NO882647L - IGE-INDUCED REGULATORY (EIR) COMPOSITIONS. - Google Patents

Description

The present invention relates to immunology in general, and specifically to compositions with activities related to the regulation of IgE antibody response, and which may be useful in the treatment of allergic diseases, or parasite infections.

Lymphocytes expressing cell surface receptors for the Fc part(s) of immunoglobulin molecules (FcR) have been instrumental in regulating the humoral immune response (J. Immunol. 130:521, Immunol. Rev. 56:51, Proe. Nati. Acad. Sci. 80: 2323, J. Immunol 133:1087). Conducted observations have considered the involvement of the FcR<+>T cells in isotype-specific regulation of antibody production. The role of lymphoid cells expressing FcR for IgE (FcRc) in regulating IgE antibody synthesis has been carefully studied (J. Immunol. 133:2821, 133:2829, 133: 2837, 133:2845, Immunol. Today 4:182) . The T-lymphocytes exhibit an enhancing isotype-specific effect on IgE antibody synthesis expressing high levels of FcRc, while those that suppress IgE synthesis lack easily detectable cell surface FcRc. The role of B cells in regulating IgE antibody synthesis has recently been discovered. B cells that regulate IgE synthesis do so by first acting on specific T subsets (J. Immunol. 133:2821, JL33:2829, 133:2837).

Exposure of normal lymphoid cells to appropriate levels of IgE, either in vivo or in vitro, stimulates enhanced expression of FcR by these cells (J. Clin. Invest. 64:714, Allergy 39: 81, Immunol. Today 4:182) . Increasing numbers of FcRc<+->lymphoid cells have been found in humans and experimental animals exhibiting elevated serum IgE concentrations. In vitro exposure of human, rat or murine lymphocytes to appropriate concentrations of IgE results in enhanced elicitation of FcRe, where a significant 3H portion is T-' cells. FcR,-<4>'T cells release IgE-binding factors (IgE-BF) which enhance IgE antibody production. T cells producing IgE-BF with IgE class-selective suppressive activity express relatively little FcRc, but express Fc receptors (FcR) for other antibody classes (eg, IgG).

The conditions of lymphocyte FcRc expression, resulting in the production of both enhancing and inhibitory forms of Ig-BF, reflecting the activation of a complex cascade of cellular and molecular interactions that act to maintain normal homeostatic regulation in the IgE antibody system, have been reported (J. Immunol. 133:2821, 133:2829, 133:2837, 133:2845, Allergy 39:81). Some of these molecular intermediates in this cascade have been termed IgE-induced regulators or EIRs. The initial initiating event in this cascade is the interaction of IgE with FcRc<+>B cells. This interaction results in the release of B cell-derived soluble mediator, designated EIRg, which can at once induce other B cells to express FcRc, and likewise induce Lyt-1<+>T cells to release IgE isotype-specific suppressor molecule , suppressive factor in allergy (SFA). SFA has been shown to suppress continued IgE synthesis both in vivo and in vitro (Allergy 39:81). SFA, which is not itself an IgE-binding factor, acts on another subset of Lyt-1<+> cells to induce the production of a special type of IgE-binding factor, termed suppressive effector molecule (SEM). SEM is thought to act directly on IgE-producing B cells to inhibit continued IgE synthesis, and has been shown to inhibit EIRg-induced FcRc expression by B cells. SEM may be identical to the suppressive IgE-BF described by others (J. Immunol. 133:803).

Enhanced expression of FcReved B cells exposed to IgE and EIRg appears to be the stimulus for the enhanced arm of this network, thus the hermostatic importance of the SEM medium the inhibition of this step described in the section above (J. Immunol. 133:2829 , 133:2837, 133:2845). The amplification arm of the cascade is formed when FcRc<+>B cells react with Lyt-2<+>T cells, an interaction that stimulates such T cells to produce EIRs. EIRfSelectively induces other Lyt-2<+>T cells to express FcRc (it has no effect on B cells), thereby producing EIRj, which is so named because of its inhibitory effect on IgE-induced FcRc- expression by the B cells. Allergy enhancing factor, EFA, which is another IgE-selective mediator identified in previous work at this laboratory, acts in a similar manner to EIR-p by inducing Lyt-2<+>T cells to release a mediator termed enhancing effector molecule, or EEM. EFA and EIR-p, on the other hand, are distinguishable molecular forms. Since both EIR and EEM are IgE-BF and their effects are biologically indistinguishable, and are probably identical constituents, we hereafter refer to the effector molecule induced by either ' EIR-p or EFA as EEM. EEM directly inhibits IgE-induced production of EIRg by FcRc<+>B cells thereby enhancing the IgE antibody system, since inhibition of EIRg release immediately reduces the production of endogenous SFA.

Monoclonal sources of each respective factor using the hybridoma technology have been developed. The biological and physiochemical properties of monoclonal (mc) factor are compared with conventional (c) factors induced by appropriate stimulation of heterogeneous lymphoid cell populations. Comparisons of conventional EIRT(cEIR-p) and monoclonal EIRT(mcEIRT) derived from a murine T-cell hybridoma are compared hereafter.EIR-p as both sides, either unfractionated or semi-purifying gel filtration, have been shown to directly and selectively , to induce FcRc expression by Lyt-2<+-> cells, and to induce production by the same cells of IgE-BF termed EEM.

During our biochemical fractionation studies, we detected in preparations containing cEIR-p, two distinct, molecular mass forms of EIRg, a finding that was unexpected compared to our previous observations using unfractionated EIR preparations. One such molecular form of biochemically-enriched EIEg was shown to have the ability to induce T cells to make SFA, whereas the other molecular form of EIRg lacked this capacity. EIRg in non-fractionated form has been shown to exhibit a suppressive effect on IgE antibody synthesis in vivo (J. Immunol.. 133:2837). A previously unidentified SFA molecule is also described. In addition to T-cell hybridomas producing EIR-p, B-cell hybridomas producing EIRg_i and EIRg_2, respectively, have also been described. A T-cell hybridoma secreting this new SFA form has also been described.

Table I summarizes the various IgE-induced regulatory agents described in the literature and those discovered by applicants. The regulatory agents produced by IgE-stimulated lymphocytes, including those described herein, are described below: I. The B cells exposed to appropriate concentrations of IgE produce B cell-derived IgE-induced regulatory agents (EIRjj ). Two forms of EIRg have been described.

eiSb-i

- - produced by B cells in response to stimulation with IgE - - also produced by a newly identified B cell hybridoma - - induces other B cells to express Fc receptors for IgE (FcRc) - - induces Lyt-1<+ >T cells to make SFA (as defined by inhibition of FcRc expression) - - inhibit IgE synthesis in vivo, presumably by inducing SFA

- - 15-20 kd in size

EJRb-2

- - produced by B cells in response to stimulation with IgE - - produced by a newly identified B-cell hybridoma

- - induce B cells to express FcRc

- - does not induce Lyt-l<+-> cells to make SFA (as defined by Inhibition of FcRc expression) - - inhibits IgF synthesis in vivo (mechanism of action unknown)

- - 30-35 kd in size

II. Enhanced expression of FcRc-induced B cells exposed to EIRB stimulates the Lyt-2<+>T cells to make T cell-selective EIR-p. The final effect of EIR-p production is suppression of continued EIR-j release, which inhibits subsequent induction of SFA and facilitates IgE synthesis.

EIRT

- - produced by Lyt-2<+>T cells in response to interaction with EIRg-activated FcRc<+->B cells

- - produced by a T-cell hybridoma line

- - induces Lyt-2<+>T cells to express FcRc

- - induces Lyt-2<+-> cells to release an IgE-binding molecule termed enhancing effector-- - molecule, EEM (EEM has also been termed EIRj) - - enhances IgE synthesis in an isotype-specific manner through the action of EEM

- - EIRt has a size of 45-60 kd

- - EIR-p is not an IgE-binding factor

EEM

- - produced by Lyt-2<+>T cells in response to stimulation with EIR^- - produced by Lyt-2<+>T cells in response to stimulation with EFA plus IgE

- - is an IgE-binding factor of 10-15 kd

- - inhibits IgE-induced EIRg_^ production, thereby inhibiting the production of the SFA synthesis that is triggered by elevated IgE levels. III. SFA acts on Lyt-l<+-> cells, in the presence of EIR-MØ, to induce the production of a suppressive effector molecule (SEM). One of the functions of SEM is to inhibit EIRg-induced FcRc expression by B cells. This SEM-mediated inhibition can therefore usurp induction of EIR-p and thus enhance continued EIRg production, all of which facilitates SFA production. Such factors can directly inhibit B cells from producing IgE. SEM-containing liquids can also act in this way. It remains to be determined whether it is the same 10-15 kd molecule described below.

SFAi (described below)

- - produced by Lyt~l<+->cells in response to stimulation with EIRg_^- - - acts on Lyt-l<+->cells to induce production of SEM - - inhibits IgE-induced FcRc expression by lymphocytes through the effect of SEM - - is believed to inhibit IgE synthesis by B cells through the effect of SEM

- - 20-30 kd in size

SFAg (described below)

- - biologically identical to SFA_^ but is larger in size (ie, SFA_2 is 3'5-40 kd, while SFA_^ is 20-30 kd) - - produced by a newly identified T-cell hybridoma

SEM

- - produced by Lyt-1<+>T cells and Lyt-1<+>B cells stimulated with SFA plus EIR-MØ

- - binds to IgE

- - Inhibits EIRB-induced FcRc expression by the B cells

- - 10-15 kd in size

EIR- MØ

- - produced by macrophages in response to stimulation with IgE - - required as a co-stimulus for the induction of SEM by SFA

Abbreviations

-The following abbreviations are used here:

B cell - bone marrow derived lymphocyte

BSA - bovine serum albumin

C - complement

DMEN - Dulbecco's modified Eagle's

medium

DNP - 2,4-dinitrophenyl

EEM - enhancing effector molecule

EFA - allergy enhancing factor EIR - IgE-induced regulatory agent EIRg - B-cell-selective IgE-induced regulatory agent

EIRj - inhibitory IgE-induced regulatory agent

EIR-r - T-cell-selective IgE-induced regulatory agent

EIR-MØ EIR DERIVED FROM MACROPHAGES

Ceir - conventional lymphoid cell-derived EIR

mcEIR-p - monoclonal T-cell hybridoma-derived EIRT

FcR - cell surface receptors for the Fe parts of immunoglobulin molecules

FcR<+>- indicates presence of

cell surface FcR

FcRc- Fc receptor for IgE

pcRc<+>- indicate presence of

cell surface FcRc

FCS - fetal calf serum

IgE-BF - IgE-binding factor

MEM - minimum essential medium PCA reaction - passive skin anaphylaxis

RAME - rabbit anti-mouse IgE antibodies SEM - suppressive effector molecule SFA - suppressive factor for allergy

-SRBC - sheep red blood cells

T cell - thymus-derived lymphocyte

TNP - trinitrophenyl

Four new molecular isolates that are novel in isolated form and unpredictable from prior knowledge are described as well as hybridoma cell lines producing each of the respective factors.

One aspect of the invention essentially includes the composition of the isolated EIRg fraction having a molecular weight of 15-20 kd, or a molecular weight of 30-35 kd, or both fractions in combination, but not separated from, e.g., as in natural liquids.

