arg Thr He Ala Tyr Glu Asn Lys Ala Leu Met Leu Cys Glu Gly

515 520 525

Leu Phe Val Ala Asp Val Thr Asp Phe Glu Gly Trp Lys Ala Ala He

530 535 540

Pro Ser Ala Leu Asp Thr Asn Ser Ser Lys Ser Thr Ser Ser Phe Pro 545 550 555 560

Cys Pro Ala Gly His Phe Asn Gly Phe Arg Thr Val He Arg Pro Phe

565 570 575

Tyr Leu Thr Asn Ser Ser Gly Val Asp 580 585

(2) INFORMATION FOR SEQ ID NO : 5 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :

ATGGCTCTCC TGTGGCTCCT CTC 23

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

CACCATGTCA GTGGAGTTAG TGAG 24

Claims

WHAT IS CLAIMED IS

1 A method for decreasing the synthesis of pro-inflammatory cytokines in a T cell population, the method comprising contacting said T cell population with a formulation comprising CYTRF as an active agent for a period of time sufficient to reduce the synthesis of pro-inflammatory cytokines

2 A method according to Claim 1 , wherein said pro-inflammatory cytokines comprise ╬│-╬╣nterferon and tumor necrosis factor ╬▒

3 A method according to Claim 1 , wherein said T cell population is synthesizing said pro-inflammatory cytokines in response to a weak antigen

4 A method according to Claim 3, wherein said weak antigen is an autoantigen

5 A method according to Claim 4, wherein said autoantigen is associated with autoimmune disease

6 A method according to Claim 1 , wherein said contacting comprises administration in vivo to an infected host

7 A method according to Claim 1 , wherein said contacting comprises administration in vitro

8 A method according to Claim 1 , wherein said CYTRF is human CYTRF

9 A method according to Claim 8, wherein said human CYTRF comprises the ammo acid sequence as set forth in SEQ ID NO 4

10 A method according to Claim 8, wherein said human CYTRF comprises an ammo acid sequence substantially similar to that set forth in SEQ ID NO 4

11. An anti-inflammatory pharmaceutical composition, comprising:

CYTRF in an amount effective to reduce the synthesis of pro-inflammatory cytokines by a T cell population; and a pharmaceutically acceptable carrier.