WO1991017759A1 - Trypanosomal immunosuppressive factor - Google Patents

Abstract

The present invention comprises a purified proteinaceous molecule (IL-2R inhibitor) derived from T. cruzi, said proteinaceous molecule having activity as an inhibitor of IL-2R expression. The present invention also comprises a partially purified proteinaceous molecule, compositions of matter containing such proteinaceous molecule and proteinaceous molecules substantially homologous to the T. cruzi derived IL-2R inhibitor. The invention also relates to pharmaceutical compositions and reagents utilizing such IL-2R inhibitors and compositions for immunologic studies.

Description

TRYPANOSOMAL IMMUNOSUPPRESSIVE FACTOR

Field Of The Invention

The present invention relates to a proteinaceous molecule secreted by the pathogenic protozoan Trγpanosoma cruzi. This invention also relates to compositions containing such proteinaceous molecule and methods for their production and use for suppressing expression of interleukin 2 (IL-2) receptors by mammalian lymphocytes. Absence (or reduced levels) of IL-2 receptors (IL-2R) prevents lymphocytes from receiving the growth factor signal that allows them to proliferate after activation. Accordingly, the compositions and methods of this invention are useful in the treatment of autoimmune diseases (e.g., arthritis, type 1 diabetes, and lupus erythematosus) , the prevention of allograft and organ transplant rejection, and in immunologic research.

References

Several publications are referenced herein. The disclosures of these publications are hereby incorporated herein by reference in their entirety, unless otherwise noted.

Background Of The Invention

In a variety of disease states, the body's immune system ceases to protect and, instead, revolts against the organism. Effective suppression of the inappropriate immune response in patients afflicted with such autoimmune diseases has long been a therapeutic dream. Similarly, allograft and transplant recipients are in need of an effective immunosuppressant to prevent rejectio of the foreign tissue or organ.

It is known that antigens interact with receptors expressed on the surface of lymphocytes and, thereafter, the activated lymphocyte cells express a specific set of genes that program the immune response. In T lymphocyte cells (T cells), these genes encode the interleukins and their receptors, which direct the cells to proliferate, differentiate, and eliminate antigen. Thus, antigen recognition initiates the immune response, and the interleukins and their receptors direct it. Accordingly, both the antigen recognition event and the interleukin-receptor interactions are potential targets for therapeutic immuno- modulation.

Interleukin-2 (IL-2) , a specific lymphocytropic hormone produced by T cells, stimulates T cells to undergo cell cycle progression via interactions with specific T cell membrane receptors, i.e., IL-2 receptors (IL-2R) . Resting T cells do not produce IL-2, nor are they capable of responding to IL-2 when it is added exogenously. Rather, signals emanating from the T cell ■ antigen-receptor complex coordinate the transcriptional activatio of both the IL-2 gene and the genes encoding IL-2 receptors (S.C. Meuer et al. , Proc. Natl. Acad. Sci. U.S.A., 81 (1984) p. 1509).

The production level of IL-2 and the period of time durin which IL-2 is available contribute to both the magnitude and extent of T cell proliferation after antigen activation. However it is the expression of functional IL-2 receptors that ultimately determines the duration of T cell clonal expansion. As the immun response proceeds and antigen is cleared, transcription of IL-2 and IL-2 receptor genes eventually declines.

Known approaches to therapeutic immunosuppression rely on the fact that IL-2 receptors are expressed solely by antigen- activated T cells. For example, antibodies reactive with the IL- receptor have been shown to prevent cardiac allograft rejection and to suppress the development of experimental autoimmune diabetes mellitus and systemic lupus erythematosus (R.L. Kirk an et al., J. Exp. Med., 162 (1988) p. 858). Bacterial toxin-IL-2 conjugates produced by genetic engineering techniques represent another approach to antigen-specific immunosuppression. Murphy e al. (J. Exp Med., 167 (1988) p. 612) have engineered diptheria toxin-IL-2 conjugates capable of deleting antigen-activated T cells. Since the vast majority of circulating T cells are quiescent (while cells recently activated express high-affinity IL-2 receptors) , such IL-2-toxin conjugates selectively kill antigen-activated cells.

Therapeutic immunosuppression can also be achieved pharmacologically with the administration of two known immunosuppressive agents, i.e., glucocorticoids or, the fungi- derived, cyclosporine. These two drugs suppress the immune system through inhibition of IL-2 production, but have little effect on the expression of IL-2 receptors (Gillis et al., J. Immunol., 123 (1979) p. 1632; Bunjes, et al., Er. J. Immunol., 11 (1981) p. 657) .

Many instances of parasite-induced impairment of the immune system have also been documented. Parasites resort to a variety of tactics to impair (or evade) the host's immune response. For example, some trematodes (e.g., schistosomes) disguise their surface by covering themselves with host antigens and some nematodes (e.g., Ascaris) express epitopes cross-reactive with those of host antigens — phenomena commonly referred to as molecular mimicry. Some cestodes have been shown to inactivate complement in order to elude antibody-mediated, complement- dependent damage.

A particularly intriguing parasite is Trypanosoma cruzi_f the causative agent of Chagas' disease that affects large segments of the population of South and Central America. The acute phase of the disease is accompanied by immunosuppression, including reduced lymphoproliteration in response to mitogenic lectins or antigens,^*impaired primary and secondary antibody responses, as well as delayed hypersensitivity reactions. In the urine model of Chagas' disease, these effects have been attributed variously to reduced levels of T cells and decreased IL-2 production.

(Hayes, M.M. and Kierszenbaum, F. , Infect. Immun. , 31 (1981) pp. 1117-1124.) In chagasic patients, manifestations of immunosuppression in the acute or subacute stage have also been described. (Voltarelli et al.. Trans R. Soc. Trop. Med. Hyg., 81 (1981) pp. 169-170.) Co-culture of human lyraphoid cells with the T. cruzi parasite markedly suppresses proliferative responses following stimulation with different mitogenic lectins or anti-CD (a monoclonal antibody (mAb) specific for an epitope of the TCR- CD3-Ti T-cell receptor complex) (Beltz and Kierszenbaum, Immunology, 60 (1987) pp. 309-315; Beltz, Sztein, and Kierszenbaum, J. Immunol., 141 (1988) pp. 289-294).

In the mouse, restoration of immune responsiveness has been systematically attained by the addition of exogenous IL-2. (Reed et al., Immunol., 133 (1984) pp. 3333-3337.) This is in sharp contrast to normal human peripheral blood mononuclear cells (PBMC) co-cultured with T. cruzi. Suppression in this case is no accompanied by reduced IL-2 production, and exogenous IL-2 does not restore responsiveness. T. cruzi inhibits mitogen-induced proliferation and expression of high- and low-affinity IL-2 receptors by human lymphocytes without curtailing IL-2 or interferon gamma production. (Kierszenbaum et al., J. Immunol., 143 (1989) p. 275.)