The invention also relates to the regulatory agent designated SFÅ2, which in isolated form consists of a molecule with a molecular weight of 30-40 kd. SFA2 acts as a molecular intermediate in the suppressive aspect of IgE class-selective regulation.

Another aspect of the present invention includes the regulatory agent EIR-p, which is a molecular intermediate in the potentiating or enhancing component of IgE class-selective regulation. EIRter is a previously unidentified molecule, which has a molecular weight of 45-60 kd.

The invention also includes compositions consisting of the aforementioned 15-20 kd EIRg fraction, the 30-35 kd EIRg fraction, the 30-40 kd SFA2 fraction, or the 45-60 kd EIRT fraction in a physiologically compatible carrier .

The invention also includes the respective hybridoma cell line from which each of the aforementioned regulatory agents can be derived, which includes 1) the B-cell hybridoma designated 99E9 which secretes EIRg_i, 2) the B-cell hybridoma designated 2C7 which secretes EIRg_2, 3) the T-cell hybridoma designated MBI-1 as

-stores SFA2 and 4) The T-cell hybridoma designated MBI-2 that produces EIR-r«

A regulatory agent also included in the invention, produced by IgE-stimulated macrophages (EIR-MØ), is a necessary co-stimulus for SFA-induced SEM production.

Figure 1 graphically depicts the results of the experiments demonstrating that both cEIR-j derived from heterogeneous lymphoid cells and mcEIR-r derived from the T-cell hybridoma MBI-2 induce only Lyt-2<+> cells to express FcRc. Normal CAFi spleen cells were treated with either C alone, monoclonal anti-o plus C, monoclonal anti-Lyt-2 plus C, or monoclonal anti-Lyt-1 plus C. Then the cells were washed and cultured in either medium alone, medium containing 50% mcEIRT derived from T-cell hybridoma MBI-2 or 50% cEIRT derived from normal lymphocytes (which in this experiment is in the form of unfractionated lymphocyte-derived cEIR). After incubation for 16 hours, the cells were harvested, washed and assayed for FcRc<+> cells by rosette formation with DNP-specific IgE-coated TNP-SRBC. Each point represents the geometric mean of rosette-forming cells for each culture condition, as determined from 3 counts of 250-300 viable lymphocytes. Horizontal lines indicate standard deviation.

Figure 2 graphically describes the results of the experiments showing that the mixture of the mediators in cEIR inhibits, while the T-cell hybridoma-derived mcEIR-p enhances the IgE antibody responses of SJL mice. SJL mouse groups were either not irradiated (group I) or irradiated with 250 rads (groups II to IV) just prior to vehicle preimmunization with 2 g KLH in 4 mg alum on day -7. On days -1 and 0, the mice in groups III and IV were injected with either RAME-adsorbed supernatant fluid from CAF, spleen cell cultures stimulated with 10 g/ml IgE (cEIR) or supernatant fluid from the cultures of T-cell hybridoma MBI-2 ( mcEIRT). Each injection consisted of 0.1 ml of supernatant fluid (diluted to

-0.5 ml in saline), given 4 times at 8-16 h intervals over a 48-h period. On day 0, all mice were immunized with 2 g DNP-KLH in 2 mg alum. Day 10 primary anti-DNP IgE responses were measured by PCA reactions, and anti-DNP IgG responses were determined by solid phase radioimmunoassay. There were no statistically significant differences in the IgG anti-DNP antibody responses among the different groups which were: 11.6 (1.20), 9.8 (1.60), 10.3 (1.30) and 8.5 (2.10) g/ml for groups I to IV, respectively. Figure 3 graphically describes the data showing that both cEIR-p and mcEIR-p have protein structure. Normal CAF^ spleen cells were cultured in either medium alone, in medium containing 2% untreated EIRT, or 2% EIRT that had been treated with the indicated enzyme. After 8 hours of incubation, the cells were harvested, washed and analyzed for FcRc<+-> cells as described above in Figure 1. Figure 4 graphically depicts molecular sieve chromatography data (AcA-54) of cEIR and mcEIR-p demonstrating that EIR-p has a molecular weight of 45-60 kd - - induction of FcRc<+->lymphoid cells, and isolation and separation of the 15-20 kd and 30-35 kd EIRg fractions which have been shown to have allergy suppressive activity. Normal CAF^^ spleen cells were either unfractionated (C only), or depleted of T cells by anti-Ø-dependent complement-mediated cytotoxicity. The cells were then cultured in either medium alone, or in medium containing 10% of the indicated column fraction of cEIR (panel A), or in medium containing 10% of the indicated column fraction of MBI-2 derived mcEIRT (panel B). After 8 hours, the cells were harvested and analyzed for FcRc<+> cells as described above in fig.1. Fig.5 graphically represents molecular sieve chromatography data (AcA-54) of cEIR and mcEIR-p demonstrating: 1) presence of SFA and SEM in cEIR, and 2) absence of such contaminants in mcEIR-j- - - inhibition of FcRc<+ ->cell induction. Normal CAF^ spleen cells either unfractionated (C only), or deprived of T cells with anti-0 plus C were cultured in one of the above-mentioned media alone, in medium containing 10 g/ml IgE, in medium containing 10 $ of the indicated column fraction of cEIR plus 10 g/ml IgE (panel A), or in medium containing 10$ of the indicated column fraction of mcEIRT plus 10 g/ml IgE (panel B). After 24 hours, the cells were harvested and analyzed for FcRc<+> cells as described in Fig.1. Data are expressed as percent inhibition of IgE-induced FcRc expression, with unfractionated and T-cell-depleted lymphoid cells cultured in the absence of IgE at 1.0 (1.41) and 3.0 (1.11) percent FcRc<+-> cells, respectively, and control IgE-stimulated cultures with 18.4 (1.03) and 29.0 (1.05) percent FcRc<+-> cells, respectively. Fig. 6 graphically presents the data showing that both EIR and hybridoma-derived mcEIR can induce Lyt-2 + cells to produce FcRc inhibitors EEM, but do not induce Lyt-1<+-> cells to produce SEM. Normal CAF^ spleen cells were deprived of T lymphocytes with anti-0 plus C, and cultured in either the medium alone, in medium containing 10 g/ml IgE plus 20% supernatant fluid from the cultures of Lyt-1<+>cell-depleted lymphoid cells stimulated with the Indicated regulatory agent (panel B), or in medium containing 10 g/ml IgE plus 10% supernatant fluid from the culture of Lyt-2<+-> cell-deprived lymphoid cells stimulated with the indicated regulatory agent (panel C). After 24 hours of incubation, the cells were harvested, washed and analyzed for FcRc<+> cells as described in fig. 1. The data are expressed as percent inhibition of FcRc induction using the values indicated in panel A as controls. Fig. 7 graphically describes the data showing that EIR and mcEIR-p have no IgE-binding affinity, while the effector molecule that they induce (EEM) is clearly an IgE-binding factor. Normal CAF-1 spleen cells, either unfractionated (panels A and B) or T cell-deprived anti-G plus C (panels C and D), were

-cultured in either bare medium, in medium containing the indicated dilution of either untreated or IgE-adsorbed cEIR-p, or mcEIR-j- (panels A and B), in medium containing 10 mg/mm IgE, or in medium containing 10 g/ml plus the indicated dilutions of either untreated or IgE-adsorbed EEM (panels C and D). IgE adsorption was accomplished by passing 2 ml of EIR onto a 2 ml IgE-Sepharose column constructed with 5.25 mg of affinity-purified anti-DNP IgE per gram Sepharose 4B. After 8 hours of incubation (panels A and B), or 24 hours of incubation (panels C and D), the cells were harvested, washed and analyzed for FcRc<+> cells as described under the number of fig. 1. Data in panels C and D are expressed as percent inhibition of IgE-induced FcRc~ expression, with lymphoid cells cultured in the absence of IgE-expressing 5.3 (1.18) percent FcRc<+-> cells and control IgE-

stimulated cultures showing 32.2 (1.04) percent FcRe<+-> cells.

Fig. 8 graphically presents data demonstrating that cell line MBI-1 produces SFA activity characteristic of SFÅ2 and that this cell line can be induced to secrete enhanced levels of the latter stimulant upon stimulation with EIRg. The MBI-1 cells were put into culture at a concentration of 2 x 10<5> cells per ml in the absence or presence of 10% supernatant fluid derived from IgE-stimulated B cells (EIRg). After 30 hours of incubation, the supernatant liquids from such cultures were harvested, and 4 ml from each culture was separately passed through an ACA 54 molecular sieve column. Normal CAF1 spleen cells either deprived of Lyt-2<+>T cells (Fig. 6) with antiI-Lyt-2 antibodies plus C, or deprived of all T cells (not shown), were cultured in either medium alone, or in medium containing various dilutions of the indicated column fraction plus 10 g/ml IgE. After 24 hours, the cells were harvested and analyzed for FcRc<+> cells as described under reference to fig. 1. The data are expressed relative to the last dilution of each fraction that could show at least 35% inhibition of IgE-induced FcRcut pressure.

Approach

Animals - SJL, BALB/C, and (BALB/C X A/ J) F1 (CAFX) mice were obtained from Jackson Laboratories, Bar Harbor, ME, and maintained at the breeding colonies of the Medcal Biology Institute, La Jolla, CA. Adult male Lewis rats were purchased from Holtzman Co., Madison, WI.

Proteins and chemicals

The reagents used were purchased or prepared as follows: Bovine serum albumin (BSA), DNase, phospholipase A2, RNase, and trypsin - - Sigma Chemical Co., St. Louis, MO, Ovalbumin (OVA) - - Miles Laboratories, Inc., Elkhart , IN, Pronase and neuraminidase - - Calbiochem-Behring Corp., La Jolla, CA, 2,4,6-trinitrobenzenesulfonic acid (TNBS) - - INC Pharmaceuticals, Cleveland, OH, CNBr-activated Sepharose 4B - - Pharmacia Fine Chemicals , Uppsala, Sweden, Ultrogel AcA-54 - - Reactifs IBF Villeneuve la Garenne, France, 2,4-dinitro-phenyl (DNP) 2s -BSA was prepared as previously described (J. Immunol. 124:2728).

Monoclonal and conventional antibodies and compliment. DNP-specific monoclonal antibodies were obtained from hybridomas constructed and characterized as reported (J. Immunol., 124:2728). The DNP-specific monomeric IgE used was isolated by affinity chromatography on DNA-BSA coupled Sepharose 4B.

Monoclonal ant-Thy 1,2 (anti-Ø), derived from the F7D5e7 hybridoma clone (Eur. J. Immunol. 9:825) was provided as an ascites (abdominal cavity) fluid and cleared by centrifugation before use. Monoclonal anti-Lyt-1,2 and monoclonal anti-Lyt-2,2 were purchased from Accurate Chemical and Scientific Corp., Westbury, NY, reconstituted as indicated, and stored at -20°C until use. Guinea pig complement (C) was obtained from Pel-Freeze Biologicals, Rogers, AR, and extensively adsorbed at 4°C with CAF, spleen cells before use.

Preparation of lymphoid cells, and removal of specific subpopulations prior to induction of FcRc.

Spleens were gently immersed in cold minimum essential medium (MEM) in a tissue grinder. Then the cells were washed twice with MEM, after which the cells were either resuspended in Dulbecco's MEM (DMEM) plus 10% fetal calf serum (FCS) and antibiotic supplements for induction of FcRc as described in the next section, or resuspended in MEM to remove lymphoid subpopulations that described below.

