WO1993019778A1 - Method for inducing tolerance to an antigen using butyrate - Google Patents

Abstract

Methods for inducing specific tolerance to an antigen in an animal by blocking Th cell cycle progression in G1a phase are disclosed. The methods comprise the administration of N-butyrate or pivalyloxymethyl butyrate in combination with the antigen.

Description

MJiTCHOD FOR INDUCING TOLERANCE TO AN ANTIGEN USING BUTΪRATE i

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for inducing antigen-specific 5 tolerance in an animal.

2. Description of Related Art

Although the immune response is often perceived as beneficial, in certain circumstances the immune response to an antigen can actually be harmful to the animal in which the immune response occurs. Examples of situations

10 where the immune response creates conditions where the animal is subject to serious pathologies sequelae are in such areas as transplantation immuπitize, such as graft versus host (GVH) rejection and host versus graft (HVG) rejection, and certain autoimmune disease, such as lupus erythematosus, insulin-dependent diabetes mellitus, muitiple sclerosis, myesthesia gravis, and

15 rheumatoid arthritis.

One possible means of preventing an immune response to an antigen would be through inducing antigen (Ag)-specific tolerance. Methods which concentrate on T cell tolerance are particularly useful, since it has been shown that tolerance induction of T cells takes less antigen and is longer lasting than 20 tolerance induction of B cells (Chiller, et al., Science, 171: 813, 1971). Ag- spec^'rfic inactivation, or tolerance, of CD4⁺ cells in vitro has been achieved by exposing cells to Ag presented by antigen presenting cells (APC) which naturally (e.g., keratinocytes) or experimentally (e.g., chemically fixed spleen cells) lack costimulatory molecules (Gaspari, et al., J. Immunol., 141:2216. 1988; Markmann, et al., Nature, 336:476. 1988; Simon, ef al., J. Immunol., 146_:485, 1991; Ashwell, ef al., J. Immunol., 141:2536, 1988; Jenkins, ef al., J. Exp. Med., 1£5302, 1987). Many of these studies have utilized cloned populations of CD4⁺ cells. The majority of long-term clones of mouse CD4⁺ cells can be assigned to one of two major subtypes, designated Th1 or Th2. Th1 clones produce IFN₇, IL-2, and lymphotoxin, mediate cytotoxic responses, and promote limited Ig production (Mosmann, ef al., Immunol. Today, Q:22, 1987; Gledlin, ef al., Cell. Immunol., 27_:357, 1986; Killar, ef al., J. Immunol., 138:1674. 1987), Th2 clones produce IL-4 and IL-5, and are thought to be more effective than Th1 in providing help for primary Ag-specific Ig secretion (Mosmann, ef al., Immunol. Today, 8:22, 1987; Coffman, ef al., Immunol. Rev., 102:5. 1988). Most studies on the induction of tolerance in CD4⁺ cell clones in vitro have been confined to Th1 clones, and have demonstrated the inability of tolerized cells to proliferate or produce IL-2 in response to Ag in secondary cultures.

Previous studies have shown that both Th1 and Th2 clones specific for human gamma globulin (HGG) can be tolerized in vitro by exposure to syngeneic spleen cells that have been incubated with HGG and subsequently fixed with paraformaldehyde (Gilbert, ef al., J. Immunol., 144:2063, 1990). Tolerance of HGG-specific Th1 clones is manifested as an inability to proliferate or produce IL-2 in HGG-stimulated secondary cultures (Gilbert, ef al., Cell. Immunol., In press, 1991; Gilbert, ef al., J. Immunol., 144:2063. 1990). In contrast, HGG- specific Th2 clones do not lose their ability to proliferate to Ag, but do lose their ability to promote Ag-spec^'rfic antibody production (Gilbert, ef al., J. Immunol., 144:2063, 1990).

In most past studies, the effects of tolerance on subsequent Th1 and Th2 proliferative activity have been examined at the level of DNA synthesis by measuring the Th cell ³H-TdR incorporation. This measurement does not provide information at the single cell level, nor does it detect effects on pre-S phase events, in this regard, studies reported herein on G₁ phase entry and progression during Ag-specific anergy are instrumental in determining the mechanism of Th cell tolerance induction, and in explaining the differences in Th1 and Th2 responses to tolerance induction. In so doing, it is now possible to utilize these findings to identify compound which can be used to induce antigen-specific tolerance and, thereby, avoid the severe drawbacks associated with prior techniques for inducing tolerance.

In the search for means which would allow the restoration or induction of a tolerogenic state existing technology relies primarily upon broad-based techniques which are not antigen specific. These techniques rely primarily on highly toxic drugs to suppress, or tolerizes, the immune response in a systemic, rather than a specific, manner. As a result, individuals receiving such therapy are immunocompromised and run a significant risk of infection, or even death, from opportunities or nosocomial infectious agents. The limitations of such therapy point to the need for tolerogenic agents which have less toxicity, but greater specificity. The present invention provides such agents and methods for these effective utilizations in achieving antigen-specific tolerance.

SUMMARY OF THE INVENTION

In the method of the invention, an animal having, or at risk of having, an undesirable immune response to an antigen is given a tolerogenicaliy effective amount of a compound which tolerizes the T helper (Th) cell repertoire which is specific for the antigen. Compounds effective in achieving this therapeutic effect are those which are capable of arresting Th cells in the G_1a phase of the cell cycle.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1. Determination of cell cycle compartments based on simultaneous staining of DNA and RNA. Contour plots representing resting Th1 (a), resting Th2(c) or Th1 or Th2 cultured for 48 h with anti-CD3 mAb (b or d, respectively) are shown. The Th cells were stained with AO and analyzed by FCF.

FIGURE 2. Effect of exposure to HGG-FAPC on Th1 cell cycle progression.

FIGURE 3. Effect of exposure to HGG-FAPC on Th2 cell cycle progression. The same procedure was followed as described for Fig. 2 except that Th2 clone 1-F2 was substituted for Th1 clone 12-11.

FIGURE 4. Expression of T cell activation molecules on Th1 , Th2, and freshly isolated CD4⁺ splenocytes.

FIGURE 5. The effect of exposure to HGG-FAPC on Th1 expression of T cell activation molecules.

FIGURE 6. The effects of exposure to HGG-FAPC on Th2 expression of T cell activation molecules. The same procedure was followed as described for Fig. 6 except that Th2 clone 1-F2 was substituted for Th1 clone 12-11.

FIGURE 7. The effects of exposure to cell cycle inhibitors on Th1 proliferation in secondary cultures. Clone Th1 cells (4HE10) from primary cultures containing APC (□) or APC + HGG (■) in medium alone, or in medium containing actinomycin D, N-butyrate or hydroxyurea. The Th cells were then isolated and recultured with HGG and freshly isolated APC (A), or with Con A CM (B). Cultures were pulsed for 2 h with ³H-TdR at the time periods indicated and incorporated radioactivity was determined by scintillation counting. The results represent the mean cpm ± the range from two experiments.