We have surprisingly discovered that T. cruzi spontaneously secrets a factor having IL-2 receptor inhibiting activity. We have termed this factor trypanosomal immunosuppressive factor (TIF) . The TIF compositions and methods of the present, invention overcome many of the hurdles confronted by prior attempts of controlled, specific immunosuppression. The IL-2R inhibitor compositions and methods of the invention suppress IL-2 receptors themselves and, thus, prevent interleukin-receptor interactions to control the responsiveness of the activated cells. Accordingly, the invention provides an effective alternative to actually killing lymphocyte cells. The novel therapeutic immunomodulation provided by the present invention — reversible suppression of IL-2 receptor expression — is also advantageous in that the invention obviates the necessity of identifying antigen, but still results in antigen-specific immunosuppression.

The present invention provides, for the first time, a specific and effective IL-2R inhibitor for use in pharmaceuticals and as a reagent in laboratory experiments to explore the biochemical interactions of the immune system. In immunologic experiments, the IL-2R receptor inhibitor of the present invention will eventually lead to the discovery of even more selective antagonists and agents that operate via the IL-2 receptor so that effective medicinal intervention will become a reality in a wide variety of disorders of the immune system.

Summary Of The Invention

We have discovered a new immunosuppressive agent that selectively inhibits expression of IL-2R by activated lymphocytes.

Accordingly, an object of this invention is to provide a method for effective therapeutic manipulation of the immune system. It is also an object of this invention to provide a pharmaceutical composition for such therapeutic treatment, and a reagent useful in specifically suppressing the expression of IL-2 receptor genes in immunologic research.

An additional object of the invention is to provide a TIF isolate having IL-2 receptor inhibiting activity. Another object is to provide a partially purified IL-2 receptor inhibitor and a purified proteinaceous molecule having such activity.

Yet another object is to provide a partially purified proteinaceous molecule, and compositions containing such proteinaceous molecule or partially purified proteinaceous molecule, for use in specifically suppressing the expression of IL-2 receptors. In a preferred embodiment, such compositions include effective pharmaceutical agents for the treatment of autoimmune diseases and the prevention of allograft and transplan rejection in medical patients.

Advantageously, the compositions and methods of the invention provide for immunomodulation, while retaining the organism's immunocompetence; the pharmaceutical agents and method of the invention selectively suppress a specific immune response without inhibiting interferon gamma production or IL-2 production and without actually killing lymphocyte cells.

Additional objects and advantages of the invention will b set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.

To achieve the objects and accordance with the purpose of the invention, as embodied and broadly described herein, the present invention comprises a purified proteinaceous molecule (IL- 2R inhibitor) derived from T. cruzi. said proteinaceous molecule having activity as an inhibitor of IL-2R expression. The present invention also comprises a partially purified proteinaceous molecule, compositions of matter containing such proteinaceous molecule and proteinaceous molecules substantially homologous to the T. cruzi derived IL-2R inhibitor. The invention also relates to pharmaceutical compositions and reagents utilizing such IL-2R inhibitors and compositions for immunologic studies.

The invention also comprises a process for making an IL-2R inhibitor by culturing T. cruzi organisms in a medium for a sufficient period of time for said organisms to secrete TIF into the medium, separating the organisms from the medium to produce an isolate containing said TIF, recovering said isolate, removing •proteins below a molecular weight of 30,000 and above a molecular weight of 100,000 from said isolate, fractionating the 30,000 to 100,000 molecular weight isolate by liquid chromatography, determing which fraction continues to have activity as an IL-2R inhibitor and recovering said fraction. The invention also relates to methods for the treatment o an autoimmune disease in a mammal, comprising administering to th mammal a pharmaceutically effective amount of the IL-2R inhibitor and to methods for suppressing an immune response in a mammal without killing lymphocytes or curtailing IL-2 or interferon gamm production.

Description Of The Figures

Figure 1 illustrates that suppression of the proliferativ response of PBMC to PHA by T. cruzi is mediated by a parasite secretion product. "PHA + fT. cruzi]" represents parasites separated from the PBMC by a Millicell-HA filter. The value for "PHA + T. cruzi" was obtained from cultures in which the PBMC and the trypanosomes were in contact and a Millicell-HA filter containing complete medium alone was present in the cultures. Th results represent the mean 3H-TdR incorporation + SD of triplicat values. Cultures were pulsed from 60 to 72 hr.

Figure 2 illustrates the effects of different TIF concentrations on PHA-induced IL-2 receptor ("IL-2R") expression and lymphoproliferation by PBMC. A: Suppression of IL-2R expression 24 hr after PHA stimulation. The reductions observed with both 75 and 90% TIF were statistically significant (p<0.05). B: Suppression of ³H-TdR incorporation; 48 to 60 hr pulse. The reduction observed with 90% TIF was statistically significant (p<0.05). Each of these sets of data is representative of three separate experiments performed with cells from different donors.

Detailed Description Of The Invention

Reference will now be made in detail to the presently preferred embodiments of the invention, which, together with the following examples, serve to explain the principles of the invention.

The present invention establishes that suppression of IL- 2R expression does not require physical parasite-cell contact and is mediated by a T. cruzi secretion product. Using culture inserts with cell-impermeable membranes, we have demonstrated significant suppression of PHA-induced lymphoproliferation whether the trypanosomes are placed in the same compartment as, or separated from, peripheral blood mononuclear cells (PBMC) . Similar effects result from using supernatants from T. cruzi suspensions. These supernatants (referred to herein as "TIF isolates") also inhibit IL-2R expression in response to PHA stimulation. The suppressive effect of the T. cruzi secretion product is reversible, as evidenced by significant recovery of the proliferative capacity of PBMC after removal of the parasite- containing inserts.

More specifically, by using flow cytometric studies and anti-Tac (an mAb against an epitope of the p55 chain of the human IL-2R (Uchiyama et al., J. Immunol., 126 (1981) pp. 1393-1403)) we have discovered that the in-vitro suppressive effect of T. cruzi is associated with a striking reduction in the proportion of huma PBMC capable of expressing Tac antigen after activation with either phytohemagglutinin (PHA) or anti-CD3 mAb. This suppressiv effect becomes more pronounced as the parasite concentration in the culture is increased, with up to 90% reduction in the proportion of Tac-positive cells in the presence of 1 X 10 parasites per ml (compared with control values obtained in the absence of parasites) . In addition, in the PBMC subpopulation that is still capable of expressing Tac antigen in the presence o T. cruzi. the cell surface density of this antigen is markedly diminished.