The T cells were removed before FcRc induction by two consecutive treatments of the lymphocytes with monoclonal anti-G plus C (J. Exp. Med. 136:455). Selective treatments with anti-Lyt-1,2 and anti-Lyt-2,2 monoclonal antibodies were performed in the same way. Control cells were treated with C alone. The efficiency of lymphocyte subpopulation removal by anti-9 treatment was greater than 95%, as determined by susceptibility to lysis with monoclonal anti-0 plus C, and immunofluorescence (J. Immunol. 133:2821 ).

Normal abdominal macrophages were obtained by washing the peritoneal cavities of mice with PBS, and were isolated by adherence to tissue culture flasks. Peritoneal cells were seeded at a density of 3 x 10 cells/ml in DMEM, and non-adherent cells were eliminated by washing after 3 hours of incubation at 37°C.

In vitro system for induction of FcRc ± lymphocytes. Unfractionated spleen cells, or cells fractionated as described above, were resuspended in DMEM plus 10% FCS and antibiotics. The amount of FCS used was specifically selected for compatibility with EIR-induced FcRc expression. Cells were adjusted in concentration so that, unless otherwise stated, a final cell density of 10 3 nucleated cells/ml was achieved after addition of monoclonal IgE, and/or EIR-containing culture supernatant fluid or column fractions. The cells were seeded in -Linbro 24-well macroplates so that a final volume of 0.5 ml/well was provided after addition of monoclonal IgE, SFA or EFA, or culture supernatant fluids or additional medium. The cultures were then incubated in a 10% CO 2 atmosphere for the indicated time period, after which the cells were harvested and analyzed for the FcRc<+-> cell frequencies, using the rosette assay described below.

Analysis for rosette formation

a) Preparation of indicator cells

Sheep red blood cells (SRBC) were obtained from the blood of a

single sheep kept in the Medical Biology Institute animal facility. TNP-derived SRBC were prepared by reaction with TNBS as previously described (J. Immunol. 127:166). IgE-coated TNP-SRBC were prepared by incubating the red

the cell suspension (1% in MEM) with subagglutinating amount of DNP-specific monoclonal antibodies (5-10 g/ml), determined from pilot experiments to provide optimal red cell sensitization for detection of a maximal number of FcRc<+->rosettes. The mixture was incubated for 30 minutes in a 37°C water bath, followed by washing twice with MEM. The Ig-coated red cells were then brought to a 1% solution in MEM.

b) Preparation of rosettes.

Fifty ;1 lymphocytes (2.5 x 10^ cells), repaired as

described above, was added to 12 x 75 mm Falcon tubes containing 100 µl appropriate indicator cells. The cell mixture was then centrifuged at 500 rpm for 5 minutes. After undisturbed incubation overnight at 4°C, the cell pellets were carefully resuspended either with a 100 µl Eppendorf or by mixing. Trypan blue was added to each tube just before counting the rosettes, the viability of the cultured cells was usually b% or more. As a rule, the number of rosettes in approximately 250-300 viable small lymphocytes in each sample mixture was counted 3 or 4 times, and the geometric mean and standard deviation were calculated for these values. When screening multiple column fractions, only a simple count of approximately 300 viable cells was performed per sample mix Since the regulatory agent was contained in multiple and consecutive column fractions, detected changes in the level of FcRc expression in consecutive cultures were considered to accurately reflect the EIR content. A rosette-forming cell is defined as a small lymphocyte that is closely surrounded by at least 3 indicator cells, although most rosettes exhibited more indicator cells than the minimum number.

Preparation of IgF-induced regulatory agents (EIR). The protocol for producing EIR has been described (J. Immunol. 133:2821). Normal spleen cells (10<7>/ml), which were either unfractionated or fractionated into different B or T lymphocyte subpopulations, or peritoneal macrophages, were cultured in the presence of 10 g/ml monoclonal IgE (monomeric form) for 24 hours as described above for IgE-induced FcRc expression. The supernatant fluids of these cultures were cultured 24 hours later, residual IgE was removed by running over RAME-Sepharose affinity columns, and the (RAME-adsorbed) elution fractions from the affinity columns were collected for biological activity testing.

Screening of T-cell hybridoma cell lines for the production of

EIR.

T-cell hybridomas were constructed by fusion of the AKR-derived T-cell line, BW5147, with alloactivated T-cell blasts from DBA/2 donors as described previously in detail. (J. Exp. Med. 152:946, 1980), and was maintained at -70°C at the Medical Biology Institute. The B-cell hybridoma was constructed by fusion of SP2/0 B-cell myeloma with in vivo antigen-treated B-lymphocytes as previously reported (J. Immuno. -124:2728). The cell lines were cloned by limiting dilution in 96-well plates (0.3 cells/well), and the supernatant fluids from these wells showing growth over 10 to 14 days were screened for the presence of EIR in the in vitro FcRc induction assay as described below for screening of molecular sieve column fraction. The hybridoma clones used in the present study were found to be free of mycoplasma by analysis with Vero cells and fluorescent DNA staining with Hoechst 33258.

Gel filtration

4 ml of EIR-containing culture supernatant fluid was applied to an Ultrogel AcA-54 column (2.6 x 97 cm) equilibrated with phosphate-buffered saline containing 0.01% PEG. Elution was performed with the same buffer at 4°C, and 2.5 ml fractions

were collected for analysis. The AcA-54 column was calibrated with ovalbumin (M.W. 43,000), chymotrypsin (M.W. 25,000) and cytochrome C (M.W. 12,300). Each fraction derived from gelf 11 trees of whole spleen cell EIR or monoclonal EIR was analyzed for the presence of FcR-induced EIR-r and EIRg, and FcRc-inhibitory SFA, EFA, and the IgE-binding factors SEM and EEM. In the first assay, 50 µl of each AcA-54 fraction (or buffer) was added to 0.45 ml of culture medium containing 5 x 10 5 of different types of lymphoid cells as indicated. After 8 h incubation, the cells were harvested and analyzed for FcRc<+> cells as described above. Significant induction of the FcRc<+> cells in both the unfractionated and T-cell-deprived (removed) lymphoid cell cultures indicates the presence of EIRg in that fraction. Induction of FcRe<+> in only the unfractionated (T cell-containing) lymphoid cell culture indicates the presence of EIR-j- in the fraction being tested. In the second assay involving inhibition of IgE-induced FcRc expression, 50:1 of each AcA-54 fraction (or buffer) was added to 0.45 culture media containing 5 µg of monoclonal IgE plus 5 x 10 3 of the indicated lymphoid cells. . In this analysis, significant inhibition of IgE-induced FcRc expression in unfractionated and Lyt-2 + cell-depleted, but not T-cell-depleted indicator cells, is indicative of the presence of SFA. Inhibition of FcRc induction only in the unfractionated lymphoid cell cultures, and not in Lyt-2<+-> cell-depleted or total T-cell-depleted cell cultures, indicates the presence of EFA. Inhibition of IgE-induced FcRc expression in a T cell-independent manner indicates the presence of the IgE-binding factors SEM or EEM.

Assessment of IgE isotype-specific immune regulation by IgE-induced regulatory agents.

The ability of EIR to selectively modulate in vivo IgE synthesis was assessed using irradiation-enhanced primary antibody responses in SJL mice (J. Immunol. 120:2050, J. Immunol., 120:2060). On day -7, SJL mice were exposed to 250 rads and then carrier primed with 2 g of KLH adsorbed on 4 mg of aluminum hydroxide gel (alum). On days -1 and 0, the animals were intravenously injected with the indicated regulatory agent, each injection consisting of 0.1 ml of supernatant fluid diluted to 0.5 ml with saline, which was given 4 times at 8-16 h intervals over a 48 hour intervals. Between these injections, on day 0 the mice were immunized with 2:g DNP-KLH in 2 mg alum. The mice were bled from the retroorbital plexus at indicated intervals thereafter and their sera analyzed for IgE and IgG anti-DNP antibody content as previously described (J. Immunol. 124:2727, J. Immunol, 117:1629).

Enzymatic cleavage of EIRf

The enzymes were used at the following final concentrations for cleavage of EIR^: trypsin, pepsin, pronase and phospholipase A2 were used at 1 mg/ml, RNase at 100 g/ml, DNase at 1 g/ml, and neuraminidase at 0.1U/ ml. 100 µl of a 10x enzyme solution was added to a tube containing 900 µl of serum-free EIR, or tissue culture medium only. The reaction mixture was incubated at 37° C. for one hour, after which 100 µl FCS

■was added. The treated EIR-r preparations, and controls, were then assayed for residual FcRc-induced activity at a final dilution of 1:50.

Summary of the molecular mediators in the IgE cascade Heterogeneous lymphoid cells from normal mouse spleen produce a mixture of IgE-selective regulatory mediators after exposure to IgE in vitro (J. Immunol. 133:2821, 133:2829, 133:2837, 133:2845 ). Because their effects are in contrast to each other, as well as synergistic effects, the presence of any of these mediators will reduce or modify the biological activity of the others, thereby necessitating the use of selective subsets of lymphoid cells to produce and /or detect the activity of each respective mediator.

We have termed this mixture of mediators EIR (cEIR). Such preparations have been shown to exhibit the following distinct biological activities: (1) EIR-p, an activity capable of inducing Lyt-2<+> cells to express FcRcog produce EEM, (2) EEM, and IgE-BF which can suppress IgE-induced EIRg production, an event manifested as an inhibition of B-cell FcRc expression, (3) SFA, a molecule that has the ability to induce Lyt-l<+-> cells to produce SEM, and (4) SEM, an IgE-BF that exhibits the ability to inhibit EIRg-induced FcRc expression by B cells, again, like EEM, detectable by its ability to inhibit IgE-induced FcRc expression by isolated B cells . As described herein, EIRg, a mediator(s) having the ability to induce B-cell FcRc expression, and the ability to induce Lyt-1<+>T cells to make SFA are also included in cEIR. [Therefore, cEIR = cEIRT+ cEIRg + cSFA + cSEM + cEEM].

Constitutive production of an EIR-like activity by T-cell hybridoma MBI-2

As indicated above, supernatant fluid derived from the cultures of IgE-stimulated lymphoid cells contains the full spectrum of EIR activities, including EIRT. For this reason, we chose to exploit the limiting heterogeneity of the product secreted by the hybridoma T cell lines to augment this analysis of EIR-p.

Examination of 200 clones of T cell hybridomas for production of EIRT showed that 8 secreted constitutively desirable amounts of regulatory agent capable of inducing expression of FcRc by unfractionated lymphoid cells. The soluble product (mcEIRT) of one such clone (MBI-2) was selected for further analyzes and comparison with cEIR-p derived from heterogeneous lymphoid cell populations.

Both cEIR-derived from heterogeneous lymphoid cells and mcEIR-β derived from T-cell hybridoma MBI-2 induce only Lyt-2<+>T cells to express FcRc.