FIGURE 8. The effects of exposure to cell cycle inhibitors on Th2 proliferation in secondary cultures. The same procedure was followed as described in Fig. 7 except that Th2 clone 1-F2 was substituted for the Th1 clone. To insure that the Th2 cells were in a G₀ state at the initiation of this experiment, the 1-F2 clone was rested 12 days after their last routine Ag-stimulated passage before use here. The results represent the mean cpm ± standard deviations from triplicate wells.

DESCRIPTION OF THE INVENTION

The present invention relates to a method of inducing antigen-specific tolerance. According to the invention, tolerance is induced using a tolerogenically effective amount of a compound, or functional analogs thereof, which arrests T-helper (Th) cell cycle progression in the G_1a phase. The term "tolerogenically effective amount" means that the amount of tolerogenic compound used is of sufficient quantity to tolerize, or suppress, the immune response of the animal to an antigen.

The method of the invention is broadly applicable to any situation where it is desirable to induce immune tolerance to antigens. Examples of situations where it would be useful to induce tolerance include autoimmune diseases and host versus graft (HVG) and graft versus host (GVH) rejection. Typically, tolerance to a particular antigen is induced by presenting the tolerizing compound substantially contemporaneously with the antigen. The term "substantially contemporaneously" means that the compound and the antigen are administered reasonably close together in time. Usually, it is preferred to administer the compound after presentation of the antigen. This preference is because it is believed that the compounds used according to the method of the invention are most effective after the particular repertoire of Th cells have been exposed to the antigen and Th clonal expansion has begun.

Under certain circumstances, such as where it is desirable to induce tolerance to an autoimmune disease, it may not be necessary to expose the animal to the antigen, since, in effect, the antigen is already present in vivo in the form of the animals' own tissues. Consequently, the clonal repertoire of Th cells which should be arrested in G_1a are already in a stimulated state with respect to the autoantigen(s). Examples of diseases associated with autoimmunity which can be ameliorated based on the method of the invention include systemic lupus erythematosus (SLE), rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and diabetes.

The antigen to which the animal, such as a human, is to be tolerized may be presented in various forms, such as a purified protein, a cell lysate, or a tissue extract or homogenate. For example, where the animal is to receive passive immunotherapy using a xenogeneic antibody, a relatively pure preparation of the xenogeneic antibody would probably be used in combination with the tolerizing compound in order to ameliorate the animals' immune response to foreign determinants associated with the xenogeneic antibody.

Additionally, where an animal is to receive an organ transplant from a donor, prior to the actual transplantation the animal can be administered a tissue homogenate or extract derived from donor tissues or cells, in combination with contemporaneously administered tolerizing compound, in order to tolerize the animal in advance of exposure to foreign antigenic determinants of the donor organ. Alternatively, the animal can be treated with the tolerogenic compound after transplantation has been done and the animal shows symptoms of GVH or HVG rejection. Such delayed administration might be preferred in certain circumstances, such as where an allogeneic bone marrow transplant has been performed and the donor bone marrow is responding immunopathogenically to antigenic determinants of the host tissues.

Compounds which are effective in inducing antigen-specific tolerance are capable of blocking Th cell cycle progression in G_1β phase. While not wanting to be bound to a particular theory, it is believed that compounds which are effective according to the invention cause hyperacetylation of histone, probably due to the inhibition of histone deacetylase. However, regardless of the mechanism of action those of skill in the art can identify compounds useful according to the invention using various screening assays disclosed herein. Compounds of particular utility are those which are derivatives of butyric acid. Thus, functional analogs, derivatives, substitution products, isomers, pro-drugs, or homologues of butyric acid which are capable of inducing antigen-specific tolerance are contemplated as equivalents. Especially preferred compounds are N-butyrate and such pro-drug forms of butyric acid as pivalyloxymethyl butyrate (Raphaeli, ef al., Int. J. Cancer, 42:66, 1991) which are degraded introcellularly to yield free butyric acid.

The invention also discloses a method of identifying a compound which induces antigen-specific tolerance. Compounds which are effective in this manner can be identified in various ways which reflect Th cell cycle arrest in the G_1a phase. Thus, a compound can be evaluated by incubating the compound, the antigen to which tolerance is to be induced, and Th cells primed to the antigen are incubated in combination under conditions sufficient to allow the components to interact, then measuring a metabolic change in the Th cells. The metabolic change which is measured may be determined either indirectly, such as by measuring accumulation of the Th cell population in G_1a phase by cytofiuoremetry, or directly, such as by measuring hyperacetylation of histone or inhibition of histone deacetylase (Chalal, ef al., Nature, 287:76. 1989). Other means, direct or indirect, for measuring Th cell arrest in G_1a phase are well known, or can readily be ascertained without undue experimentation, by those of ordinary skill in the art.

The dosage ranges for the administration of the tolerizing compound according to the method of the invention are those large enough to produce the desired effect in which the symptoms of the immune response show some degree of tolerance to the antigen (s). The dosage should not be so large as to cause adverse side effect, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex, and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary from about 10 mg/kg/dose to about 1000 mg/kg/dose, preferably about 100 mg/kg/dose to about 800 mg/kg/dose, most preferably about 300 mg/kg/dose to about 700 mg/kg/dose in one or more dose administrations daily, for one or several days as may be needed.

The tolerizing compound can be administered parenterally, by injection or by gradual perfusion over time, or enterally. The parenteral administration of the composition can be by such routes as intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous or non- aqueous solutions, suspension, and emulsions. Examples of non-aqueous solvents are propyiene glycol, polyethylene glycol, vegetable oils such as sesame oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishes, electrolyte replenishes (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobial, anti-oxidants, chelating agents, and insert gases and the like.

Preparations for enteral administration may be in a liquid, lyophilized, or gel form. In solid dosage forms, the preparation may comprise the tolerizing composition together with the antigen(s) to which it is desired to induce tolerance. The pharmaceutical carrier may be in the nature of an aqueous or nonaqueous liquid or a solid. In solid dosage forms, the preparation may contain such inert diluents as sucrose, lactose, starch, or vermiculite as well as a lubricating agent. These lubricating agents aid in the passage of the compositions through the gut. In the case of capsules, tablets, and pills, the unit dosage forms may also comprise buffering agents. Other forms of oral administration may also be prepared with an enteric coating which would prevent dissolution of the composition until reaching the intestines. Liquid dosage forms for oral administration will generally comprise an enterally coated capsule containing the liquid dosage form. Suitable forms include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water, sugars, polysaccharides, silicate gels, gelatin, or an alcohol. These inert diluents do not actively participate in the therapeutic effect of the invention. Besides the inert diluents, such compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, perfuming agents.