Medium absorbed for four days (under culture conditions) with four times as many organisms than required to induce suppression still supports optimal proliferation in response to mitogenic stimulation. Thus, the suppressive effect of the parasite is not due to the consumption of essential medium nutrients. Similarly, neither mitogen removal by the trypanosome nor loss of PBMC viability due to co-culture with the organisms are significant factors in the production of the suppressive effects of T. cruzi. Under optimal stimulatory conditions, ___ cruzi does not impair IL-2 production by the PBMC and does not use, absorb or inactivate this lymphokine. Because exogenous recombinant IL-2 does not. correct the suppressive effect of the parasite, an insufficient level is not a causative factor. This is a major difference between the manner in which mouse and human lymphocytes are modified by the presence of T. cruzi. Under non-optimal stimulatory conditions (i.e., supra-optimal mitogen concentration and cell crowding) , both IL-2 production and IL-2R expression by human PBMC are suppressed by the presence of T. cruzi. However, under optimal conditions, only suppressed IL-2R expression occurs. In sum, the inhibitory effect of the parasite on I -2R expression is the common denominator.

The high-affinity IL-2R, responsible for IL-2 internalization and signal transduction, is a complex of at least two proteins-p55 and p70. (Tsudo, et al. Proc. Natl. Acad. Sci. USA, 83 (1986) pp. 9694-9698.) Anti-Tac mAb recognizes an epitope on the p55 protein and does not react with the p70 component. Therefore, use of this antibody does not clarify whether T. cruzi affects the expression of p55 alone or of both p55 and p70. However, by chemically cross-linking 125l-IL-2 to IL-2R expressed by activated human PBMC exposed to T. cruzi. we have determined that the expression of both proteins is similarly affected. We derived independent evidence for the suppression of the high-affinity IL-2R by this parasite from radiobinding experiments performed under high-affinity conditions.

Reduced numbers of lymphocytes expressing IL-2R and decreased surface density of IL-2R on the cells that still express Tac antigen renders them unable and less capable, respectively, of reacting with IL-2. Inhibited lymphoproliferation due to an inability to use IL-2 also explains the multifaceted aspects of the suppression that accompanies the acute phase of T. cruzi infection — no effective immune response can be mounted if clonal expansion does not reach a significant level.

We have established that T. cruzi suppresses the expression of IL-2R by human lymphocytes even when separated from the lymphocyte cells by a filter whose pore size prevents direct trypanosome-human cell contact. These observations reveal for the first time the existence of a soluble mediator released by T. cruzi that has specific suppressive properties. We have termed this mediator the trypanosomal immunosuppressive factor (TIF) . Clearly, such a factor has enormous potential as a biological reagent for studying the mechanisms of regulation of IL-2R expression by human lymphocytes.

That suppression by T. cruzi of PHA-induced IL-2R expression and proliferation is mediated by a secretion product was initially revealed by the occurrence of these effects despite separation of the PBMC and the parasites by a cell-impermeable filter (Figure 1) . These observations, which also indicated that direct PBMC-T. cruzi contact is not required to cause suppression, was independently confirmed by the presence of suppressive activity in T. cruzi suspension supernatants. TIF is spontaneously released by the parasite, as its production does not require the presence of either mitogen-stimulated PBMC or substances released by normal or activated cells. Gradual decrease in the level of suppression results from extending the time elapsed between removal of the parasite¬ 's containing inserts and the initiation of the 12-hr H-TdR pulse, denoting that TIF activity is reversible. When the inserts are removed at 72 hr and ³H-TdR pulses (started at 72 hr) are extended from 6 to 24 hr, the degree of suppression decreases as the pulse time is lengthened. These results indicate that PBMC can recover from T__s_σruzj.-induced suppression. The rate of reversibility is relatively fast as marked recovery occurs in the last 12 hr of the

72-96-hr pulse. The reversibility of the suppressive effect is demonstrated even when the parasite-containing inserts are replaced with new ones containing the parasite-free diffusate of parallel cultures set up with identical concentrations of PBMC, parasites and PHA placed outside the insert. Therefore, the reversibility of the suppressive effect is not the result of any sudden decrease in the concentration of the suppressive factor upon insert exchange. In a second approach, we used TIF isolates instead of inserts containing living parasites. The suppressive effect subsided with time and was virtually undetectable during the last 12 hr of 84- or 96-hr culture periods. Reversibility of the suppression caused by TIF isolates indicates the presence of

• adequate culture medium components to support effective lymphoproliferation and confirms that reduced proliferation does not result from mitogen or cytokine absorption or consumption by the parasite. The TIF molecule was identified as a protease-sensitive molecule with a molecular weight between 30,000 and 100,000 daltons. Accordingly, T. cruzi-induced suppression of IL-2R expression and lymphoproliferation is mediated by a secretory product of protein nature (i.e., a proteinaceous material) with a molecular weight greater than 30,000 daltons, but smaller than 100,000 daltons, and is reversible. This trypanosomal immunosuppressive factor (TIF) is clearly a useful tool in the treatment of autoimmune disease states, the suppression of inappropriate immune responses to allografts and transplants, as well as exploration of the mechanisms that regulate the expression of IL-2R and other surface molecules playing key roles in lymphocyte activation. That the suppressive effects are mediated by a secretion product should greatly simplify the study of the relevant mechanisms of action. While not wishing to be bound by theory, we believe that such mechanisms involve alterations in transcription of the genes coding for receptor proteins, messenger RNA stability or translation and/or receptor protein transport to the lymphocyte membrane.

Accordingly, one embodiment of the present invention comprises an IL-2R inhibitor derived from T. cruzi. which inhibitor is isolated from T. cruzi in culture and is either partially purified or purified to homogeneity. The term "partially pure" as used herein shall be understood to mean a TIF composition resulting from removing the proteins below 30,000 and above 100,000 molecular weight from a TIF isolate and preferably a further purified TIF composition obtained by subjecting such first composition to high pressure liquid chromatography to fractionate the composition into components and recovering the fraction with IL-2R inhibiting activity. The terms "pure" or "purified" as used herein shall mean substantially free of other proteinaceous molecules that are not inhibitors of IL-2 receptor expression. The purified inhibitor of the invention is at least 90% pure, preferably 95% pure, and most preferably 99% pure by weight. The TIF isolate, the partially pure IL-2R inhibitor, and the purified IL-2R inhibitor of the invention all have specific activity as inhibitors of IL-2 receptor expression.

In accordance with the present invention, there is also provided a process for producing an IL-2R inhibitor, by suspending T. cruzi organisms to produce a culture medium containing the inhibitor and recovering the IL-2R inhibitor from the medium. The inhibitor may be purified or substantially purified on the basis of its physical and chemical characteristics, particularly its hydrophobicity, charge and molecular weight. In a preferred embodiment, the inhibitor is produced by suspending T. cruzi organisms in a serum-free culture medium, separating the medium from the cells, and subjecting the separated medium to high pressure liquid chromatography.