The experiment summarized in Figure 1 illustrates that the cellular targets involved in the expression of FcRc resulting from exposure to either cEIRT or mcEIR-p are completely identical in both cases. Similar to our previously reported results obtained with cEIR-β alone, both cEIRT and mcEIRT induced FcRc expression in both unfractionated (groups II and III), but not in completely T-cell-depleted (groups V and VI), lymphoid cells. When lymphoid cells selectively deprived of the T cell subset were examined, both cEIR-p and mcEIR-p readily induced FcRc expression in Lyt-1<+>cell-deprived (groups VIII and IX) but not in Lyt-2<+ >cell-deprived (groups XI and XII), lymphoid cells. These results illustrate that both cEIR-p and mcEIR-p induce Lyt-2<+>T cells to express FcRcmembrane markers.

The mixture of mediators in cEIR inhibits, while T-cell hybridoma-derived mcEIR-β enhances, IgE antibody response to SJL mice.

EIR-p should have an enhancing effect on the IgE antibody response. However, when cEIR-p was administered in vivo, we observed a considerable suppression of IgE antibody production, this suppression appeared to reflect the presence of a desirable amount of SFA (also cEIRg) in the cEIR>p preparation. To document the selective existence of EIR-p in supernatant fluids derived from T-cell hybridoma, MBI-2, we compared the in vivo effects of such putative mcEIR-p with cEIR-p.

The experiment in Figure 2 illustrates the well-described transformation of low IgE response SJL mice (group I) into high responders when low dose irradiation precedes antigenic stimulation (group II). Administration of cEIR - - containing a mixture of cEIR-p, cEIRg and SFA - - reversed this irradiation-enhanced IgE response (group III). In contrast, administration of mcEIR-p derived from hybridoma MBI-2 did not have the ability to reverse the high response state of such mice, but even further enhanced such response 4-fold (group IV), and these effects were selective for the IgE responses, while the IgG responses were not affected. These results thus demonstrate that this mcEIR-p preparation, in contrast to cEIR, does not contain any detectable SFA (or other inhibitory factors) that could exhibit inhibitory effects on the in vivo IgE antibody response, thus exhibiting the hypothesized enhanced effects of EIR- p.

Preliminary biochemical and physiochemical characterization of cEIR-p and mcEIR-p

To document the existence of comparable molecular properties of the active constituents present in cEIR-p and mcEIR-p preparations, the following experimental procedures were performed: (a) Both cEIR-p and mcEIR-p are protein structures. cEIR-β preparations were made under serum-free conditions and tested, respectively, for sensitivity to different forms of enzymatic cleavage. As shown in Figure 3, the ability of both cEIR-p and mcEIR-p to induce FcRc expression was inactivated after treatment with all three proteolytic enzymes used - - trypsin, pepsin and pronase - - (groups III, IV, V and XII, XIII, XIV). In contrast, neither cEIR-p nor mcEIRp was inactivated by RNase, DNase, neuraminidase or folifase A2 (groups VI, VII, VIII, IX and XV, XVI, XVII, XVIII). None of these enzymes could directly induce the expression of FcRc at any of the concentrations used b) Molecular sieve chromatograph in (AcA-54 ) of cEIR and mcEIR-p demonstrates that EIR-p has a molecular weight of 45-60 kd -induction of FcRc<+>lymphoid cells .

Serum-free preparations of cEIR (containing cEIR-p, mcEIR-p, SFA and other mediators) and of MBI-2 hybridoma-derived mcEIR were subjected to ACa-54 molecular sieve chromatograph1. The resulting fractions of both cEIR and mcEIRT were assayed for FcRc-inducing activity on unfractionated and T-cell-deprived lymphoid cell populations. This allows us to differentiate between the target cell specificities of such activities, particularly in the presence of EIRg (which induces FcRc expression selectively on B lymphocytes) in such preparations. The results of this comparative analysis are summarized in figure 4.

As shown in panel A of Figure 4, three FcRc-inducing activity peaks can be rare among the different AcA-54 fractions of cEIR when unfractionated lymphoid cells are used as indicator targets, a peak in the 45-60 kd range (the fractions 30- 34 ), another 'in the 30-35 kd range (fractions 41-45), and the last in the 15-20 kd range (fractions 49-52). On T cell-deprived indicator cells, fractions 30-34 (40-60 kd) were inactive, indicating that the FcRc-inducing activities of molecules in this size range are independent of the presence of T cells, which is characteristic of EIR-p . This conclusion was confirmed by demonstrating that the FcRc-inducing activity of fractions 30-34 of cEIR was completely lost upon removal of Lyt-2<+->o.

This conclusion was further documented by the chromatogram of mcEIR-p on the same AcA-54 column (panel B). On unfractionated lymphoid cells, significant induction of FcRctil was achieved with fractions 28-32, which correspond to a molecular weight of approximately 45-60 kd, the same exact size range denoted in panel A for cEIR. These same fractions (28-32) from mcEIR-p could induce FcRc expression on lymphoid cell populations that did not contain Lyt-1<+>, but not on Lyt-2<+> (data not shown) or total T cell deprived (panel B) populations.

The other relevant observations in Figure 4 will relate to the biological activities of certain fractions provided from

AcA-54 column from T-cell-deprived target cells. As shown in panel A, two peaks with such activity were provided on the chromatogram of cEIR - - one corresponding to fractions 40-45, (30-35 kd) and the other corresponding to fractions 49-56, (15-20 kd) - - which thereby indicates the presence of EIRg in such fractions. This clearly illustrates that it is possible to clarify a heterogeneous molecular population in fractions exhibiting activity (eg EIRg) that is not detectable in the non-fractionated starting population (eg cEIR, see Fig. 1) . The lower molecular weight forms of EIRg (eg, 15-20 kd EIRg) were shown to induce T cells in vitro to secrete SFA, and the activity was confirmed using the selective in vitro FcRc assay system not shown graphically ). In contrast, the chromatographic profile provided from mcEIR-p (panel B) lacked any FcRc-inducing activity on T cell-depleted populations thereby further confirming the absence of EIRg. Other studies confirm that EIRg produced in conventional lymphoid cell populations and by EIRg-secreting B-cell hybridomas fall into the same size category as those designated in panel A 1 Figure 4. We thereby conclude that EIR-p belongs to the size range of 45-60 kd and - that EIRg belongs to the size range of 30-35 kd (fractions 40-45) and 15-20 kd (fractions 49-56).

The discovery of these EIRg fractions is very important in the present invention. These isolated fractions have been shown to provide allergy suppressive activity (ie, these molecules suppress IgE antibody synthesis when used together) and are considered to be valuable in therapy.

In one form, the present invention may include the isolated EIRg fractions with a molecular weight of 15-20 kd, or another isolated EIRg fraction with a molecular weight of 30-35 kd, or, equivalently, a mixture of these two isolated fractions.

As shown above, one or both of the isolated fractions have an allergy suppressant activity which was not present in the starting liquids and which has never before existed in any composition whether natural or synthetic. Such isolated EIRg fractions have therapeutic activity and utility not previously known or suggested by previous work.

These isolated fractions are usually used in the form of the EIRg fraction in physiologically active amounts of, for example, a few nanograms per 100 pg. dose in a biologically compatible carrier, such as normal saline, and may be injected or may be prepared in digestible form.

When isolated from the natural fluid, or prepared by hybridoma expression or synthetically, for example using Merrifield's method, the composition which is entirely new separately from intervening fluid constituents, described herein, can be formulated for therapy using conventional pharmaceutical practice.

As used herein, a composition consisting essentially of one or both of the above-described EIRg fractions contains a physiologically active amount of either the 15-20 kd or the 30-35 kd fractions (or a mixture thereof) without intervening natural or other constituents , such as for example the 45-60 kd fraction described above. The composition may, but need not, also contain physiologically compatible diluents, adjuvants, carriers, preservatives, etc.

c) Molecular sieve chromatograph in (AcA-54 ) of cEIR and mcEIR-p demonstrates (1) presence of SFA and SEM in cEIR, and (2)

the absence of such contaminants in mcEIRT- - inhibition of FcRc<+>cell induction.

The same AcA-54 fractions of cEIR and mcEIR-β were also analyzed for the presence of inhibitors of activity on IgE-induced FcRc<+> cells, such activity would be associated with the presence of SFA, EFA, SEM or EEM. As shown in Figure 5, panel A, three inhibitory activity peaks were observed with fractionated cEIR, in a range of 35-40 kd (fractions 38-42), 20-25 kd (fractions 48-51) and 10-15 kd (fractions 57-60), respectively. Identical results were obtained when Lyt-2 cell-deprived indicator cells were used, indicating that EFA alone is not responsible for the activity present in any of these fractions. When total T-cell-depleted indicator cells were used, only the cEIR fractions in the 10-15 kd range (fractions 57-60) exhibited inhibitory activity (panel A). FcRc inhibitory activity of fractions 57-60 was specifically removed by adsorption with IgE-Sepharose, indicating that such activity is caused by the presence of the IgE-binding factors, SEM and/or EEM. These data also document that SFA-like mediators are present in cEIR preparations and migrate as molecules of size 20-25 and 35-40 kd, respectively. -None of the mcEIR-β fractions exhibited inhibitory activity on IgE-induced FcRc expression either on unfractionated or T-cell-deprived lymphoid cell cultures (Fig. 5, panel B). This allows us to determine that only mcEIRT activity is present in the supernatant fluid derived from hybridoma MBI-2 and that neither SFA, EFA nor EIRg are produced by these cells.

Both cEIR-p and hybridoma-derived mcEIR-p can induce Lyt-2 + cells to produce FcRc-inhibitory EEM, but do not induce Lyt-1<+> cells to produce suppressive effector molecule (SEM).

The experiment summarized in figure 6 represents a comparative analysis of the ability of cEIR^ and mcEIR-p - - and likewise appropriately fractions of corresponding activities provided from chromatograms illustrated in figures 4 and 5 - - to induce production of final effector molecules in the cascade , specifically EEM and/or SEM. It should be noted that EIR-p induces Lyt-2<+>T cells to produce EEM, while SFA induces Lyt-1<+>T cells to produce the suppressive effector molecule, SEM. Both SEM and EEM can directly inhibit IgE-induced FcRc expression by the B cells, but at different sites in the cellular cascade, and can thus be differentiated with respect to this.

Lyt-1<+>and Lyt-2<+>cell-depleted primary cultures were exposed to various cEIR-p, mcEIRp, and SFA preparations, and the supernatant fluids from these primary cultures were harvested and analyzed for their ability to directly inhibit IgE-induced FcRc expression by B cells. As illustrated in panel A of Figure 6, IgE-stimulated B cells exhibit enhanced expression of FcRc. But the data presented in panel B illustrate that EEM was produced by Lyt-2<+>T cells exposed to cEIR-p (group I) or mcEIR-p (group IV). Fraction 31 of both cEIR-p and mcEIR-p-Induced production of EEM as indicated by an approximately 50$ Inhibition of FcRc expression (groups II and -V). In contrast, EIR-p-f ratatte fraction 41 to cEIR and mcEIR-p could not induce Lyt-2<+-> cells to make EEM (groups III and VI). One should also note that similar to the data presented in the next experiment (Figure 7), the EEM-like activities induced by cEIR-p or mcEIRT-enriched fraction 31 were completely removed by adsorption onto IgE affinity columns.