The tolerogenic compounds of the invention are especially suited for use in targetable delivery systems such as synthetic or natural polymers in the form of macromolecular complexes, nanocapsules, microspheres, or beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, liposomes, and resealed erythrocytes. These systems are known collectively as colloidal delivery systems. Typically, such colloidal particles containing the dispersed compound are about 50 nm - 2 μm in diameter. The size of the colloidal particles allows them to be administered intravenously such as by injection, or as an aerosol. Materials used in the preparation of colloidal systems are typically sterilizable via filter sterilization, nontoxic, and biodegradable, for example albumin, ethylcellulose, casein, gelatin, lecithin, phospholipids, and soybean oil. Polymeric colloidal systems are prepared by a process similar to the coacervation of microencapsulation. Preferred as a targeted delivery system for the compounds used in the method of the invention are liposomes. When phospholipids are gently dispersed in aqueous media, they swell, hydrate, and spontaneously form muttilamellar concentric bilayer vesicles with layers of aqueous media separating the lipid bilayer. Such systems are usually referred to as muttilamellar liposomes or muttilamellar vesicles (MLVs) and have diameters ranging from about 00nm to about 4um. When MLVs are sonicated, small unilamellar vesicles (SUVs) with diameters in the range of from about 20 to about 50 nm are formed, which contain an aqueous solution in the core of the SUV.

In preparing liposomes containing the compounds of the invention, such variables as the efficiency of compound encapsulation, lability of the compound, homogeneity and size of the resulting population of liposomes, drug-to-lipid ratio, permeability instability of the preparation, and pharmaceutical acceptability of the formulation should be considered. (Szoka, ef al., .Annual Reviews of Biophysics and Bioengineering, 9_:467, 1980; Deamer, ef al., in Liposomes, Marcel Dekker, New York, 1983, 27; Hope, ef al., Chem. Phys. Lipids, 40:89, 1986).

The targeting of liposomes has been classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be further distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticula-endothelial system (RES) in organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves the alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposomes themselves in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization. Altematively, liposomes may physically localize in capillary beds such as the lung or may be given by site-specific injection.

Another targeted delivery system which can be used with the compounds of the invention is resealed erythrocytes. When erythrocytes are suspended in a hypotonic medium, swelling occurs and the cell membrane ruptures. As a consequence, pores are formed with diameters of approximately 200-500 A which allow equilibration of the intracellular and extracellular environment, if the ionic strengths of this surrounding media is then adjusted to isotonic conditions and the cells incubated at 37 °C, the pores will close such that the erythrocyte reseals. This technique can be utilized with the compounds used in the method of the invention to entrap the compound inside the resealed erythrocyte. The resealed erythrocyte containing the compound can then be used for targeted delivery.

The surface of the targeted delivery system may be modified in a variety of ways. Non-lipid material may be conjugated via a linking group to one or more hydrophobic groups, for example, alkyl chains from about 12-20 carbon atoms. In the case of a liposomal targeted delivery system, lipid groups can be incorporated in to the lipid bilayer of the liposome in order to maintain the compound in stabile association with the liposomal bilayer. Various linking groups can then be used for joining the lipid chains to the compound.

Whether a ligand or a receptor, the number of molecules bound to a liposome will vary with the size of a liposome, as well as the size of the molecule, the binding of affinity the molecule to the target cell receptor or ligand, as the case may be, and the like. In most instances, the bound molecules will be present on the liposome in from about 0.05 to about 2 moI%, preferably from about 0.1 to about 1 mol%, based on the percent of bound molecules to the total number of molecules in the outer membrane bilayer of the liposome. ln general, the compounds to be bound to the surface of the targeted delivery system will be ligands and receptors which will allow the targeted delivery system to actively "home in" on the desired tissue. A ligand may be any compound of interest which will specifically bind to another compound, referred to as a receptor, such that the ligand and receptor form a homologous pair. The compounds bound to the service of the targeted delivery system may vary from small haptens of from about 125-200 molecular weight to much larger antigens with molecular weights of at least about 6000, but generally of less than 1 million molecular weight. Proteinaceous ligands and receptors are of particular interest.

In general, the surface membrane proteins which bind to specific effector molecules are referred to as receptors. As presently used, however, most receptors will be antibodies. These antibodies may be monoclonal or polyclonal and may be fragments thereof such as Fab, and F(ab')₂, which are capable of binding to an epitopic determinant.

The compounds used in the method of the invention can be utilized as therapeutic agents when incorporated in a solid phase matrix. The matrix can then be implanted in an animal to be tolerized to allow gradual in vivo release of the compound over time. In traditional delivery, it has long been recognized that tablets, capsules, and injections may not be the best mode of administration. These conventional routes often involve frequent and repeated doses, resulting in a "peak and valley" pattern of therapeutic concentration. Using a polymeric carrier is one effective means to deliver the compound locally and in a controlled fashion (Langer, et al., Rev.Macro.Chem.Phys., C23(1), 61, 1983). As a result of less total compound required, systemic side effects can be minimized. Polymers have been used as carriers of therapeutic compounds to effect a localized and sustained release (Controlled Drug Delivery, Vol. I and II, Bruck, S.D., (ed.), CRC Press, Boca Raton, FL, 1983; Novel Drug Delivery Systems, Chien, Y.W., Marcel Dekker, New York, 1982). These therapeutic delivery systems simulate infusion and offer the potential of enhanced therapeutic efficacy and reduced systemic toxicity.

For a non-biodegradable matrix, the steps leading to release of the therapeutic tolerogenic compound are water diffusion into the matrix, dissolution of the therapeutic, and out-diffusion of the therapeutic through the channels of the matrix. As a consequence, the mean residence time of the therapeutic existing in the soluble state is longer for a non-biodegradable matrix than for a biodegradable matrix where a long passage through the channels is no longer required. Since many pharmaceuticals have short half-lives it is likely that the compound is decomposed or inactivated inside the non-biodegradable matrix before it can be released. This problem is largely alleviated by using a biodegradable matrix which allows controlled release of the compound.

Biodegradable polymers differ from non-biodegradable polymers in that they are consumed or biodegraded during therapy. This usually involves breakdown of the polymer to its monomeric subunits, which should be biocompatible with the surrounding tissue. The life of a biodegradable polymer in vivo depends on its molecular weight and degree of cross linking; the greater the molecular weight and degree of cross linking, the longer the life. The most highly investigated biodegradable polymers are polylactic acid (PLA), polyglycolic acid (PGA), copolymers of PLA and PGA, polyamides, and copolymers of polyamides and polyesters. PLA, sometimes referred to as polylactide, undergoes hydrotytic deesterification to lactic acid, a normal product of muscle metabolism. PGA is chemically related to PLA and is commonly used for absorbable surgical sutures, as is PLA/PGA copolymer. An advantage of a biodegradable material is the elimination of the need for surgical removal after it has fulfilled its mission. The appeal of such a material is more than simply for convenience. From a technical standpoint, a material which biodegrades gradually and is excreted overtime can offer many unique advantages.