The IL-2R inhibitor may also be purified by other techniques well-known in the art. Such techniques include electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (including ion exchange chromatography, affinity chromatography, immunoadsorbent affinity chromatography, reverse-phase high performance liquid chromatography, and gel permeation high performance liquid chromatography) , isoelectric focusing, and variations and combinations thereof. One or more these techniques are employed sequentially in a procedure design to separate molecules according to their physical and chemical characteristics. These characteristics include the hydrophobicity, charge and the molecular weight of the inhibitor. The various fractions of materials obtained after each technique are tested for their ability to inhibit IL-2R or cellular processes involved in IL-2R expression. These tests are describe in Examples 4 and 5 below. Those fractions showing anti-IL-2R activity are then subjected to the next technique in the sequential procedure, and the new fractions are tested again. Th process is repeated until only one fraction having the ability to inhibit IL-2R remains and that fraction produces only a single band when subjected to polyacrylamide gel electrophoresis.

The discovery and purification of IL-2R inhibitor from T. cruzi cells will allow the production of antibodies to the inhibitor. Antibodies are highly specific and have high affinities for the proteinaceous molecules they have been raised against. When attached to an insoluble matrix, they allow the easy and efficient separation of the proteinaceous molecule they have been raised against from a complex mixture of proteinaceous and other substances. The methods of using such antibodies are well-known in the art and are disclosed in R. Scopes, Protein Purification: Principles and Practice (New York: Springer Verlag 1982) , pp. 132-136, which is incorporated herein by reference. Thus, it is now possible for the first time to recover purified IL-2R inhibitor from any cell that produces it by using antibodie to the IL-2R inhibitor derived from T. cruzi organisms and techniques well-known in the art. Therefore, such purified IL-2R inhibitors are within the scope of this invention.

In a preferred embodiment, purified IL-2R inhibitors are recovered from any type of cell that produces such an inhibitor and is capable of being cultured. The method comprises:

(a) culturing cells that produce an IL-2R inhibitor and are capable of growing in a culture medium;

(b) separating the inhibitor-containing medium from the cells;

(c) bringing the medium into contact with immobilized antibodies to an inhibitor so as to bind the inhibitor in the medium to the antibodies;

(d) removing the medium minus the bound inhibitor; and

(e) separating the inhibitor from the immobilized antibodies so as to recover it in a purified form.

Cells may be evaluated for their ability to produce the IL-2R inhibitor of the invention by many techniques known in the art, including those disclosed in the instant specification. Those types of cells that are capable of being grown in a culture medium can be cultured by well-known techniques, including those disclosed in the instant specification. However, antibodies may also be used to recover IL-2R inhibitor from cells that are not capable of growing in culture through known extraction or recover techniques.

Antibodies to IL-2R inhibitors may be made by various techniques well-known in the art. Polyclonal antibodies may be made by injecting an inhibitor into rabbits, goats, horses, or other animals. The animals are then bled, and the presence of antibodies can be determined by such methods as double diffusion or detection of antibody-antigen aggregates using 125 I-labeled protein A. The antibodies to inhibitor are then recovered from the serum. Generally, it is necessary only to partially purify the antibodies. In an alternative embodiment, monoclonal antibodies may be used instead of polyclonal antibodies.

The antibodies potentially need not be made by using as the antigen the particular inhibitor sought to be recovered or produced. Since IL-2R inhibitors produced by different types of cells may^'be substantially homologous to each other, an antibody to an inhibitor such as that recovered from T. cruzi cells may bind to an IL-2R inhibitor produced by a different type of cell.

The present invention also relates to biologically active analogs of the T. cruzi IL-2R inhibitor. The biologically active analogs are proteinaceous molecules with at least one active site having IL-2R inhibitor activity, which site may exhibit substantial homology to the natural IL-2R inhibitor produced by T- cruzi or may function in a manner biologically equivalent to such inhibitor. The site may also be altered to form a proteinaceous molecule having enhanced IL-2R inhibitor activity.

It is possible to conceive of a class of IL-2R inhibitors having common elements of structure and mechanism of action and differing from one another in only a few amino acid residues. In addition to being isolated from cells, members of such class could be produced by chemical modifications of existing members by techniques well-known in the art once such members were identified by the teachings of the present invention. Such modifications may enhance the activity of the original inhibitor or may have no effect on such activity. In addition, the present invention will allow the cloning of the gene coding for the active inhibitor. Once this gene is cloned, numerous modifications of the active material can be made via base substitution and introduction of the modified gene into a variety of hosts. Thus, it is contemplated that such a class of IL-2R inhibitors is within the scope of the present invention.

It is also contemplated that the IL-2R inhibitors of the ^'present invention may contain one or more amino acid sequences that are not necessary to their activity. Such sequences can be removed by techniques well-known in the art. Unnecessary amino acid sequences could be readily removed via limited proteolytic digestion using enzymes such as trypsin or papain or related proteolytic enzymes. Thus, such inhibitors are within the scope of the present invention.

T. cruzi is not the only protozoan known to be capable of suppressing IL-2R expression by lymphocytes. Trvpanosoma brucei infection can affect IL-2R expression by mouse lymphocytes. (Sileghem et al., Eur. J. Immunol. 17 (1987) pp. 1417-1421.) Sileghem et al. have shown that lymph node cells from infected animals have a reduced IL-2R expressing capacity following stimulation with con-canavalin A. Reduced expression of IL-2R after stimulation with Mycobacterium leprae antigen has also been noted in lymphocytes from patients with lepromatous leprosy. (Mohagheghpour et al., J. Immunol., 135 (1985) pp. 1443-1449.) We have also found that T. rhodesiense suppresses the expression of T cell surface components, including the IL-2R.

Nor is IL-2R the only surface molecule whose expression is affected by T. cruzi. The expression of certain key surface molecules involved in lymphocyte activation, such as CD3, CD4 and CD8 and receptor for transferrin is also suppressed. (Beltz, Kierszenbaum, and Sztein, Infect. Immun. , 57 (1989) p. 2301; Sztein, Cuna and Kierszenbaum, J. Immun., 144 (1990) pp. 3558- ^• ₃5₆2₎. Consequently, TIF should help clarify the regulatory mechanisms governing the expression of human lymphocyte surface molecules playing key roles in lymphocyte activation.