The reciprocal experiment in panel C demonstrated that cEIR clearly induces Lyt-2<+>cell-deprived lymphoid cells to produce SEM that can inhibit FcRc induction in the B cells, such as the SFA-enriched (and EIR-β-depleted) fraction 41 (groups VII and IX). However, neither non-fractionated mcEIR-p (group X) nor EIRT-enriched fraction 31 to cEIRT (group VIII) or mcEIR-p (group XI) could induce Lyt-2<+>cell-deprived lymphoid cells to release SEM. However, fraction 41 of mcEIRT (group XII), which corresponds to the EFA-enriched fraction from cEIR (group IX), also lacks the ability to induce production by the SEM bed Lyt-1 cells. We thus conclude that EIR-r-enriched fraction 31 is free of detectable SFA-like activity and that the only EIR-like activity present in these two EIR-r-enriched preparations, and likewise the non-fractionated material from hybridoma MBI-2, it is for EIRT.

cEIR and mcEIR-r do not exhibit IgE-binding affinity, while the effector molecule that they induce (EEM) is clearly an IgE-binding factor.

According to the IgE cascade that has been proposed, EFA stimulates Lyt-2<+>T cells to release EEM, an enhancer molecule that acts in part by (1) inhibiting the development of FcRc<+>B cells, (2 ) block the production of EIRg which entails (3) reducing the synthesis and/or secretion of SFA (J. Immunol. 133:2845). In contrast to EFA (which does not bind to IgE), EEM is an IgE-BF. Since EIRTs are significantly involved in the formation of EEM, it is important to determine whether the relevant -EIR-p activity component is an IgE-BF - - a question that can be asked unambiguously, now that mcEIR-p is available.

The experiment summarized in Figure 7 asks these questions in a comparative way. Panels A and B of Figure 7 illustrate the effects of IgE-Sepharose adsorption on FcRc-inducing activity of cEIRp and mcEIRT, respectively. Significant induction of FcRc expression was observed with a dilution as small as 10"<2> of either untreated cÉIRT or mcEIRp. Stimulation of lymphoid cells with IgE-adsorbed preparations from both regulatory agents provided identical titration curves. We thus conclude from these data that cEIR-p and mcEIRp, like EFA, do not show affinity for IgE and are not IgE-binding factors.

Panels C and D in Figure 7 conclude that the cEIR-p and mcEIR-p preparations can induce the production of an effector molecule that can inhibit IgE-induced FcRc expression in the B cells, and similar to EEM, is an IgE-BF. Thus, Lyt-1 cell-deprived lymphoid cells were cultured for 48 h in the presence of either 10% cEIR-p or mcEEIR-p. The resulting supernatant fluids were divided into two portions, one of which was subjected to IgE adsorption. The various preparations were then tested for the ability to directly inhibit FcRc expression by B cells.

Unabsorbed regulatory agent, induced by either cEIR-p (panel C) or mcEIR-p (panel D) was clearly inhibitory to IgE-induced FcRg expression, causing a 40-45$ suppression of this response at dilutions of 1 :50. The parallel preparations subjected to IgE adsorption, whether induced with cEIR-p or mcEIRp, completely lacked FcRc inhibitory activity. In addition, the supernatant fluid from unstimulated Lyt-1 cell-deprived lymphoid cell cultures, as well as the original EIRβ preparations, similarly lacked FcRc inhibitory activity. We thus conclude that the regulatory agent induced by EIR-p is an IgE-BF, and that probably homologous to that which is induced

-of EFA (ie, EEM).

The untreated and IgE-absorbed EIR-β preparations described in panels A and B of Figure 6 were also compared for the ability to induce Lyt-2 cells to produce EEM. Adsorption of either cEIR-p or mcEIR-p with IgE-Sepharose did not alter the subsequent induction of EEM by either regulatory agent.

When analyzing the different fractions obtained by passing the cEIR over a molecular sieve column, two SFA-like activity peaks were quite unexpectedly observed. The fact that SFA were present in cEIR was not very surprising, since we have previously demonstrated that supernatant fluids derived from IgE-stimulated lymphocyte cultures contain these activities (J. Immunol. 133:2837), but there was no reason to predict two separate SFA - similar functions. The original observations were confirmed by in vivo suppression of IgE antibody responses (Fig. 2), and in vitro by Lyt-1<+>cell-dependent inhibition of IgE-induced FcRc expression (Fig. 5) and induction of Lyt- l<+->cells to produce IgE-BF, SEM (Figure 6). However, the fact that two distinct molecular weight SFA forms were present in such supernatant fluids was unexpected.

As shown in Figure 8, a T-cell hybridoma (designated MBI-1) has been identified that secretes SFA molecules exhibiting a molecular weight characteristic of SFA2, and that such secretion can be dramatically enhanced by stimulation of such cells with EIRg. It can thus be seen in figure 8 that unstimulated MBI-1 cells produce SFA molecules with 35-40 kd, but in low amounts. EIRg-stimulated MBI-1 cells produce this same molecular component, however, to a greatly enhanced extent.

Fractions that could inhibit IgE-induced FcRc expression by isolated B cells were also observed by molecular sieve fractionation of cEIR. The peak of this activity showed an elution pattern typical of molecules between 10-15 kd. The mediator present in these fractions could be removed by being passed through an IgE-Sepharose column indicating that it is an IgE-BF. It is likely that both SEM and EEM were present in this peak of activity.

Lack of detectable B-cell FcRc-inducing activity (EIRg) in unfractionated lymphoid cell-derived EIR observed in Figure 1 is caused by the simultaneous presence of SEM and SFA in these preparations. From the data illustrated in panel A of Figure 4, EIRg exists as molecules of 15-20 kd and as molecules of 30-35 kd. Reconstitution experiments showed that SFA and IgE-BF containing fractions illustrated in Figure 5 could suppress expression of FcRg induced with the EIRg-containing fractions illustrated in Figure 4. The effect of EgF-BF and SFA present in cEIR on the activity of EIRg also present in these preparations, can be distinguished by analysis of the different fractions in Figures 4 and 5. The shoulder in FcRc induction on unfractionated lymphoid cells at fractions 40-42, illustrated in panel A in Figure 4, corresponds to the presence of SFA-like activity in the same fractions as illustrated in panel A of Figure 5. When T cells are removed from the indicator cell population In panel A of Figure 4, a single large EIRg activity peak incorporates the earlier shoulder, recall that for SFA to inhibit FcRc- expression, the Lyt-1+ T cells must be present to produce the final effector molecule SEM. A similar relationship between the activities of SFA and EIRg, illustrated in fractions 49-51 in Figures 4 and 5, is also evident. They thus conclude that EIRg activity is present in non-fractionated cEIR, but that this activity is disrupted by the SFA-like mediators and IgE-BF also present. Since the activity of EIR-β is not altered by the presence of these FcRc inhibitory mediators, only EIR-β-induced FcRe expression is observed with whole spleen cell-derived cEIR (Figure 1).

Table II presents a summary of the experiments relating to the characterization of the secreted product derived from selected B-cell hybridomas. The B-cell hybridoma secreting EIRg produces regulatory agents with biological activity characteristic of either EIRg_ or EIRg_2« B-cell hybridoma 99E9 which was selected on the basis of induction of B-cell FcRc expression may also be shown to stimulate Lyt- 1<+>T cells to produce SFA in vitro. 99E9 cannot be biologically distinguished from EIRg_^produced by polyclonal B lymphocytes upon stimulation with IgE at this time. B-cell hybridoma 2C7, which was also selected on the basis of its ability to induce B-cell FcRc expression, does not induce T-cell secretion of SFA in vitro. EIRg derived from cell line 2C7 has a molecular weight of 30-35 kd, and is thus at the present time not possible to distinguish from EIRg.g derived from IgE-stimulated B cells.

Macrophages play a central role in the EIR cascade

We have reported that SFA acts on Lyt-1<+>T cells to induce production of the suppressive Ige-BF termed SEM ( J. Immunol. 133:2845 ). These original studies were performed with SFA derived from CFA-induced ascites, a preparation expected to exhibit considerable molecular heterogeneity. When we attempted to induce the production of SEM with SFA derived from the in vitro culture of MBI-1 cells, these attempts were unsuccessful. Since MBI-1 cell-derived SFA could inhibit IgE-induced FcRc expression, probably through the induction of SEM in situ, it appeared that IgE may act on another cellular population to initiate the production of an EIR that has the ability to act as co-stimulus with SFA for the induction of SEM-outski11 ing. Since ascites-derived SFA (a preparation capable of inducing secretion of SEM) was provided from a macrophage-rich environment, macrophage-derived EIR was tested for its ability to act on monoclonal SFA to induce secretion of SEM.

Purified T cells (G-10 "passed and panned" on anti-Ig coated plates) were cultured in the presence or absence of SFA, with or without EIR derived from macrophages, and subsequent secretion by SEM followed by application of inhibition of IgE-induced FcRc expression by the B cell as for Figure 6. As shown in Table III, unstimulated T cells could not produce desirable amounts of SEM, whereas T cells stimulated with ascite-derived SFA could (group I vs .group II). Monoclonal SFA (mcSFA) could not mediate SEM production directly (group III), nor could EIR derived from macrophages (group IV). However, when mcSFA was used together with EIR-MØ, SEM production was detected. WE conclude that SFA is necessary, but not sufficient, to induce SEM production, EIR-MØ is necessary as a co-stimulus.

The BALB/c spleen cells were fractionated to enrich T lymphocytes by sequential G-10 Sepharose passage and plating on anti-Ig coated plates. Then the cells were grown to 10<7> cells/ml for 24 hours in either medium alone, in medium containing 10% ascites-derived SFA, 10% supernatant fluid from the MBI-1 cell cultures (eg, SFA). 10$ EIR derived from macrophages [formed by culturing normal peritoneal macrophages for 48 hours in the presence of 10 ; g/ml IgE], or in medium containing 10$ mcSFA and 10$ EIRMØ. Supernatant fluids derived from such stimulated T cell cultures were then tested for a SEM content as described in the text of Figure 6.

The compositions according to the present invention are usually administered to the individual by injection in suitable carrier compositions and adjuvants which are usually known in immunology and pharmacology. Adjuvants from several classes that may be suitable for use in the present invention are described in many publications, see, e.g., Klein, J., IMMUNOLOGY, pp. 361 et. seq.

The following examples illustrate the general compositional criteria that can be used by the person skilled in the art for formulating diagnostic and therapeutic compositions to produce suitable compositions.

Composition No. 1. An isolated EIRg fraction having a molecular weight of 15-20 kd in a solution consisting of normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferably from 1 to 25 ;g /ml, of the isolated fraction, and optionally containing physiologically acceptable preservatives and/or known, physiologically compatible immunological adjuvants, is injected in amounts from 0.1 ml to 5 ml or more intravenously into the individual for allergy response therapy or diagnostics.

Composition No. 2. An isolated EIRg fraction having a molecular weight of 30-35 kd in a solution consisting of normal saline, containing from about 0.01 ;g/ml to about 100 ;g/ml, preferably from 1 to 25 ; g/ml, of the isolated fraction and optionally containing physiologically acceptable preservatives and/or known, physiologically acceptable immunological adjuvants, is injected in amounts from 0.1 ml to 5 ml or more intravenously into the individual for allergy response control therapy or diagnosis.

Composition No. 3.

An isolated SFA2 fraction having a molecular weight of 35-40 kd in a solution consisting of normal saline containing from about 0.01 ;g/ml, to about 100 ;g/ml, preferably from 1 to 25 ;g/ml, of the isolated fraction, and optionally containing physiologically acceptable preservatives and/or known, physiologically compatible immunological adjuvants, is injected in amounts from 0.1 ml to 5 ml or more, intravenously into the subject for allergy response control therapy or diagnosis.