A biodegradable delivery system has several additional advantages: 1) the therapeutic release rate is amenable to control through variation of the matrix composition; 2) implantation can be done at sites difficult or impossible for retrieval; 3) delivery of unstable therapeutic is more practical. This last point is of particular importance for compounds with short in vivo hatf-lives and low Gl tract absorption which renders them unsuitable for conventional oral or intravenous administration.

In its simplest form, a tolerogenic compound delivery system consists of a dispersion of the compound in a polymer matrix. The compound is released as the polymeric matrix decomposes, or biodegrades into soluble products which are excreted from the body. Several classes of synthetic polymers, including polyesters (Pitt, ef al., in Controlled Release of Bioactive Materials, R. Baker, Ed., Academic Press, New York, 1980); polyamides (Sidman, ef al., Journal of Membrane Science, 7:227, 1979); polyurethanes (Maser, ef al., Journal of Polymer Science, Polymer Symposium, g6_:259, 1979); polyorthoesters (HeMer, etal.,PotymerEngineering Science, 21:727, 1981); and polyanhydrides (Leong, ef al., Biomaterials, 7:364, 1986) have been studied for this purpose.

By far most research has been done on the polyesters of PLA and PLA/PGA. Undoubtedly, this is a consequence of convenience and safety considerations.

These polymers are readily available, as they have been used as biodegradable sutures, and they decompose into non-toxic lactic and glycolic acids. Polyorthoesters and polyanhydrides have been specifically designed for controlled release purposes. By taking advantage of the pH dependence of the rate of orthoester cleavage, preferential hydrolysis at the surface is achieved by either the addition of basic substances to suppress degradation in bulk, or the incorporation of acidic catalysts to promote surface degradation.

The invention also relates to a method for preparing a medicament or pharmaceutical composition comprising the tolerogenic compounds according to the invention, the medicament being used for therapy to induce antigen specific tolerance.

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.

EXAMPLE 1

EFFECT OF EXPOSURE TO HGG-FAPC ON Th CELL CYCLE ENTRY AND PROGRESSION

To determine the effects of tolerance induction on early phases of the Th cell cycle, HGG-specific Th1 and Th2 clones were preincubated wfth HGG-FAPC or FAPC in primary cultures and then recultured as follows: (1) in the absence of antigen challenge (with naive APC), (2) in the presence of Ag (with HGG- presenting APC), or (3) in the presence of exogenous IL-2 (with Con A CM). Male A/J Mice at 4 to 5 weeks of age were purchased from the Jackson Laboratory (Bar Harbor, ME). Experiments were performed by using 8 to 12 week-old mice. Female Lewis rats were purchased from the Jackson Laboratory.

Soluble HGG was purified from Cohn fraction II of human plasma by DEAE- cellulose chromatography (Parks, ef al., J. Exp. Med., 148:625. 1978). Anti-Thy 1.2 mAb was purchased from New England Nuclear (Boston, MA). Hybridoma cells secreting 145-2C11 (hamster anti-mouse CD3e) were provided by Dr. J.A. Bluestone (University of Chicago, Chicago, IL) and the mAb was purified as described (Ernst, ef al., J. Immunol., 142:1413. 1989). The sources and purification procedures for anti-RL388Ag mAb (RL388), anti-mouse 55 KDa IL-2R mAb (PC61), anti-mouse L3T4/CD4 mAb, (YTS 191.1) were described previously (Ernst, ef al., J. Immunol., 142:1413. 1989).

The HGG-specific CD4⁺ Th1 and Th2 clones were developed and characterized as previously described (Gilbert, ef al., J. Immunol, 144:2063.

1990). Th lines and clones were maintained by weekly feedings with antigen, Con A conditioned medium and irradiated APC. Con A conditioned medium was prepared by collecting the supernatant from cultures of spleen cells from Lewis rats incubated at 10⁶ cells/ml with 5μg/ml Con A for 24 hours.

In order to prepared fixed APC's, splenocytes in RPMI-1640 medium (M.A. Bioproducts, Walkersville, MD) supplemented with 2mM L-glutamine, 0.1 mM nonessential amino acids, 1mM sodium pyruvate, 100 units penicillin/ml, 100μg streptomycin/ml, 5 x 10^"5 M 2-mercaptoethanol and 5% fetal bovine serum (M.A. Bioproducts, Walkersville, MD) were incubated at 2 x 10⁷/ml in medium alone, or with 1 mg/ml HGG for 3 hrs at 37 *C. The spleen cells were then washed in RPMI, passed over a Ficoll-Hypaque (FH) gradient to remove the dead cells, and fixed by using a modification of a previously described method (Roska, ef al., J. Immunol. 135:2953. 1985). Briefly, the spleen cells were incubated in 1 ml of 0.15% paraformaldehyde in Hank's BSS for 1 min at 37 ^βC. The reaction was stopped by adding 7 ml of cold 0.5% glyclyglycine. The fixed APC were washed 3 times in medium prior to use.

In preparing primary cultures for inducing Th cell unresponsiveness, Th1 and Th2 cells were harvested from culture 5 to 12 days after the last antigenic stimulation. They were passed over a FH gradient to remove irradiated APC, and incubated at 5 x 10⁴ cells/well in 0.2 ml medium consisting of 1 part Click's medium, 1 part supplemented RPMI 1640 medium, and 10% FBS. In some experiments 5 x 10⁵ HGG-FAPC were added to the wells. The control cultures received FAPC prepared in the absence of HGG. Following preincubation (18- 20 hr at 37 ^βC), the cells in these cultures were harvested, washed, passed over a Ficoll-Hypaque gradient to remove fixed APC, and the Th cells recultured in secondary cultures. In other experiments irradiated (3000R) T cell-depleted spleen cells in the presence or absence of HGG (100 μg/ml were added to the wells together with various cell cycle inhibitors. Following preincubation, viable Th cells were isolated and recultured in secondary cultures. For monitoring tolerized Th cells, secondary cultures of Th cells were harvested from primary cultures were recultured at 5 x 10⁴ cells/well with 10^s irradiated (3000 R of γ-radiation) naive APC, with HGG-presenting APC, or with 20% Con A CM in Costar 3596 plates in a 0.2 ml final volume. In these experiments APC consisted of adherent peritoneal exudate cells which had been preincubated overnight with or without HCC (1mg/ml). Similar results have been obtained in experiments in which the APC consisted of irradiated T cell-depleted spleen cells and the HGG was added directly to the secondary cultures. Cultures were incubated for various time intervals at 37 * C. To measure DNA synthesis, cultures were pulsed for 2 or 4 hours with ³H-TdR, and incorporated radioactivity was determined by scintillation counting. Th cells were isolated from parallel cultures and stained with F1 -labeled mAb or acridine orange (AO) for subsequent flow cytofluorometric (FCF) analysis.