In one embodiment, the IL.-2R inhibitors of the invention, either as an isolate, or in partially pure or purified form. comprise a specific reagent for use in immunologic research. By specifically inhibiting expression of the IL-2 receptor, the biomolecular mechanisms of the immune system can be explored. IL- 2R protein measurements in cells inhibited by TIF, compared to normal control values, provide information about the ability of the suppressed cells to synthesize the receptor components. Measurements of IL-2R messenger RNA in cells cultured in medium alone or containing TIF provide information about the ability of the cells to transcribe the IL-2R receptor genes. Similarly, other cell surface components and their biomolecular mechanisms can also be explored by the use of the IL-2R inhibitor of the invention. Techniques for measuring a specific protein or mRNA in cells are well-known in the art. New therapeutic agents can thus be anticipated.

This invention also relates to compositions of a therapeutically effective amount of IL-2R inhibitor in admixture with a pharmaceutically acceptable carrier.

The IL-2R inhibitors of the present invention are contemplated for human and veterinary uses in the form of pharmaceutical products possessing IL-2R inhibitor activity. Such pharmaceutical preparations contain, as at least one of the active ingredients, the present IL-2R inhibitor and also appropriate, pharmaceutically acceptable carriers, dilutents, fillers, binders, and other excipients depending on the dosage form contemplated. For oral administration, steps must be taken to prevent degradation of the active proteinaceous molecule in the digestive track. Enteric coated dosage forms are contemplated as one form suitable for oral administration. It is also contemplated that pharmaceutical preparations containing an IL-2R inhibitor can be administered locally, as by injection or topical application, intravenously, intraocularly, suconjuήctiveally, intramuscularly, and intrathecally. The mode of administration will necessarily depend upon the disease or ailment involved.

In another embodiment, the invention comprises a method for altering an immune response. The method comprises administering an effective amount of IL-2R inhibitor admixed with an acceptable pharmaceutical carrier. When administered, the pharmaceutical composition functions as an immunomodulator, suppressing the inappropriate immune response without deleting lymphocyte cells or inhibiting interferon gamma production. Accordingly, the method provided by this embodiment is particularly useful in preventing rejection of allograft and tissue or organ transplants.

The IL-2R inhibitors of the present invention can be used to treat any disorder in which autoimmunity plays a role. For example, they can be used to inhibit the immune response to self cells and tissues as seen in autoimmune disease states. In addition, they can be used to inhibit the immune response to foreign cells and tissues and, thus, they can be used to prevent the rejection of allografts and organ transplants. The inhibitors can also be used to control other disorders in which actuated lymphocytes cause problems, such as occurs in tumor management.

In accordance with the present invention, a process is provided for treating disorders in humans and other mammals or animals in which IL-2 plays a role by administering an effective amount of a purified IL-2R inhibitor in admixture with a carrier. The method of administration may be intravenous, topical, intraocular, subconjunctival, intramuscular, or intrathecal administration or by direct injection. The disorders that may be treated include arthritis, type 1 diabetes, systemic lupus erythematosus, and other autoimmune diseases, e.g., myasthenia gravis. In a preferred embodiment, a therapeutically effective amount of the IL-2R inhibitor in admixture with a pharmaceutically acceptable carrier is administered intravenously.

The amount of the IL-2R inhibitor to be administered would depend upon the particular disorder being treated. Such a determination is routinely made by those of ordinary skill in the art in determining therapeutic dosages and is within the scope of tasks routinely performed by them without undue experimentation.

EXAMPLES

Materials and Methods:

Parasites: T. cruzi trypomastigotes (Tulahuen isolate) were purified from the blood of Crl-CDl(ICR) Swiss mice (Charles River Laboratories, Portage, MI) infected subcutaneously 9-11 day previously with 4 to 5 x 10 5 organisms. The flagellates were separated from blood cells by centrifugation (400 X g, 20*C, 45 min) over a mixture of Ficoll-Hypaque of density 1.077 (Budzko an

Kierszenbaum, J. Parisitol., 60 (1974) pp. 1037-1038) followed by chromatography through diethylaminoethyl-cellulose (Mercado and

Katusha, Prep. Biochem. , 9 (1979) p. 97). After being washed with

RPMI 1640 medium (GIBCO, Grand Island, NY) containing 100 IU of penicillin and 100 ug of streptomycin per ml, the parasites were resuspended at the desired concentration in the same medium either without serum supplement (referred to as serum-free medium) or containing 5% heat-inactivated (56*C, 20 min) FCS (referred to as complete medium) . The suspensions consisted of 100% trypomastigotes (>99.9% viable, i.e., displaying mctility) .

The parasites can also be obtained by culture. The organism is grown in cultures of rat heart myoblasts (Dulbecco's modified minimal essential medium, supplemented with 10% heat inactivated fetal bovine serum and penicillin (100 ILU/ml) plus 100 ug/ml streptomycin) . The organisms in the culture medium are harvested, washed by centrifugation and purified by chromatography through diethlyaminoethyl-cellulose, as described above.

Preparation of PBMC: Blood was collected from healthy donors, ages 25 to 49. PBMC were purified by density gradient centrifugation. over Ficoll-Hypaque under the same conditions as described above. After being washed with serum-free medium, the cells were resuspended in complete medium, counted microscopically using a hemacytometer and adjusted to the desired concentration in the same medium. In all cases, cell viability, determined by trypan blue dye exclusion, was >99%.

Preparation of T. cruzi isolates: Suspensions of T. cruzi

7 at 1 to 2 X 10 parasites per ml in serum-free or complete medium were incubated at 37°C and 5% CO- for 24 or 48 hr and filtered through a 0.45-um-pore-size sterile filter. This material was aliquoted and stored at -20*C until used, and is referred to as

TIF. Some TIF preparations were dialyzed against 100 volumes of serum-free medium (4*C, 5 hr) before use. In these cases, an aliquot was kept under the same conditions but was not dialyzed.

Co-cultures of PBMC with T. cruzi: Parasites added to cell cultures were allowed to establish physical contact with the

PBMC or were separated by using a 0.45-um-pore-size filter. PBMC (6.25 X 10 5 cells in 0.5 ml of complete medium) were placed in

24-well culture plates, and a sterile Millicell-HA filter insert

(Millipore Corp., Bedford, MA) into which 0.4 ml of complete medium containing or lacking 4.5 X 10 T. cruzi trypomastigotes was placed. Where appropriate, 4.5 ug of PHA (Sigma Chemical Co.,

St. Louis, MO) was added to the PBMC suspension so that the final concentration in the 0.9-ml total culture volume would be 5 ug/ml.

When T. cruzi was allowed to be in direct contact with PBMC, both the organisms and the PBMC were placed in the culture well and an insert containing 0.4 ml of complete medium was present in the culture, all other conditions remaining unchanged.

In some of the examples in which inserts were removed at various times, new ones containing only complete medium alone were used to replace them. In other examples, insert containing 0.4 ml of conditioned medium (i.e., the diffusate of parallel cultures containing identical concentrations of PBMC, parasites and PHA, all placed outside of the insert) replaced the ones being removed (the outside surfaces of these filters were flushed with serum-free medium to detach any parasites) .