Composition No. 4. An isolated EIR-β fraction having a molecular weight of 45-60 kd in a solution consisting of normal saline containing from about 0.01 µg/ml, to about 100 µg/ml, preferably from 1 to 25 ;g/ml, of the isolated fraction and possibly including physiologically acceptable preservatives and/or known, physiologically compatible immunological auxiliaries, is injected in amounts from 0.1 ml to 5 ml or more, intravenously into the individual in allergy response control therapy or diagnosis.

Composition No. 5. An isolated EIR-MØ fraction, which has the ability to act synergistically with SFA to induce secretion of SEM in a solution consisting of normal saline containing from about 0.01 µg/ml to about 100 µg /ml, preferably from 1 to 25 ;g/ml, of the isolated fraction and optionally containing physiologically acceptable preservatives and/or known, physiologically compatible immunological adjuvants, is injected in amounts from 0.1 ml to 5 ml or more intravenously into the individual by allergy response control therapy or diagnosis.

Composition No. 6. An allergic reaction suppressive composition comprising biologically compatible carriers and diluents and a physiologically effective amount of a composition consisting essentially of an EIRg fraction having a molecular weight of 15-20 kd. The composition typically contains normal saline containing from about 0.01 g/ml to about 100 g/ml, preferably from 1 to 25 g/ml, of the isolated fraction and optionally containing physiologically acceptable preservatives and/or known, physiologically compatible immunological adjuvants, are injected in amounts from 0.1 ml to 5 ml or more intravenously into the individual for allergy response control therapy or diagnosis.

Composition No. 7. An allergic reaction suppressing composition comprising biologically compatible carriers and diluents and a physiologically effective amount of a composition consisting essentially of an EIRg fraction having a molecular weight of 30-35 kd. The composition consists of normal saline containing from about 0.01 g/ml to about 100 g/ml, preferably from 1 to 25 g/ml, of the isolated fraction, and optionally including physiologically acceptable preservatives and/or known physiologically compatible immunological adjuvants which are injected in amounts from 0.1 ml to 5 ml or more intravenously into the subject for allergy response control therapy or diagnosis.

Composition No. 8. An allergic reaction developing composition comprising biologically compatible carriers and diluents and a physiologically effective amount of a composition consisting essentially of the EIR-MØ fraction wherein the composition comprises normal saline containing from about 0.01 ;g/ml to about 100 ; g/ml, preferably from 1 to 25 ;g/ml, of the isolated fraction and possibly including physiologically acceptable preservatives and/or known, physiologically compatible immunological adjuvants, is injected in amounts from 0.1 ml to 5 ml or more intravenously into in the individual by allergy response control therapy or diagnosis.

Composition No. 9. An allergic reaction suppressive composition comprising biologically compatible carriers and diluents and a physiologically effective amount of a composition consisting essentially of an SFAg fraction having a molecular weight of 35-40 kd. The composition includes normal saline containing from about 0.01 g/ml to about 100 g/ml, preferably from 1 to 25 g/ml, of the isolated fraction and optionally includes physiologically acceptable preservatives and/or known physiologically compatible immunological adjuvants , is injected in amounts from 0.1 ml to 5 ml or more intravenously into the individual for allergy response control therapy or diagnosis.

Composition No. 10.

An IgE class-selective potential composition capable of suppressing allergic reactions by virtue of polyclonal stimulation of IgE secretion and thus saturation of mast cell-FcRcmed IgE that does not recognize the allergen in question, comprising biologically compatible carriers and diluents and a physiologically effective amount of a composition essentially consisting of an EIR-p fraction with a molecular weight of 45-60 kd. The composition includes normal saline containing from about 0.01 µg/ml to about 100 µg/ml, preferably from 1 to 25 µg/ml, of the isolated fraction and optionally includes physiologically acceptable preservatives and/or known, physiologically compatible immunological adjuvants, are injected in amounts from 0.1 ml to 5 ml or more intravenously into the individual in allergic response control therapy or diagnosis.

Composition No. 11. A composition consisting essentially of a physiologically compatible carrier and an allergy-suppressing EIRg factor having a molecular weight of 15-20 kd and biological characteristics equivalent to non-IgE-binding EIRg_^ produced by the B cells in response to IgE stimulus and which induces Lyt-1<+> cells to make SFA and Induce B-cell FcRc expression, wherein the composition includes normal saline containing from about 0.01 µg/ml to about 100 µg/ml, preferably from 1 to 25 ;g/ml, of the isolated fraction and optionally including physiologically acceptable preservatives and/or known physiologically compatible immunological adjuvants, which are injected in amounts from 0.1 ml to 5 ml or more intravenously into the individual by allergy response control energy or diagnosis.

Composition No. 12. A composition consisting essentially of a physiologically compatible carrier and an allergy-suppressing EIRg factor having a molecular weight of 30-35 kd and biological characteristics equivalent to non-IgE-binding EIRg2 produced by the B cells in response to IgE- stimulus and which induces B-cell FcRc expression wherein the composition comprises normal saline containing from about 0.01 µg/ml to about 100 µg/ml, preferably from 1 to 25 µg/ml, of the isolated fraction and comprises optionally physiologically acceptable preservatives and/or known, physiologically compatible physiological adjuvants, which are injected in amounts from 0.1 ml to 5 ml or more intravenously into the individual for allergy response control therapy or diagnosis.

Composition No. 13. A composition consisting essentially of a physiologically compatible carrier and an allergy suppressive SFA factor having a molecular weight of 35-40 kd and biological characteristics equivalent to non-IgE binding SFA2 produced by the T cells in response to EIRg_^ stimulus, and which suppresses FcRc expression in vitro and suppresses IgE antibody response in vivo, the composition comprising normal saline containing from about 0.1 µg/ml, to about 100 µg/ml, preferably from 1 to 25 ;g/ml, of the isolated fraction and optionally includes physiologically acceptable preservatives and/or known, physiologically compatible immunological adjuvants, which are injected in amounts from 0.1 ml to 5 ml or more intravenously into the individual for allergy response control therapy or diagnosis.

Composition No. 14. A composition consisting essentially of the EIRg fraction with a molecular weight of 15-20 kd. The composition includes normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferably from 1 to 25 ; g/ml, of the isolated fraction and possibly including physiologically acceptable preservatives and/or known physiologically compatible immunological adjuvants, which are injected in amounts from 0.1 ml to 5 ml or more intravenously into the individual for allergy response control therapy or diagnosis.

Composition No. 15. A composition consisting essentially of EIRg fraction with a molecular weight of 30-35 kd, wherein the composition includes normal saline containing from 1 to 25 ;g/ml to about 100 ;g/ml, preferably from 1 to 25 g/ml, of the isolated fraction and possibly including physiologically acceptable preservatives and/or known, physiologically compatible immunological adjuvants, which are injected in amounts from 0.1 ml to 5 ml or more intravenously into the Individual for allergy response control therapy or diagnosis.

Composition No. 16.

A composition consisting essentially of SFÅ2~ fraction with a molecular weight of 35-40 kd, where the composition contains normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferably from 1 to 25 ;g/ml, of the isolated fraction and optionally includes physiologically "acceptable" preservatives and/or known physiologically compatible immunological adjuvants, which are injected in amounts from 0.1 ml to 5 ml or more intravenously into the subject for allergy response control therapy or diagnosis.

Composition No. 17. A composition consisting essentially of EIR-p-fraction with a molecular weight of 45-60 kd, wherein the composition includes normal saline containing from about 0.1 ;g/ml, to about 100 ;g/ml, preferably from 1 to 25 ;g/ml, of the isolated fraction and optionally includes physiologically acceptable preservatives and/or known, physiologically compatible immunological adjuvants which are injected in amounts of from 0.1 ml to 5 ml or more intravenously into the subject for allergy response control therapy or diagnosis .

Composition No. 18. A lymphocyte-derived composition exhibiting SFA-like activity consisting essentially of EIRg having a molecular weight of 15-20 kd, the composition comprising normal saline containing from about 0.01 µg/ml to about 100 µg/ml, preferably from 1 to 25 ;g/ml, of the isolated fraction and possibly including physiologically acceptable preservatives and/or known, physiologically compatible immunological adjuvants, which are injected in amounts from 0.1 ml to 5 ml or more intravenously into the individual by allergy response control therapy or diagnosis.

Composition No. 19. A lymphocyte-derived composition exhibiting SFA-like activity consisting essentially of EIRg having a molecular weight of 30-35 kd, the composition comprising normal saline containing from about 0.01 µg/ml to about 100 µg/ml ml, preferably from 1 to 25 ;g/ml, of the isolated fraction, and optionally including physiologically acceptable preservatives and/or known physiologically compatible immunological adjuvants which are injected in amounts from 0.1 ml to 5 ml or more intravenously into the individual in allergy response control therapy or diagnosis.

Composition No. 20. A macrophage-derived composition exhibiting EIR-MØ activity upon induction of SEM, wherein the composition comprises normal saline containing from about 0.01 µg/ml to about 100 µg/ml, preferably from 1 to 25 ;g/ml, of the isolated fraction, and optionally includes physiologically acceptable preservatives and/or known, physiologically compatible immunological adjuvants, which are injected in amounts from 0.1 ml to 5 ml or more, intravenously into the individual by allergy response control therapy or diagnosis.

Composition No. 21. A lymphocyte-derived or T-cell hybridoma-derived composition exhibiting IgE class-selective suppressive activity consisting essentially of SFA having a molecular weight of 35-40 kd, the composition comprising normal saline containing from about 0.01 ; g/ml to about 100 ;g/ml, preferably from 1 to 25 ;g/ml, of the isolated fraction, and optionally including physiologically acceptable preservatives and/or known physiologically compatible immunological adjuvants which are injected in amounts from 0.1 ml to 5 ml or more, intravenously into the individual for allergy response control therapy or diagnosis.

Composition No. 22. A lymphocyte-derived or T-cell hybridoma-derived composition exhibiting IgE class-selective enhancing activity, consisting essentially of EIRf having a molecular weight of 45-60 kd, the composition comprising normal saline containing from 0.01 µg/ml to about 100 ;g/ml, preferably from 1 to 25 ;g/ml, of the isolated fraction, and optionally including physiologically acceptable preservatives and/or known, physiologically compatible immunological adjuvants, which were injected in amounts from 0.1 ml to 5 ml or more Intravenously into the individual for allergy response control therapy or diagnosis.

The methods of the invention can be carried out in accordance with good medical and immunological practice using various diluents, carriers, adjuvants, etc. The following methods can be used by the person skilled in the art as general guidelines for preparing the compositions and using the compositions effectively.

Method No. 1. Method for suppressing allergic reactions comprises administering a composition selected from the group consisting of EIRjj_i ,<E>IRg_2, SFA2 or EIR-MØ, in a diluent composition consisting of normal saline containing from about 0.01 ;g/ml to about 100 µg/ml, preferably from 1 to 25 µg/ml, of the isolated fraction, and optionally including physiologically acceptable preservatives and/or known, physiologically compatible immunological adjuvants, which are injected in amounts from 0.1 ml to 5 ml or more intravenously into the individual for allergy response therapy or diagnosis.