At various time intervals, the Th cells were stained with AO and analyzed by FCF analysis for cell cycle distribution. The formulation of staining medium and procedures for two stage IF staining were described previously (Ernst, ef al., J. Immunol., 142:1413. 1989). The mAb and polyclonal IgG were conjugated with fiuorescein or phycoerythrin, and were prepared for use by airfuging to eliminate aggregates (Ernst, ef al., J. Immunol., 142:1413. 1989). Cells from secondary cultures were harvested at 18 h and, viable Th cells were isolated by Ficoll-Hypaque density centrifugation and 2-color IF stained for CD4 versus membrane RL388 Ag, IL-2R, TcR and TfR. This time interval was selected since previous studies (Ernst, ef al., J. Immunol., 142:1413. 1989) revealed that expression of these antigens on stimulated CD4⁺ splenocytes or cloned Th cells reached maximal levels at 18 h and remained constant until 24 h. The stained cells were examined by flow cytofluorometry using a FACS IV Flow Cytometer (Becton Dickinson), and optical arrangements and machine settings for the FACS IV were previously described in detail (Ernst, ef al., J. Immunol., 142:1413. 1989). Non-CD4⁺ cells (e.g., B cells or macrophages) were excluded from analysis by gating on CD4⁺ cells. Each analysis consisted of 10,000 events counted. For two color analyses using F1 reagents, F1 frequency distributions histograms obtained from PE-gated viable cells were generated by computer and the total number of positively stained cells or mean fluorescence intensity (MFI) values of cell populations were determined. Markers used to calculate percent positive cells were set so that <3% of the cells stained with FITC rat IgG control were scored as positive. F1 histograms were normalized by electronic reseating of gates to facilitate comparisons between F1 histograms.

For cell cycle analysis, cells from secondary cultures were harvested and viable Th cells were isolated by FH purification. The Th cells were then stained with metachromatic fluorescent dye, acridine orange (Polysciences Inc., Warrington PA), as previously described (Ernst, etal., Cell. Immunol., 114:161 , 1988). The fluorescence intensities of individual cells were measured using a FACS IV with optical arrangements and machine settings as previously described (Ernst, ef al., Cell. Immunol., 114:161, 1988). Dead cells and debris were gated out of the analyses based on low levels of forward light scatter and green fluorescence. Correlated red and green fluorescence was displayed as a contour plot. The marker settings used to identify cells in G₀, G_1a, G_lb and S/G₂ + M phases of the cell cycle were based on the levels of green and red fluorescence of individual Th cells as compared to the staining patterns of freshly isolated resting splenic T cells and splenic T cells stimulated 48 hr previously with 0.5 μg/ml anti-mouse CD3 mAb.

Resting or anti-CD3-activated Th cells, which had not been preincubated, were also stained and used for controls; representative contour plots showing the distribution of these Th cells in the various phases of the cell cycle are displayed in Figure 1. Shown in Figure 2 are the cell cycle distributions of the experimental groups. Cloned Th1 cells (12-11) from primary cultures containing FAPC (□) or HGG- FAPC (■) were isolated and recultured with APC (A), with HGG-pulsed APC (B), or with Con A CM (C). The Th cells were removed from the secondary cultures at the times indicated, stained with AO and subjected to FCF analysis. The results are represented as the percentage of viable cells/well found in G₀, G_1a, G_1b or S/G_g/M phases of the cell cycle. Some cultures were pulsed with ³H-TdR for 4 hours at the times indicated, and the radioactivity incorporated is represented as cpm. The results were reproduced in a second experiment with the same clone.

Th1 preincubated with FAPC (control Th1) and recultured with naive APC remained predominantly in G₀, (Fig. 2A). Similarly recultured Th1 which had been preincubated with HGG-FAPC appeared somewhat activated; with 30% of the Th1 cells being found in G_1a, or beyond.

If recultured with HGG-presenting APC, instead of naive APC, the pattern of cell cycle phase transitions exhibited by Th1 preincubated with HGG-FAPC differed markedly from that of control Th1 (Fig. 2B). In response to Ag challenge the majority of control Th1 entered cell cycle after 42 h with cells being found in all phases of the cycle. The same pattern of cell cycle progression was observed if untreated Th1 instead of control Th1 were cultured with Ag-presenting APC. Tolerized Th1 moved readily from G₀ to G_1a in the two early time points, but unlike control, Th1 did not progress beyond G_ta. At the later time point, the tolerized Th1 seemed to fall back from G_1a into G₀. Almost no tolerized HGG- stimulated Th1 were found in G_1b at any time interval. The percentage of tolerized Th1 in S phase or beyond in HGG-stimutated secondary cultures remained small and fairly constant throughout the culture period. It is likely that the number of tolerized Th1 cells in S phase and beyond is even smaller than the data shown. Cell clumping was particularly noticeable in cultures containing tolerized Th1 cells. These cell clumps appeared within the gates set to delineate individual cells in S phase or G_g/M phase, and may have artificially elevated the measurement of tolerized Th1 cells in late cell cycle.

In contrast to the block in cell cycle transition displayed by tolerized Th1 when recultured with Ag, these Th1 showed no inhibition in cell cycle transition when recultured with Con A CM (Fig. 2C). The evaluations of cell cycle progression in all these groups correlated well with simultaneous measurements of ³H-TdR incorporation.

Unlike the differences observed between tolerized and control Th1, Th2 preincubated with HGG-FAPC were similar to control Th2 in the degree to which their cell cycle progression was stimulated in secondary cultures containing naive APC (Fig. 3A), HGG-presenting APC (Fig. 3B), or Con A CM (Fig. 3C). The Th2 used in this experiment appeared to be initially somewhat activated as reflected by the fact that even in those cultures stimulated with naive APC the majority of Th2 had entered cell cycle. The observation that the preponderance of Th2 existed in G_1a rather than G₀ was noted regardless of whether the Th2 were preincubated with FAPC or HGG-FAPC, and a similar observation was made when untreated Th2 were examined (Fig. 1).

Taken together, the results obtained using AO-stained Th showed that exposure to HGG-FAPC stimulates Th1 movement from G₀ to G_1a phase of the cell cycle in the absence of subsequent Ag challenge. However, when challenged with HGG-presenting APC, the majority of the tolerized Th1 , unlike control Th1, were unable to move beyond G_1a. In contrast to Th1, Th2 exposed to HGG-FAPC were not inhibited in their progression from G_1a to G_1b or beyond. Similar results were obtained in experiments that used a second Th1 and Th2 clone. Effect of exposure to HGG-FAPC on expression of activation molecules on Th1 and Th2. The differential expression of certain early T cell activation molecules can be used as an indication of cell cycle progression (Ernst, ef al., J. Immunol., 142:1413. 1989). Accordingly, two color IF staining and flow cytofluorometric analysis were used to examine the expression of TcR, RL388 ag, IL-2R, and TfR by Th1 and Th2 clones. The expression of these markers on resting CD4⁺ spleen cells and unstimulated Th1 and Th2 cells, are shown in Fig. 4. In this study, Th1 and Th2 cells harvested 7 days after their last antigenic stimulation, and A/J splenocytes were stained by two-color IF with PE-anti-CD4 mAb, and either (1) FITC-rat Ig, (2) FITC-anti-TcR, (3) FITC-anti-RL388 Ag, (4) FITC anti-IL- 2R, or (5) FITC anti-TfR mAbs. Stained cells were analyzed by flow cytofluoremetry and the F1 histograms of PE-gated (CD4⁺) cells are shown. Th1 and Th2 express high levels of TcR and RL388 Ag, moderate to high levels of IL-2R, and low to uπdetectable levels of TfR as compared to Ig control staining levels. These staining patterns were reproducible using other Th1 and Th2 clones, and were stable through 24 hr of culture in the absence of antigen or mitogen stimulation.