The cultures were incubated at 37"C and 5% CO- for various

₃ periods of time and pulsed with 5 uCi of H-TdR (specific activity

= 2 Ci/mmol, Amersham, Arlington Heights, IL) for either a fixed or increasing amount of time. For determination of 3H-TdR incorporation, 100-ul aliquots of each culture were transferred to

96-well plates for automated harvesting and processing for liquid scintillation counting. In no case where parasites had been placed in inserts or washed off before placing an insert in a culture was any trypanosome detected in the surrounding medium upon extensive microscopic examination.

TIF effects, on PHA-induced PBMC proliferation: Cultures of PBMC (1.25 X 10⁵/well) were set up in triplicate or quadruplicate in 96-well flat-bottom plates (final volume = 0.1 ml) . In some of these cultures, varying proportions of medium were replaced by TIF isolate. The TIF preparations used in this type of experiment were made in complete medium. Incubation with or without PHA (5 ug/ml) at 37*C and 5% CO- took place for various periods of time and lymphoproliferation was determined as

₃ described above except that H-TdR pulses consisted of 1 uCi/well.

In examples relating to the reversibility of the suppressive

₃ effect, of TIF, the cultures were pulsed with H-TdR either for increasing amounts of time or during various time intervals after culture initiation. Of the various TIF preparations used to study reversibility some were made in serum-free and others in complete medium. In all cases, the cultures were terminated by automated harvesting and processed for liquid scintillation counting.

TIF effect on IL-2R expression: Cultures of PBMC were performed in 24-well plates (1.25 X 10 /well; final volume = 1 ml) . In these cultures, TIF isolate (made in complete medium) substituted for varying proportions of medium; incubation with or without PHA (5 ug/ml) at 37'C (5% Co-) took place for various periods of time. IL-2R expression was determined by flow cytometry. Cells were collected and washed with PBS containing 1% BSA (PBS+BSA) . Cells were incubated with anti-Tac (which recognizes an epitope on the p55 component of human IL-2R; kindly provided by Dr. T. A. Waldmann, National Institutes of Health) for 30 min at 4'C, followed by one wash with PBS+BSA, and incubation with 25 ul of a 1:6 dilution of fluorescein-labeled F(ab^,)₂ goat anti-mouse I G antibody (Tago Immunodiagnostics, Burlingame, CA) for 30 min at* 4*C, Stained cells were fixed in 1% formaldehyde and stored at 4"C in the dark until analyzed using a FACS IV flow cytometer (Becton-Dickinson, Mountain View, CA) . A minimum of 10,000 cells, gated to exclude erythrocytes, platelets, non-viabl cells and T. cruzi was accumulated for each histogram. The percentage of positive cells was estimated against a background o cells stained with normal mouse I_G. Mean channel numbers of the logarithm of fluorescence intensities of the positive cell populations (MFCh) were used to compare the relative density of the relevant lymphocyte marker in the presence or absence of T. cruzi.

Treatment of TIF isolate with proteases: TIF preparation made in serum-free medium were incubated with trypsin- or pronase-agarose bead conjugates (Pierce, Rockford, IL) or with agarose beads .alone at room temperature for 210 min with frequent shaking. The preparations were then filter-sterilized and used immediately.

Estimation of molecular weight: Centricons of different molecular weight cut-offs (10,000; 30,000 and 100,000 daltons; Amicon Corporation, Danvers, PA) were washed several times with serum-free medium; the last washing filtrate was sterilized by filtration and used as control. The Centricons were then used to filter aliquots of either serum-free TIF or TIF made with complet medium. The TIF filtrates were sterilized by filtration and used immediately. Cultures set up with the filtrates received heat- inactivated FCS to a final concentration of 5%. Statistics: The statistical significance of differences between means was determined by Student's "t" test.

EXAMPLE I

SUPPRESSION OF THE RESPONSE OF PBMC TO PHA BY T. CRUZI DOES NOT

REQUIRE CELL-PARASITE CONTACT

To establish whether the immunosuppression produced by T. cruzi resulted from direct contact with the PBMC, we used a culture system in which both cell types were either on the same or separate compartments created by the presence of a Millicell-HA insert. As can be seen in Figure 1, significant suppression, in terms of reduced 3H-TdR incorporation during the last 12 hr of

72-hr cultures, occurred whether or not physical parasite-PBMC contact was allowed. Although the extent of . cruzi-induced suppression varied among repeat experiments, even when cells from the same donor were used, the suppressive effect was always demonstrable and statistically significant (p<0.05). In the various repeat experiments, suppressed responses represented 10 to

60% of the control values (PBMC plus PHA) . The example exemplified in Figure 1 depicts comparable suppression with parasites inside or outside of the insert. EXAMPLE II

SUPPRESSION BY T. CRUZI ISOLATES OF PHA-INDUCED EXPRESSION OF IL-2 RECEPTORS AND LYMPHOPROLIFERATION

The demonstration of the production of a filterable suppressor factor(s) by T. cruzi made it possible to explore whether such production was spontaneous or was induced by the presence of PBMC or PBMC products. To this end, we added to the PBMC cultures TIF isolate instead of trypanosome suspension and monitored IL-2R expression by PHA-stimulated PBMC. Concentrations of TIF isolate representing 75 or 90% of the culture volume significantly reduced IL-2R expression (Figure 2, panel A). Suppression by 50% TIF was seen only in some of the repeat experiments, and 25% TIF never displayed detectable suppressive

₃ activity. Similar observations were made when we monitored H-TdR incorporation (Figure 2, panel B) . Significant suppressive activity was demonstrable in the various TIF isolates, whether or not serum-free or complete medium had been used in their preparation, and also in TIF preparations tested after dialysis versus serum-free medium.

EXAMPLE III

REVERSIBILITY OF THE SUPPRESSIVE EFFECT OF T. CRUZI

The two-compartment culture system was also used to initially study whether the suppressive effect of T. cruzi was reversible. In a first approach, Millicell-HA inserts containing parasites were removed at different times after culture initiation and 3H-TdR incorporation was measured during the last 12 hr of 72- hr cultures. As can be seen in Table I, as the time interval between insert removal and initiation of the 3H-TdR pulse increased, the extent of the suppressive effect decreased. This reversibility of the TIF effect was demonstrable not only when the

Millicell-HA inserts containing T. cruzi were removed and replaced with another insert containing complete medium alone (Table I) but also when parasite-containing inserts were removed and replaced with another insert which had been kept in a parallel co-culture where all cells had been placed outside the insert (and whose outer surfaces had been flushed with complete medium to wash off any parasites) (Table I, footnote a) .