Method No. 2. Method for enhancing IgE antibody responses, comprising administering a composition consisting of EIR-r in a diluent composition consisting of normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferably from 1 to 25 ;g/ml, of the isolated fraction, and optionally including physiologically acceptable preservatives and/or known, physiologically compatible immunological adjuvants, which are injected in amounts of from 0.1 ml to 5 ml or more intravenously into the individual by allergy response control therapy or diagnosis.

The compositions of the present invention which are suitable for therapeutic administration will essentially consist of an effective amount of the active ingredient described herein in the general range of from 0.001 ;g/ml to 1000 ;g/ml, or more, usually from one to one hundred micrograms per milliliters of carrier or diluent compositions such as normal saline, distilled water, etc., optionally an effective amount of from 0.0001 to 0.1 adjuvant such as Freund's adjuvant, alum, etc.

In general, administration of the compositions according to the present invention can be carried out by any of the methods described in natural science and clinical literature in which the solution containing the active, isolated fraction is injected into the individual. Procedures such as those described by: Humprey, J.H. & White, R.G., IMMUNOLOGY, For Medical Students, Blackwell, Oxford (1970 ): Natelson, S., Pescve, A. J. , Dietz, A.A. , CLINICAL IMMUNOCHEMISTRY, Am. Assoc. Clin. Chem., Washington, D.C., 1978: Sheldon, J.M., Lovell, R.G., Mathews, K.P., A MANUAL OF CLINICAL ALLERGY, W.B. Saunders, Philadelphia

(1967): Katz, D.H., Benacerraf, Baruj, IMMUNOLOGICAL

TOLERANCE, Mechanisms and Potential Therapeutic Applications, Academic Press, New York, (1982), and Alving, B.M., Finlayson,

J.S., Editors, IMMUNOGLOBULINS: CHARACTERISTICS AND USES OF

INTRAVENOUS PREPARATIONS, U.S. Dept. H&HS, Food and Drug Administration, DHHS Publication No. (FDA )-80-9005, can be used as a basis for special methods within the present invention.

The isolated compositions of the present invention shown in Table 1, for example, are useful for producing polyclonal antibodies according to conventional immunological practice and for producing monoclonal antibodies according to the method of Kohler and Milstein, Nature (256:495) (see also Kennett , R.H., McKearn, T.J. & Bechtol, K.B., MONOCLONAL ANTIBODIES, Plenuk Press, New York, 1980) which have both therapeutic and diagnostic application according to the principles described in the above-cited reference. These antibodies can, for example, be used to purify the compositions according to the present invention and likewise in diagnosis.

The compositions according to the present invention are described in specific detail so that the best method for carrying out and applying the invention is fully described, and the specific criteria and methods should not be restrictive. Active fragments of these compositions are known or expected to exist, and are included within the respective definitions of the inventive compositions.

Industrial application

The present invention is useful for the production of pharmaceutical and diagnostic preparations.

REFERENCES

The following references describe background methods and procedures and underlying principles. 1. Hoover, R., and R. Lynch. 1983. Isotype-specific suppression of IgA: Suppression of IgA responses in BABL/c mice by Tc cells. J. Immunol. 130:521. " 2. Fridman, W. H. , C. Rabourdin-Combe, C. Neauport-Sautes, and R. H. Gisler. 1981. Characterization and function of T cell FcY receptor. Immunol. Rev. 56:51.<*>3. Lowy, I ., C. Brezin, C. Neuport-Sautes, J. Theze, and W. H. Fridman. 1983. Isotype regulatlon of antibody production: T cell hybrids can be selectively induced to produce IgG^and IgGg subclass-specific suppressive immunoglobulin-binding factors. Proe. Nati. Acad. Sei. USA 80:2323.<*>4. Kiyono, H. , J. U. Phillips, D. E. Colwell, S. M. Michalek, W. J. Koopman, and J. R. McGee. 1984. Isotype-specificity of helper T cell clones: Fcc receptors regulate T and B cell collaboration for IgA responses. J. Immunol. 133:1087.<*>5. Adachl , M. , J. Yodoi , N. Noro, T. Masuda, and H. Ochlno. 1984. Murine IgA binding factors produced by FccR<+>T -cell: Role of FcYR<+>cells for the induction of FccR and formation of IgA-binding factor in Con A-activated cells. J. Immunol. 133:65. 6. Marcellettl , J. F., and D. H. Katz 1984. FcRc + lymphoc performance and regulation of the IgE antibody system. I. A new class of molecules termed IgE-induced regulators (EIR), which modulate FcRc expression by lymphocytes. J. Immunol. 133:2821.<*>7. Marcellett, J.F., and D.H. Katz. 1984. FcRc<+>lymphocytes and regulation of the IgE antibody system. II. FcRc<+>B cells initiate a cascade of cellular and molecular interactions which control FcRc expression and IgE production. J. Immunol. 133:2829. * 8. Marcellett, J.F., and D.H. Katz. 1984. FcRc + lymphocytes and regulation of the IgE antibody system. III. Suppressive factor of allergy (SFA) is produced during the in vitro FcRc expression cascade and displays corollary physiological activity in vivo. J. Immunol. 133:2837.<*>9. Marcellett, J.F., and D.H. Katz. 1984. FcRc + lymphocytes and regulation of the IgE antibody system. IV. Delineation of target cells and mechanisms of action of SFA and EFA in inhibiting in vitro induction of FcRc expression. J. Immunol. 133:2845. 10. Ishizaka, K., J. Todoi, M. Suemure, and M. HIrashima. 1983. Isotype-specific regulation of the IgE response by IgE-binding factors. Immunol. Today 4:192.<*>11. Yodoi, J., M. HIrashima, and K. Ishizaka. 1980. Regulatory role of IgE-binding factors from rat T lymphocytes. II. Glycoprotein nature and source of IgE potentiation factor. J. Immunol. 125:1436. 12. Uede, T., K. Sandberg, B. R. Bloom, and K. Ishizaka. 1983. IgE-binding factors from mouse T Lymphocytes. I. Formation of IgE-binding factors by stimulation with homologous IgE and interferon. J. Immunol. 130:649. 13. HIrashima, M., J. Yodoi, and K. Ishizaka. 1980. Regulatory role of IgE-binding factors from rat T lymphocytes. III. IgE-specific suppressive factor with IgE binding activity. J. Immunol. 125:1442. 14. Yodoi, J., M. Suemura, and T. Klshimoto. 1983. FccR and FciR expressed on murine IgE-specific suppressor T hybridomas: Dissociation between suppressor activity and FccR expression. Microbiol. Immunol. 27:877. 15. Katz, D.H., C.A. Bogowltz, and L.R. Katz. 1984. The IgE antibody system: Mature, peripheral B. lymphocytes exert regulatory Influences on the IgE systems of self-reconstituted, sublethally irradiated mice. J. Mol. Cell Immunol. 1:83. 16. Splegelberg, H. , G. Boltz-Nitulescu, J. Plummer, and F. Melewlcz. 1983. Characterization of IgE Fc receptors on monocytes and macrophages. Fed. Pro. 42:126. 17. Splegelberg, H.L., R.D. 0'Conner, R.A. Simon, and D.A. Mathison. 1979. Lymphocytes with immunoglobulin E Fc receptors in patients with atopic disorders. J. Clin. Invest. 64:714. * 18. Hoover, R.G., H.M. Gebel, B.K. Dichgraefe, S. Hickman, N.F. Rebbe, N. Hirayama, Z. Ovary, and R.G. Lynch. 1981. Occurrence and potential significance of increased number of T cells with Fc receptors in myeloma. Immuno.Rev. 56:115. 19. Katz, D. H. 1984. Regulation of the IgE system: Experimental and clinical aspects. Allergy 39:81.<*>20. parker, C. W., T. Schechtel, S. Falkenhein, and M. Huber. 1983. Induction of IgE receptors on human lymphocytes. Immunol. Comm. 12:1. 21. Yodoi, J., T. Ishizaka, and K. Ishizaka. 1979. Lymphocytes bearing Fc-receptors for IgE. II. Induction of Fcc-receptor bearing rat lymphocytes by IgE. J. Immunol. 123:455. 22. Chen, S.-S., J.W. Bohn, F.-T. Liu, and D.H. Katz. 1981. Murine lymphocytes expressing Fc receptors for IgE (FcRc). I. Conditions for Inducing FcRc<+>lymphocytes and Inhibition of the inductive events by suppressive factor of allergy (SFA). J. Immunol. 127:166.<*>23. Tung, A.S., N. Chiorazzi, and D.H. Katz. 1978. vRegulation of IgE antibody production by serum molecules. I. Serum from complete Freund's adjuvant immune donors suppresses radiation-enhanced IgE production in low responder mouse strains. J. Immunol. 120:2050. 24. Katz, D.H., and A.S. Tung. 1978. Regulation of IgE antibody production by serum molecules. II. Strain-specificity of the suppressive activity of serum from complete Freund's adjuvant-immune low responder mouse donors. J. Immunol. 120:2060.<*>25. Katz, D. H. 1978. The allergic phenotype: Manifestations of "allergic breakthrough" and imbalance in normal "damping" of IgE antibody production. Immunol. Fox. 41:77. 24. Katz, D. H. 1979. Regulation of IgE antibody production by serum molecules. III. Induction of suppressive activity by allogeneic lymphoid cell interactions and suppression of IgE synthesis by the allogeneic effect. J. Exp. Med. 149:539. 27. Katz, D.H., R.F. Bargatze, C.A. Bogowitz and L.R. Katz. 1980. Regulation of IgE antibody production by serum molecules. VII. The IgE-selective damping activity of suppressive factor of allergy (SFA) is exerted across both strain and species restriction barriers. J. Immunol. 124:819. 28. Katz, D.H., R.F. Bargatze, C.A. Bogowitz, and L.R. Katz. 1979. Regulation of IgE antibody production by serum molecules. IV. Complete Freund's adjuvant induces both enhancing and suppressive activities detectable in the serum of low and high responder mlce. J. Immunol. 122:2184. 29. Katz, D. H. 1980. Recent studies on the regulation of IgE antibody synthesis in experimental animals and man. Immunology 41:1. 30. Zuraw. B. L., M. Nonaka, C. H. 0'Hair, and D. H. Katz. 1981. Human IgE antibody synthesis in the cellular interactions required for enhancement of antibody production by the graft-versus-host reaction. J. Exp. Med. 136:455.<*>38. Katz, D.H., T.E. Bechtold, and A. Altman. Constructlon of T cell hybridomas secreting allogeneic effect