In parallel with the analysis of cell cycle progression described above, as illustrated in Figs. 2 and 3, Th1 and Th2 preincubated with HGG-FAPC and recultured with naive APC, HGG-presenting APC, or Con A CM for 24 hr were examined for expression of activation markers.

Studies were also done on the effect of exposure to HGG-FAPC on Th1 and Th2 expression of T cell activation molecules. As shown in Fig. 5, cloned Th1 cells (12-11) from primary cultures containing FAPC (D) or HGG-FAPC (■) were isolated and recultured with naive APC, with HGG-pulsed APC, or wfth Con A CM. After 18 hours in secondary cultures, the Th1 were reisolated and stained by two-color IF wfth PE-anti-CD4 mAb, and either FITC-anti-TcR, FITC- anti-RL388 Ag, FΪTC-anti-IL-2R or FITC-anti-TfR mAbs. Stained cells were analyzed by FCF and the F1 histograms of PE-gated (CD4⁺) cells are presented. F1 histograms from unstimulated cells were similar to the staining patterns of control Th1 recultured with APC. Some secondary cultures were pulsed for 4 hours with ³H-TdR and incorporated radioactivity represented as cpm.

When Th1 recultured with naive APC were examined, it was seen that preincubation of Th1 wfth HGG-FAPC, as compared to FAPC, increased Th1 expression of RL388 Ag (MFI of 137 compared to 70), but not IL-2R or TfR. Addition of HGG-presenting APC to the secondary cultures stimulated expression of RL388Ag, IL-2R, and TfR on control Th1 (Δ MFI of 76, 165 and 14 respectively). In contrast to control Th1, similarly recultured Th1 which had been preincubated with HGG-FAPC exhibited no further HGG-induced increase in the RL388 Ag expression, a small increase in IL-2R, expression (ΔMFI 52), and no increase in TfR expression. Reculturing with Con A CM stimulated similar patterns of RL388 Ag, IL-2R and TfR expression on Th1 regardless of whether they were preincubated with FAPC or HGG-FAPC. Expression of the TcR by Th1 remained fairly constant regardless of pretreatment regimen or secondary stimulation.

When Th2 recultured with naive APC were examined (Fig. 6), it was seen that, similar to Th1, Th2 preincubated with HGG-FAPC as compared to FAPC, exhibited increased expression of RL388 Ag (MFI of 314 compared 203).

When recultured with HGG-presenting APC, control Th2, exhibited HGG- induced increases in RL388 Ag and IL-2R. Similarly recultured Th2, which had been preincubated with HGG-FAPC, exhibited no HGG-induced increase in an already elevated expression of RL388 Aag, and less HGG-induced expression of IL-2R than control Th 2 (ΔMFI of 27 compared to 91). The decreased ability of tolerized Th2 to express IL-2R was also observed if the cells were recultured with Con A CM instead of Ag-presenting APC. Unlike the IL-2R, expression of TfR was similar on both control Th2 and Th2 preincubated with HGG-FAPC, regardless of whether the T cells were recultured in the presence of Ag or Con A CM. Similar to Th1, Th2 expression of the TcR was not altered by preincubation regimes or in response to secondary stimulation. For both Th1 and Th2 clones, parallel measurements of ³H-TdR incorporation in secondary cultures correlated well with the expression of activation markers by Th cells in secondary cultures.

The results reported were delineate the effects of tolerance induction on Th cell cycle progression. Cell cycle progression was monitored both as a function of RNA/DNA content and of expression of certain early activation molecules. The increased expression of these activation molecules has been shown to accompany the movement of activated T cells through the cell cycle. For example, increased expression of the RL388 Ag coincides with lymphocyte blastogenesis and G₁ phase entry, and is maintained on cycling lymphocytes (Ernst, ef al., J. Immunol., 142:1413. 1989; Bran, ef al., J. Immunol., 137:397. 1986). Transcription of IL-2 and IL-2R genes are also early activation events (Krone, ef al., J. Exp. Med., 161:1593. 1985), and increased expression of the low affinity IL-2R component occurs early in G, phase (Stern, ef al., Science, 223:203, 1986). The interaction of IL-2 with the IL-2R is required for G, phase progression and results in increased expression of membrane TfR late in G, phase (Stern, ef al., Science, 233:203. 1986; Neckers, ef al., Proc. Natl. Acad. Sci. USA, £Q:3493, 1983). Appearance of TfR and its subsequent interaction with iron-loaded transferrin is needed for S phase entry (Neckers, ef al., Proc. Natl. Acad. Sci. USA, Sf2:3493, 1983; Herzberg, ef al., J. Immunol., 129:998, 1987).

Exposure to tolerogenic APC initiated Th entry into cell cycle, but did not promote the further cell cycle progression associated wfth exposure to immunogenic APC. An FCF examination of AO-stained Th1 revealed that exposure to HGG-FAPC stimulated Th1 to enter G_la. Th1 from primary cultures containing FAPC instead of HGG-FAPC were not stimulated to enter cell cycle. The stimulatory effect of HGG-FAPC on Th1 was also illustrated as a function of increased expression of RL388 Ag on Th1 exposed to HGG-FAPC as compared to FAPC. The increased level of RL388 Ag expression on these Th1 was similar to that induced on control Th1 in response to Ag and APC. The fact that exposure of Th1 to HGG-FAPC did not increase significantly Th1 expression of IL-2R or TfR corresponded well with the relatively small level of ³H-TdR incorporation in these cultures, and with the fact that AO staining detected only a small number of these Th1 in cell cycle phases beyond G_1a. These results suggest that HGG-FAPC are capable, similar to HGG-pulsed nonfixed APC, of stimulating Th1 entry into cell cycle, but unlike nonfixed APC, lack the capability to stimulate Th1 beyond G_1a.