To independently confirm that the suppressive effect of T. cruzi was indeed reversible, and to monitor the rate of reversibility, we removed the parasite-containing inserts at 72 hr

₃ and measured the level of H-TdR incorporation during increasing amounts of time starting immediately after insert removal. As shown in Table II, the level of suppression decreased as the length of the pulse was extended but did not vary significantly if the parasite-containing inserts were allowed to remain during the entire culture period. In an alternative approach, we used TIF isolate; this was added to the cultures immediately prior to the addition of PHA, and replicate sets of cultures were pulsed with

³H-TdR during various time intervals. In the various repeat experiments, the degree of suppression, usually maximal for H-Td pulse from 48 to 60 hr, was less pronounced or minimal at later times (Table III) .

TABLE I

SUPPRESSION OF PHA-INDUCED PBMC PROLIFERATION BY A T. CRUZI SECRETION PRODUCT: EXTENT OF SUPPRESSION AFTER PARASITE

REMOVAL AT VARIOUS TIMES

Material Time of insert cpm X 10 -3 % Suppression tested removal (hr)

Complete medium Not removed 0.6 ± 0.1

PHA Not removed 66.6 ± 0.4

PHA + [T. cruzil 12 56.7 ± 2.1 14.8

PHA + CT. cruzi] 24 52.0 ± 1.2 21.9

PHA + fT. cruzil 48 44.2 ± 1.9 33.6

PHA + [T. cruzi] Not removed 41.9 ± 1.1 37.1

PHA + T. cruzi Not removed 39.2 ± 1.1 41.1

Mitogen-stimulated PBMC were incubated with T. cruzi or T. cruzi-containing Millicell-HA inserts placed in the cultures immediately after PHA addition. Inserts were removed at the indicated^"times. Control cultures received an insert containing complete medium instead of parasite suspension. Removed inserts were replaced with new inserts containing complete medium, which were left in the cultures until termination. The cultures were pulsed with ³HTdR during the last 12 hr of a 72-hr culture period. This set of results is representative of two separate repeat experiments. In a representative experiment in which parasite- containing inserts were replaced with new ones containing conditioned medium (see Materials and Methods) the % suppression after exchanging inserts at 12, 24 and 48 hr, was 7.4, 47.7, and 57.4%, respectively; where the parasite-containing insert was not removed, the % suppression was 74.5%.

_v. fT. cruzil = parasite-containing insert.

^~ These reductions in cpm with respect to the values obtained with PHA-stimulated cultures without T. cruzi were statistically significant (P<0.05).

TABLE II

SUPPRESSION OF PHA-INDUCED PBMC PROLIFERATION BY T . CRUZI AT VARYING ³ H-TDR PULSE TIME INTERVALS

Time of insert Percentage of suppression after pulsing removal with ³H-TdR from: 72 to 78 hr 72 to 84 hr 72 to 96 hr

72 hr 81.3 75.8 52.6

Not removed 86.3 88.0 85.0

Mitogen-sti ulated PBMC were incubated with T. cruzi- containing inserts placed in the cultures immediately after PHA addition. The values (expressed as cpm X 10 -3 ') obtained with stimulated PBMC in the presence of inserts without parasites were

61.4 ± 6.4, 61.7± 10.2 and 77.3 ± 9.2, for the 6-, 12- and 24-hr. pulses, respectively. These control cultures received an insert containing complete medium alone. This set of results is representative of four separate repeat experiments. All percentages of suppression represent statistically significant reductions in cpm (p<0.05) with respect to the values obtained with PHA-stimulated cultures without T. cruzi. TABLE III

REVERSIBILITY OF THE SUPPRESSIVE EFFECT OF TIF³

3 H-TdR pulse (hr) Percentage of Suppression

48 to 60 52'

60 to 72 23< 72 to 84 17⁽ 84 to 96 0

^a Mitogen-stimulated PBMC were incubated at 37* C and 5% CO, with medium alone or containing 90% TIF. Cultures were

3 . . . . pulsed with lu Ci H-TdR per well during the indicated periods of time.

This level of suppression corresponded to a statistically significant (p<0.05) reduction in cpm with respect to the value obtained with PHA alone.

^c This level of suppression did not correspond to a statistically significant reduction in cpm. EXAMPLE IV

CHEMICAL NATURE AND MOLECULAR SIZE To determine whether suppressive activity is mediated by a molecule of protein nature, we incubated TIF isolates with agarose beads with or without conjugated trypsin or pronase. The results, shown in Table IV, indicated that digestion with either protease significantly reduced or abolished the suppressive capacity of TIF.

The approximate molecular size of the TIF molecule(s) was estimated by filtration through A icon filters of different molecular weights cut offs. Suppression was readily demonstrable with filtrates containing molecules <100,000 daltons but not with those <10,000 or <30,000 daltons (Table V).

TABLE IV

TIF ACTIVITY IS LOST UPON DIGESTION WITH PROTEOLYTIC ENZYMES³

Material tested cpm X 10~³

Exp. 1 Exp. 2

Medium alone 0.3 ± 0.1 O.S ± o.O

PHA alone 37.4 ± 2.3 32.6 1 3.4

PHA + mock-treated TIF 7.8 ± 1.0 6.1 ± 0.3

PHA + trypsinized TIF 26.8 ± 3.3 16.0 ± 0.3

PHA + pronase-treated TIF 39.4 ± 2.2. 14.8 ± 1.5

^a Mitogen-stimulated PBMC were incubated with medium alone or containing 90% TIF isolate for 60 hr at 37'C (5% C0-). Cultures were pulsed with 3H-TdR during the last 12 hr.

Experiments 1 and 2 were performed separately using different TIF isolates and cells from different donors. TIF was incubated with agarose-1inked enzymes or mock-treated with agarose beads alone at room temperature for 210 min. TABLE V

APPROXIMATE MOLECULAR SIZE OF THE TIF ACTIVE MOIETY'

^a Mitogen-stimulated PBMC were incubated with fresh medium, medium used to wash the Centricons (medium filtrate) or medium containing 90% TIF (before and after) filtration through Centricons (molecular weight cut offs indicated in parentheses) . Cultures were pulsed with ³H-TdR during the last 12 hr of a 60-hr incubation period. Experiments 1 and 2 were performed separately using different TIF isolates. This reduction with respect to the corresponding control was statistically significant (p<0.05).

EXAMPLE VI

PURIFICATION AND FURTHER CHARACTERIZATION

We fractionated a TIF isolate by high-pressure liquid chromatography (HPLC) on a Mono-Q column consisting of l ml of io exchange resin. TIF activity eluted at 7 ml from the Mono-Q column with a 0 to 1 M KCl gradient (in Hepes pH 7.2). The activ fraction displayed suppressive activity similar to that of the unfractionated isolate.