-factor (AEF). J. Exp. Med. 152:956, 1980.<»>

39. Chiorazzi, N., D.A. Fox, and D.H. Katz. 1976. Hapten-specific IgE antibody responses in mice. WE. Selective enhancement of IgE antibody production by low doses of X-Irradiation and by cyclophosphamide. J. Immunol. 117:1629.<*>40. Huff, T.F., T. Uede, M. Iwata, and KJ. Isahlza-ka. 1983. Modulation of the blologic activities of IgE-suppressice factor to the formation of IgE-potentlating factor. III. Switching of a T cell hybrid clone from formation of IgE-suppressive factor to formation of IgE potentiating factor. J. Immunol. 133:1090. 41. Uede, T., and K. Ishizaka, 1984, IgE-binding factor from mouse T lymphocytes. II. Strain differences in the nature of IgE binding factor. J. Immunol. 133:359. 42. Manouvriez, P., and H. Bazin. 1984. in vivo kinetics and nature of rat IgE-bearing lymphocytes after IgE stimulation. J. Immunol. 133:3274. 43. Jardieu, P., T. Uede, and K. Ishizaka. 1984. IgE-binding factors from mouse T lymphocytes. III. Role of antigen-specific suppressor T cells in the formation of IgE-suppressive factor. J. Immunol. 133:3266. 44. Katona, I.M., J.F. Urban, J.A. Titus, D.A. Stephany, D.M. Weegal, and F.D. Flnkelman. 1984. Characterization of murine lymphocyte IgE receptors by flow microfluorometry. J. Immunol. 133:1521. 45. Vender-Mallie, R., T. Ishizaka, and K. Ishizaka. 1982. Lymphocytes bearing Fc receptors for IgE. VIII. Affinity of mouse IgE for FccR on mouse B lymphocytes. J. Immunol. 128:2306. 46. Humphrey, J. H & White, R. G., IMMUNOLOGY, For Medical Students, Blackwell, Oxford (1970).<*>47. Natelson, S., Pesce, A.J., Dietz, A.A., CLINICAL IMMUNOCHEMISTRY, Am. Assoc. Clin. Chem., Washington, D.C, 1978.<*>48. Sheldon, J. M. , Lovell, R. G. , Mathews, K. P., A MANUAL OF CLINICAL ALLERGY, W. B. Saunders, Philadelphia .(1967). * 49. Katz, D. H., Benacerraf, Baruj, IMMUNOLOGICAL TOLERANCE, Mechanisms and Potential Therapeutic Applications, Academic Press, New York (1974).<*>50. Klein, J., IMMUNOLOGY, John Wiley, New York,

(1982).<*>51. Alvlng, B. M., Finlayson, J. S., Editors,

IMMUNOGLOBULINS: CHARACTERISTICS AND USES OF INTRAVENOUS

PREPARATIONS, U. S. Dept. H&HS, Food and Drug Administration, DHHS Publication No. (FDA )-80-9005.<*>52. Kennett, R.H., McKearn, T.J. & Bechtol, K.B., M0N0CL0NAL ANTIBODIES, Plenum Press, New York, 1980.

<*>References Incorporated by reference and upon as disclosing supportive compositions and/or techniques. All references are background references which are incorporated by

reference for disclosure of related compositions and/or techniques, as well as disclosing general background technology.

Claims (46)

1. jisolated EIRB fraction, characterized in that it has a molecular weight of 15-20 kd. 2. Isolated EIRg fraction, characterized in that it has a molecular weight of 30-35 kd. 3. Isolated SFÅ2~ fraction, characterized in that it has a molecular weight of 35-40 kd. 4. Isolated EIR-p-fraction, characterized in that it has a molecular weight of 45-60 kd. 5 . Isolated EIR-MØ fraction, characterized in that it has the ability to act synergistically with SFA to induce flushing of SEM. 6. Allergic reaction suppressing composition, characterized in that it includes biologically compatible carrier and diluents and a physiologically effective amount of a composition consisting essentially of an EIRB fraction with a molecular weight of 15-20 kd. 7. Allergic reaction suppressing composition, characterized in that it includes biologically compatible carriers and diluents and a physiologically effective amount of a composition consisting essentially of an EIRg fraction having a molecular weight of 30-35 kd. 8. Allergic reaction suppressing composition, characterized in that it includes biologically compatible carriers and diluents and a physiologically effective amount of a composition consisting essentially of an EIR-MØ fraction. 9. Allergic reaction suppressing composition, characterized in that it includes biologically compatible carriers and diluents and a physiologically effective amount of a composition consisting essentially of an SFA2~ fraction with a molecular weight of 35-40 kd. 10. IgE class-selective potentiation composition, characterized in that it can suppress allergic reactions by virtue of polyclonal stimulation of IgE secretion, and thus saturation of mast cell FcRc with IgE that does not recognize the allergens in question, including biologically compatible carriers and diluents and physiologically effective amount of a composition consisting essentially of a -EIR-p fraction with a molecular weight of 45-60 kd. 11. Composition, characterized in that it essentially includes a physiologically compatible carrier and an allergen-suppressing EIRg factor with a molecular weight of 15-20 kd and biological characteristics equivalent to non-IgE-binding EIRg_i produced by the B cells in response to IgE stimulation and which induces Lyt-1 + cells to make SFA and induces B-cell FcRc expression. 12. Composition, characterized in that it essentially includes a physiologically compatible carrier and an allergen-suppressing EIRg factor with a molecular weight of 30-35 kd and biological characteristics equivalent to non-IgE-binding EIRjj2 produced by the B cells in response to IgE stimulation and which induce B-cell FcRc expression. 13. Composition, characterized in that it essentially includes a physiologically compatible carrier and an allergen-suppressing SFA factor with a molecular weight of 35-40 kd and biological characteristics equivalent to non-IgE-binding SFA2 produced by the T cells in response to EIRg_^ -stimulant, and which suppresses FcRc expression in vitro and suppresses IgE antibody response in vivo. 14. Composition, characterized in that it essentially consists of EIRg fraction with a molecular weight of 15-20 kd. 15 . Composition, characterized in that it essentially consists of EIRg fraction with a molecular weight of 30-35 kd. 16. Composition, characterized in that it essentially consists of SFA2~ fraction with a molecular weight of 35-40 kd. 17. Composition, characterized in that it essentially consists of EIRp fraction with a molecular weight of 45-60 kd. 18. Lymphocyte-derived composition, characterized in that it exhibits SFA-like activity consisting essentially of EIRg with a molecular weight of 15-20 kd. 19. Lymphocyte-derived composition, characterized in that it exhibits SFA-like activity consisting essentially of EIRg with a molecular weight of 30-35 kd. 20. Macrophage-derived composition, characterized in that it exhibits EIR-MØ activity upon induction of SEM. 21. Lymphocyte-derived or T-cell hybridoma-derived composition, characterized in that it exhibits IgE class-selective suppressive activity consisting essentially of SFA2 with a molecular weight of '35-40 kd. 22 . Lymphocyte-derived or T-cell hybridoma-derived composition, characterized in that it exhibits IgE class-selective enhancing activity consisting essentially of EIR-r with a molecular weight of 45-60 kd. 23. - Method for suppressing allergic reactions, characterized by administering a composition consisting of one or more of the compositions selected from the group consisting of EIRg^ , EIR-g_2 , SFA2 and EIR-M0. 24 . Method for enhancing the IgE antibody response, characterized in that a composition consisting of EIRj is administered. 25 . B-cell hybridoma line characterized by the fact that it is designated 99E9 which secretes EIRg_^ . 26. B-cell hybridoma line designated 1 99E9, characterized in that it secretes EIRg_2 - 27. B-cell hybridoma line designated 2C7, characterized in that it secretes EIRg_2« 27. The cell hybridoma line is designated MBI-1, characterized by the fact that it secretes SFA2 • 28. T-cell hybridoma line designated MBI-2, characterized by the fact that it secretes EIRf 29. EIR characterized in that it is a non-IgE-binding composition with a molecular weight of 15-20 kd produced by the B cells in response to IgE, which induces Lyt-l <+-> cells to make SFA and induce B cell -FcRc expression. - 30. EIRg_2" characterized in that it is a non-IgE-binding composition with a molecular weight of 30-35 kd produced by the B cells in response to IgE, and which induces B-cell FcRc expression. 31. SFAi , characterized in that it is a protein which is non-IgE binding and which has a molecular weight of 20-30 kd, and which is produced by the T cells in response to EIRg_i and induce Lyt-l <+-> cells to to create SEM. 32. SFA2, characterized in that it is a non-IgE-binding composition with a molecular weight of 35-40 kd produced by Lyt-l <+-> cells in response to EIRg that induces Lyt-l <+-> cells to make SEM . 33. EIRf, characterized in that it is a protein that is non-IgE binding, with a molecular weight of 45-60 kd produced by Lyt-2 <+-> cells in response to enhanced expression of FcRc caused by EIRg that induces Lyt-2 <+-> cells to express FcRc and induce Lyt-2 <+-> cells to make EIRj. 34. EIRj, characterized in that it is an IgE-binding composition with a molecular weight of 10-15 kd produced by the Lyt-2 <+-> cells in response to EIR-p which potentiates IgE synthesis by suppressing continued EIRg.^ production and which thus suppresses endogenous SFA synthesis. 35 . EFA, characterized in that it is a non-IgE-binding composition with a molecular weight of 15-20 kd produced by the T cells that induces the Lyt-2 <+-> cells to -create EEM if IgE is added as a co-stimulant. 36. EEM, characterized in that it is an IgE-binding composition with a molecular weight of 10-15 kd produced by the cells in response to Lyt-2 <+> which potentiates IgE synthesis by suppressing continued EIRg_^ production and thus suppresses endogenous SFA synthesis. 37. EIR-MØ, characterized in that it is a composition produced by macrophages in response to IgE which is necessary as a co-stimulant for the induction of SEM production. 38. Polyclonal or monoclonal antibody to EIRg_i, characterized in that it is a non-IgE-binding composition with a molecular weight of 15-20 kd produced by B cells in response to IgE, which induces the Lyt-l <+-> cells to make SFA and Induce B-cell FcRc expression. 39. Polyclonal or monoclonal antibody to EIRg.g" characterized in that it is a non-IgE binding composition with a molecular weight of 30-35 kd produced by the B cells in response to IgE, which induces B cell FcRc expression. 40. Polyclonal or monoclonal antibody to SFA^ , characterized in that it is a protein that is non-IgE binding and has a molecular weight of 20-30 kd, and is produced by the T cells in response to EIRg_^ and induces Lyt-l <+-> the cells to make SEM. 41. -Polyclonal or monoclonal antibody to SFA2, characterized in that it is a non-IgE-binding composition with a molecular weight of 35-40 kd produced by the Lyt-l <+-> cells in response to EIRg which induces Lyt-l <+- > the cells to make SEM. 42. Polyclonal or monoclonal antibody to EIR-p, characterized in that it is a protein that is non-IgE binding, with a molecular weight of 45-60 kd produced by the Lyt-2 <+-> cells in response to enhanced expression of FcRc caused of EIRg which induces the Lyt-2 <+-> cells to express FcRc and induces the Lyt-2 <+-> cells to make EIRj. 43. Polyclonal or monoclonal antibody to EIRj, characterized in that it is an IgE-binding composition with a molecular weight of 10-15 kd produced by Lyt-2 <+-> cells 1 response to EIRp which potentiates IgE synthesis by suppressing continued EIRg. ^ -production and which thus suppresses endogenous SFA synthesis. 44 . Polyclonal or monoclonal antibody to EFA, characterized in that it is a non-IgE-binding composition with a molecular weight of 15-20 kd T-produced with the T cells inducing the Lyt-2 <+-> cells to make EEM if IgE is added as a co-stimulant. 45 . Polyclonal or monoclonal antibody to EEM, characterized in that it is an IgE-binding composition with a molecular weight of 10-15 kd produced by the cells in response Lyt-2 <+> which potentiates IgE synthesis by suppressing continued EIRg_^ production and which thus suppresses endogenous SFA synthesis. 46. Polyclonal or monoclonal antibody to EIR-MØ, characterized in that it is a composition produced by macrophages in response to IgE which is necessary as a co-stimulant for the induction of SEM production.