Similar to Th1, Th2 exposed to HGG-FAPC in primary cultures exhibited increased expression of RL388 Ag. Evaluating the stimulatory effects of tolerogenic APC on Th2 was complicated by the fact that, unlike control Th1 and freshly isolated CD4⁺ spleen cells, the control Th2 used in this experiment were not found predominantly in G₀ phase: the majority of these Th2 were found in G_1a or beyond even when recultured with APC alone. A similar pattern was observed w^'rth untreated Th2 harvested 6 days after their last antigenic stimulation and recultured in medium alone. Evidence suggests that this is due to the fact that these Th2 clones take longer than the Th1 clones to return to a "resting" state following antigenic stimulation. An examination of the RNA/DNA content of Th1 clones stimulated as recently as 5 days before with HGG and APC revealed that these Th1 to be almost entirely in G₀ phase. Only after 7 or 8 days post Ag stimulation did the Th2 clones attain this resting state. It seemed possible, therefore, that the use of Th2 not in a completely rested state could account for the inability to inhibit Th2 Ag-specific proliferation by exposure to HGG-FAPC. This possibility can be dismissed, however, since many experiments in this and other studies conducted using Th2 harvested 7 or 8 days after their last antigen stimulation, and presumably existing in G₀, showed the same inability to inactivate Th2 proliferative activity.

The inhibitory effects of tolerance induction on Th1 cell cycle progression were revealed when Th1 exposed to HGG-FAPC or FAPC in primary cultures, were recultured wfth HGG-presenting APC. HGG-stimulated control Th1 could be found in all phases of the cell cycle, with the percentage found in S phase or beyond correlating with ³H-TdR incorporation in these cultures. These results also correlated wfth the increased expression of RU388 Ag, IL-2R, and TfR on control Th isolated from HGG-stimulated secondary cultures. In contrast to control Th1, Th1 exposed to HGG-FAPC and recultured with HGG-presenting APC did not show a steady progression through the cell cycle. The tolerized Th1 start to enter G_1a phase in numbers similar to or greater than control Th1. However, these tolerized cells do not progress into G_1b, and some appear to fall back into G₀ by 42 hours in culture. The small percentage of tolerized Th1 found in S phase or beyond correlates well with the low, but significant, ³H-TdR incorporation measured in these cultures. The inability of tolerized Th1 to progress beyond G_1a is underlined by the lack of HGG-induced upregulation of IL-2R and TfR.

In contrast to the differences between control and tolerized Th1 , Th2 from primary cultures containing either FAPC or HGG-FAPC exhibited similar patterns of cell cycle procession when recultured in the presence of Ag. Similarly, with the exception of a lower level of IL-2R expression, Th2 exposed to HGG-FAPC exhibited the same HGG-induced expression of T cell activation markers as control Th2. EXAMPLE 2

EFFECT OF INHIBITORS ON TOLERANCE

Previous experiments had shown that Th1 cells exposed to HGG-FAPC lose functional activity (i.e, ability to progress through the cell cycle in response to Ag stimulation) in G_1a. If tolerance induction of Th1 results from initial Th cell activation which is aborted in G_1a phase, then it seemed possible to reproduce this effect using a cell cycle inhibitor and Th1 cells exposed to Ag and immunogenic APC. Accordingly, Th1 cells were incubated for 20 h with non- fixed APC and HGG in the presence of actinomycin D (50 ng/ml, SIGMA), N- butyrate (1mM, SIGMA), or hydroxyurea (2mM, SIGMA) which halt cell cycle progression in G₀, G_1a, and G_1b phase, respectively. These concentrations of inhibitors were chosen on the basis of results of preliminary experiments testing the effectiveness and reversibility of the drugs. Th1 and Th2 clones responded similarly to the effects of these inhibitors in primary cultures.

The Th cells were then isolated and recultured with APC and HGG or Con A CM, in the absence of cell cycle inhibitors, and their proliferation was examined. Proliferation in the presence of Con A CM was monitored as a control for the toxicity of the various drugs. The results are presented as a measurement of ³H-TdR incorporation 24-48 h after initiation of secondary cultures, when proliferation as monitored over three days was maximal. Exposure of Ag- activated Th1 cells to N-butyrate in primary cultures inhibited by 91% Th1 Ag- specific proliferative activity in secondary culture (Fig. 7). This inhibition was Ag-driven: Th1 from primary cultures containing N-butyrate and APC, but no HGG, did not differ significantly from Th1 preincubated without N-butyrate in terms of their ability to proliferate in Ag-stimulated secondary cultures. In contrast to the effect of N-butyrate, preincubation of Ag-activated Th1 in the presence of actinomycin D or hydroxyurea did not suppress the ability of Th1 to proliferate subsequently to HGG and APC. None of the pretreatment regimes inhibited the ability of Th1 to proliferate in the presence of Con A CM in secondary cultures.

Similar to Th1 , Ag-stimulated Th2 preincubated with N-butyrate lost their ability to proliferate in response to Ag in secondary cultures (Fig. 8). However, unlike Th1, Th2 from primary cultures containing N-butyrate and APC, but no HGG, also exhibited a decrease in subsequent Ag-specific proliferation. This result was repeated using a second Th2 clone, and in other experiments with the same clone. Preincubation of Ag-activated Th2 in medium alone, or with actinomycin-D or hydroxyurea for 20 h, did not inhibit subsequent Ag-induced Th2 proliferation. High levels of proliferation in all secondary cultures stimulated with Con A CM instead of APC and HGG revealed that the inhibitory effects of N-butyrate were not due to drug-induced toxicity.

The trends observed were not altered if Th1 or Th2 were preincubated with actinomycin D or N-butyrate for 48 h instead of 24 h. Th cells preincubated with hydroxyurea for 48 h showed a decrease in viability and could not be evaluated.

The data presented herein indicates that tolerance induction of Th1 following exposure to HGG-FAPC in primary cultures is associated with sequestration of Th1 in G_1a phase. Th2 exposed to HGG-FAPC are not locked in G_1a phase, and are consequently not inhibited with regard to Ag-activated proliferation. However, addition of N-butyrate, an inhibitor G_1a/G_1b phase transition to primary cultures containing Th cells, HGG, and immunogenic APC, inhibited the subsequent Ag-specific proliferative capacity of both Th1 and Th2 in secondary cultures. The inhibitory effect of pretreatment wfth N-butyrate was also observed in the Th2 primary cultures which contained APC but not HGG. This effect is most probably due to the fact that in this system Th2 are more sensitive than Th1 to activation, and can be stimulated to enter cell cycle by APC in the absence of added HGG. In contrast to the effect of N-butyrate, exposure of Th cells to inhibitors of G₍JG_1a or G_1b/S phase transitions did not inactivate subsequent Th prolrferative capacity. The inability of actinomycin-D and hydroxyurea to inactivate Ag-specific Th cell proliferation in secondary cultures indicates that Th cells must progress to G_1a in order to be tolerized, and that they cannot be tolerized once they have progressed beyond G_1a. This finding also eliminates the possibility that tolerance induction following exposure to N-butyrate is a non-specific effect of cell cycle inhibitors.