The eluted fractions are analyzed by sodium-dodecyl- sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . The molecular weight(s) of the identified band(s) are determined by comparison with molecular weight markers run side-by-side. The molecular weight of TIF determined in this manner falls within th previously established range (30,000 to 100,000 daltons).

If more than one ban is identified by SDS-PAGE in an eluted fraction of the HPLC column, antibodies are raised in rabbits and mice against each of the bands by repeated injection of homogenized gel bands (the gel is known to act as an adjuvant, enhancing the antibody response) . Rabbit antibodies against each band are used for several major purposes, as outlined below. RABBIT ANTIBODIES

Rabbit antisera raised against each gel band is utilized in inhibition assays designed to establish whether they can block TIF biological activity. An inhibitory serum identifies the corresponding antigen as being the TIF molecule.

After purification by ammonium sulfate precipitation followed by diethylaminothyl-cellulose column chromatography, the inhibitory antibody(ies) is polymerized by cross-linking, and made into an immunosorbent matrix to be used in the purification of TIF from crude T. cruzi extracts by affinity chromatography. The purified antigen (TIF) is then used to establish the amino acid sequence of some segments of the molecule. From these limited amino acid sequences, the corresponding gene codes are deduced and oligonucleotides probes are synthesized for the identification of the corresponding gene(s) in expression c-DNA libraries of Trypanosoma cruzi. The identified genes are cloned into expression vectors by well-known recombinant-DNA techniques. (The clones make it possible to produce larger amounts of TIF, produced by the transformed hosts, and circumvent use of the parasite as the source of TIF.) Furthermore, sequencing of the cloned gene •makes it possible to deduce the entire amino acid sequence of TIF.

The specific (inhibitory) rabbit antibody is utilized in Western blotting analysis, simplifying the identification of TIF in any subsequent purification steps, if required. MOUSE ANTIBODIES

The serum from mice immunized with the SDS-PAGE bands is tested for inhibitory activity in the aforementioned TIF assay. The spleens of animals producing the inhibitory antibodies are used in the production of hybridomas secreting monoclonal antibodies against epitopes of the TIF molecule. These monoclonal antibodies may be used for the same purpose outlined above for rabbit antibodies, including Western blotting analysis, affinity chromatography and purification of TIF. Western blotting analysis using these monoclonal antibodies identifies the same band as the inhibitory rabbit antibody(ies) .

It will be apparent to those skilled in the art that various modifications can be made to processes and products of the present invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

Claims

WE CLAIM;

1. A purified proteinaceous molecule derived from T. cruzi or T. brucei. said proteinaceous molecule having an activity as an inhibitor of IL-2 receptor expression.

2. A partially purified proteinaceous molecule derived from T. cruzi or T. brucei. said proteinaceous molecule having activity as an inhibitor of IL-2 receptor expression.

3. A composition of matter having activity as an inhibitor of IL-2 receptor expression, comprising a proteinaceous material secreted by T. cruzi or T. brucei and in isolation from T. cruzi or T. brucei organisms.

4. The composition of claim 3, wherein said proteinaceous material is secreted by and in isolation from T. cruzi.

5. The composition of claim 3, wherein said proteinaceous material is secreted by and in isolation from T. brucei.

6. The composition of claim 5, wherein said T. brucei is T. brucei brucei.

7. The composition of claim 5, wherein said T. brucei is T. brucei rhodesiense.

8. The composition of claim 5, wherein said T. brucei is T. brucei gambiense.

9. The composition of claim 4 wherein said proteinaceous material has a molecular weight between 30,000 and 100,000 daltons.

10. A proteinaceous molecule substantially homologous to the proteinaceous molecule of claim 1, wherein said homologous proteinaceous molecule has an activity as an inhibitor of IL-2 receptor expression.

11. A pharmaceutical composition comprising the purified proteinaceous molecule of claim 1 in a pharmaceutically acceptable carrier.

12. An experimental reagent for examining IL-2 receptor function comprising the composition of matter of claim 3.

13. A process for making an inhibitor of IL-2 receptor expression, comprising: culturing T. cruzi or T. brucei organisms in a medium for a sufficient period of time for said organisms to secrete TIF into the medium; separating the organisms from the medium to produce an isolate containing said TIF; and recovering said isolate.

14. The process of claim 13 further comprising: removing proteins below a molecular weight of 30,000 and above a molecular weight of 100,000 from said isolate.

15. The process of claim 14 further comprising: fractionating said 30,000 to 100,000 molecular weight isolate by chromatography, determining which fraction continues to have activity as an IL-2R inhibitor, and recovering said fraction.

16. An IL-2R inhibitor prepared by the process of claim 15.

17. The IL-2R inhibitor of claim 16, wherein said cultured organisms are T. cruzi.

18. A method for the treatment of an autoimmune disease in a mammal comprising administering to said mammal a pharmaceutically effective amount of the proteinaceous molecule of claim 1.

19. The method of claim 18, wherein said proteinaceous molecule is derived from T. cruzi.

20. A method for suppressing an immune response in a mammal without curtailing IL-2 or interferon gamma production, comprising administering an effective amount of the proteinaceous molecule of claim 1.

21. The method of claim 20, wherein said proteinaceous molecule is derived from T. cruzi.

22. The method of claim 20, wherein said immune response is the result of an autoimmune disease, an allograft or a transplant.

23. The method of claim 20, wherein said mammal is a human being.

24. In a method for the biochemical or immunologic investigation of an immune response in a mammal, the improvement comprising the use of the composition of claim 3.

25. In a method for the biochemical or immunologic investigation of an immune response in a mammal, the improvement comprising the use of the inhibitor of claim 16.

26. A process for producing an IL-2R inhibitor comprising the steps of: culturing organisms that produce an IL-2R inhibitor and are capable of growing in a culture medium to produce a culture medium containing said IL-2R inhibitor; separating said medium from said cells; bringing said medium into contact with immobilized antibodies to an IL-2R inhibitor so as to bind said IL-2R inhibitor in said medium to said immobilized antibodies; and separating said IL-2R inhibitor from said immobilized antibodies.

27. The process of claim 26 wherein said antibodies are polyclonal antibodies.

28. The process of claim 26 wherein said antibodies are monoclonal antibodies.

29. The method of claim 20, wherein said suppression of immune response is reversible.

30. In a method for the biochemical or immunologic investigation of the expression of a lymphocyte cell surface component, the improvement comprising the use of the proteinaceous molecule of claim 1.

31. The method of claim 30, further comprising the step of measuring the amount of said surface component present in said lymphocyte cell.

32. The method of claim 30, further comprising the step of measuring the amount of the mRNA for such surface component present in said lymphocyte cell.