In line with the finding that both Th1 and Th2 responded similarly to exposure to N-butyrate, it has been shown that calcium ionophore inhibited IL-2- dependent proliferation of both subsets (Gajewski, ef al., J. Immunol., 144:4110, 1990). The inability of HGG-FAPC to inhibit both subsets may be due to the fact that exposure to HGG-FAPC does not cause an elevation in intracellular Ca²⁺ in Th2 cells. Such an elevation is required for tolerance induction in Th1 cells (Jenkins, ef al., Proc. Natl. Acad. Sci. USA., fi4:5409, 1987). Immobilized anti-CD3 antibody inhibits IL-2-induced proliferation of Th1 clones, but not Th2 clones (Williams, ef al., J. Immunol., 144:1208, 1990), and stimulates elevated Ca²⁺ in Th1 clones but not Th2 clones (Gajewski, et al., J. Immunol., 144:4110. 1990). Evidence has also been presented that following TcR stimulation Th1 and Th2 differ in their generation of inositol phosphates (Gajewski, ef al., J. Immunol., 144:4110, 1990), and sensitivity to an elevation in cAMP (Munoz, ef al., J. Exp. Med., 172:95, 1990). Thus, it appears that although Th1 and Th2 may share some parts of their regulatory pathway, they differ in several signals triggered by TcR-ligand interaction.

It seems unlikely that tolerance-induced G_1a blockade is attributable solely to tolerance-induced effects on IL-2 production. The synthesis of IL-2 is required for G, progression, and is inhibited by exposure of Thl to tolerogenic APC (Gilbert, ef a/., Cell. Immunol., 1991; Jenkins, ef al., Proc, Natl. Acad. Sci. USA., 84:5409, 1987; Quill, ef al., J. Immunol., 122:3704, 1987). Ag-induced upregulation of IL-2R on tolerized Th1, as compared to control Th1, was decreased in this system. It has been previously shown that while IL-2R expression on Th1 system was elevated slightly following anergy, this increase was much less than that observed or Th1 following exposure to immunogenic APC and Ag (Jenkins, ef a/., Proc. Natl. Acad. Sci. USA., 24:5409, 1987). Whether these other inhibitions are the cause of Th1 tolerance (e.g., inhibit the receptor-ligand interactions required for S phase entry), or are merely the byproducts of some other inactivating mechanism cannot be ascertained from the experiments reported here. However, ft has been reported in another system that while IL-2 induces proliferation of tolerized Th1, it does not inhibit tolerance induction, and does not reverse tolerance except at high doses (Quill, ef al., J. Immunol., 138:3704. 1987; Schwartz, R.H., Science, 242:1349, 1990). In addition, IL-2 secretion and Th1 anergy induction differ in their susceptibility to inhibition of tyrosine-specif ic protein kinase activation, suggesting that these two responses are mediated by distinct biochemical pathways (Norton, ef al., J. Immunol., 146:1125, 1991).

Ag-mediated T cell activation to cell division can be divided into at least two phases. The first phase is initiated by signaling through the T cell Ag receptor, and results in the increased expression of certain lymphocyte activation molecules, and activation of certain lymphokine genes and cellular oncogenes required for cell division (Crabtree, G.R., Science, 243:355. 1989). The second phase requires a second receptor-ligand interaction such as that provided by the interaction between IL-2 and the IL-2R (Stern, ef al., Science, 233:203. 1986), and may also be Ag-driven (Miller, ef al., J. Immunol., 132:977, 1986). Most probably G_la represents the portion of the cell cycle during which the first phase of T cell activation occurs. It has been shown that G_1a encompasses a phase in which an initially very heterogeneous population of cycling cells acquires a highly uniform level of RNA and protein in preparation for G_1b entry. This "equalization" period appears to be particularly susceptible to inhibitory influences: the proliferative suppression induced by a variety of different reagents or cells including dexamethasone, calcttriol, TGF-/3, and Ts has been pinpointed to the G_1a phase (Morris, ef al., Exp. Cell. Res., 125:529, 1989; Loertscher, ef al., Transplant, 45:194, 1988; Rigby, ef al., Cell, Immunol., 125:396, 1990; Bettens, ef al., J. Immunol., 132:261, 1984). Once cells have entered G_1b ft is much more difficult to inhibit their proliferation (Bettens, ef al., J. Immunol., 132:261, 1984). Taken together, these findings support the conclusion that tolerance induction occurs in G_1a phase. The results reported here indicate that inactivated Th cells induced by exposure to HGG-FAPC, or to HGG and immunogenic APC together with N-butyrate, represent Th cells which are activated, but prevented from achieving a critical threshold of RNA or protein required for progression into G_1b and S phase. This incomplete activation may trigger a checkpoint mechanism which shuts down subsequent A_g-iπduced cell cycle progression.

A number of embodiments of the present invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, it is to be understood that the invention is not to be limited by the specific illustrated embodiment, but only by the scope of the appended claims.

Claims

CLA1MS

1. A method of inducing tolerance to an antigen in an animal which comprises: administering to the animal a tolerogenically effective amount of a compound which arrests blocks Th cell cycle progression in G_1a phase.

2. The method of claim 1 , wherein the antigen is a self-antigen.

3. The method of claim 2, wherein the self-antigen is associated an autoimmune disease.

4. The method of claim 3, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, myasthenic gravis, and diabetes.

5. The method of claim 1 , wherein the animal is administered the antigen substantially contemporaneously wfth the compound.

6. The method of claim 5, wherein the antigen is a non-self-antigen.

7. The method of claim 6, wherein the non-self-antigen is selected from the group consisting of an antibody, an allogenic tissue antigen, and an allergen.

8. The method of claim 1 , wherein the compound causes hyperacetylation of histone.

9. The method of claim 8, wherein the compound inhibits histone deacetylase.

10. The method of claim 1, wherein the compound is butyric acid or a derivative thereof.

11. The method of claim 10, wherein the compound is N-butyrate.

12. The method of claim 10, wherein the compound is pivalyloxymethyl butyrate.

13. The method of claim 1, wherein the administration is parenteral.

14. The method of claim 13, wherein the parenteral administration is by subcutaneous, intramuscular, intraperitoneal, intracavity, transdermal, or intravenous injection.

15. The method of claim 1, wherein the administration is enteral.

16. The method of claim 1, wherein the animal is human.

17. A method for identifying a compound which induces antigen-specific tolerance, the method comprising:

a. incubating components comprising the compound and the antigen in the presence of Th cells primed to the antigen, wherein the incubating is carried out under conditions sufficient to allow the components to interact; and

b. measuring a metabolic change in the Th cells.

18. The method of claim 17, wherein the metabolic charge is indicated by the arrest of the Th cells in G_1a phase.

19. The method of claim 17, wherein the metabolic charge is indicated by hyperacetylation of histone.

20. The method of claim 19, wherein the metabolic change is indicated by inhibition of histone deacetylase.

21. A pharmaceutical composition comprising a tolerogenically effective amount of a compound which induces antigen-specific tolerance together with a pharmaceutical carrier.

22. The pharmaceutical composition of claim 21, wherein the compound is butyric acid or a derivative thereof.

23. The pharmaceutical composition of claim 22, wherein the compound is N-butyrate.

24. The pharmaceutical composition of claim 22, wherein the compound is pivalyloxymethyl butyrate.