This invention relates to immunology generally and specifically to compositions having activity related to the regulation of IgE antibody responses, which may be useful in the treatment of allergic diseases, or parasitic infections.

Background of the Invention Lymphocytes expressing cell surface receptors for the Fc portion(s) of immunogl obul i n molecules (FcR) have been implicated in the regulation of the humoral immune response (J . Immunol . 130:521, Immunol . Rev . 56:51, Proc Nat! . Acad. Sci . 80: 2323, J. Immunol 133 : 1087) . Consistent observations have highlighted the involvement of FcR ⁺ T cells in the i sotype-speci fic regulation of antibody production. The role of lymphoid cells which express FcR for IgE (FcR _e ) in the regulation of IgE antibody synthesis have been extensively studied (J. Immunol. 133 : 2821, 133:2829, 133: 2837, 133:2845, Immunol. Today 4:182). T lymphocytes exert an enhancing isotype-specific influence on IgE antibody synthesis express high levels of FcR _£ , while those which suppress IgE synthesis lack readily detectable cell surface FcR _£ . The role of the B cell in the regulation of IgE antibody synthesis has been recognized recently. B cells which regulate IgE synthesis appear to do so by first acting on distinct subsets of T (J. Immunol. 133 ;2821, 133:2829, 133:2837) .

The exposure of normal lymphoid cells to suitable levels of IgE, either xxx vivo or _in vitro, stimulates enhanced expression of FcR by these cells (J. Clin. Invest. .64.:714, Allergy 39: 81, Immunol. Today 4.: 182) . Increased proportions of FcR _e ⁺ lymphoid cells have been found in humans and experimental animals exhibiting elevated concentrations of serum IgE. JEn Vitro exposure

of human, rat or urine lymphocytes to appropriate concentrations of IgE results in enhanced expression of FcR _e , a significant proportion- of which are, T cells. FcR _e ⁺ T cells release IgE-binding factors (IgE-BF) which potentiate IgE antibody production. T cells which produce IgE-BF with IgE class-selective suppressive activity appear to express relatively little FcR _g , but do express Fc receptors (FcR) for other classes of antibody (e.g., IgG) . The events surrounding FcR _e expression by lymphocytes, which result in the ultimate production of both potentiating and inhibitory species of Ig-BF, reflect activation of a complex cascade of cellular and molecular interactions which, function to maintain normal homeostatic regulation in the IgE antibody system have been reported (J.- ^' Immunol. 133:2821, 133:2829, 133:2837, 133:2845 _f Allergy J39.:81). Some of the molecular intermediaries in this cascade have been termed IgE- Induced Regulants or EIR. The initial inductive event in this cascade is the interaction of IgE with FcR _e ⁺ B ^• cells. This interaction results in the release of the B cell-derived soluble mediator, termed EIR _B , which can directly induce the expression of FcR _e by other B cells, as well as induce Lyt-1 ⁺ T cells to release the IgE isotype-specific suppressor molecule, suppressive factor of allergy (SFA) . SFA has been shown to suppress continued IgE synthesis both ji vivo and in vitro fAllergy 39: 81) . SFA, which itself is not an IgE- binding factor, functions by acting on yet another subset of Lyt-1 ^"1" cells to induce production of a distinct species of IgE-binding factor, denoted suppressive effector molecule (SEM) . SEM is believed to act directly on IgE-producing B cells to inhibit continued IgE synthesis, and has been shown to directly inhibit EIR _B -induced FcR _£ -expression by B cells. SEM

may be identical to the suppressive IgE-BF described by others (J. Immunol. 133:803).

The enhanced expression of FcR _e by B cells exposed to IgE and EIR _B '-also appears to be the stimulus for the enhancing arm of this network, hence, the homeostatic significance of SEM-mediated inhibition of this step described in the previous paragraph (J. Immunol. 133:2829, 133:2837, 133:2845) . The enhancing arm of the cascade is generated when FcR _e ⁺ B cells interact with Lyt-2 ⁺ T cells, an interaction which stimulates such T cells to produce EIR _T . EIR _T selectively induces the expression of FcR _£ by other Lyt-2 ⁺ T cells (it exerts no effects on B cells), which, in turn, produce EIR , so termed because of its inhibitory effects on IgE-induced FcR _g expression by B cells. Enhancing factor allergy (EFA) , another IgE-selective mediator identified by earlier work in this laboratory, functions similarly to EIRrp by inducing Lyt-2 ⁺ T cells to release a mediator termed enhancing effector molecule, or EEM. EFA and EIR _T are nevertheless distinguishable molecular species. Since both EIR _j and EEM are IgE-BF whose actions are biologically indistinguishable, and are likely to be identical entities, we hereafter refer to the effector molecule induced by either EIR _T or EFA as EEM. EEM directly inhibits IgE-induced production of EIR _B by FcR _e ⁺ B cells thereby enhancing the IgE antibody system, since inhibition of EIR _B release ultimately diminishes the production of endogenous SFA.

Monoclonal sources of each respective factor using hybridoma technology have been developed. The biological and physicochemical properties of the monoclonal (mc) factor is compared to those of the conventional (c) factors induced by appropriate stimulation of heterogenous lymphoid cell populations. Comparisons of conventional EIR _T (cEIR _τ ) and monoclonal

EIRrp (mcEIRi _j i) derived from a murine T cell hybridoma are compared hereinafter. EIR _T from either source, either unfractionated or semi-purified by gel filtration, is shown to directly, and selectively, induce FcR _e - expression by Lyt-2 ⁺ cells, and to induce production by these same cells of the IgE-BF termed EEM.

More importantly, in the course of our biochemical fractionation studies, we discovered in preparations which contained cEIRip, two distinct molecular mass species of EIR _B , a finding which was unexpected given our previous observations using unfractionated EIR preparations. One such molecular species of biochemically-enriched EIR _B was shown to have the capacity to induce T cells to make SFA, whereas the other molecular species of EIR _B lacked such capacity. EIR _B in unfractionated form has been shown to exert a suppressive influence on IgE antibody synthesis _in vivo (J . Immunol.. 133:2837) . A heretofore unidentified SFA- like molecule is also disclosed. Finally, in addition to the T cell hybridoma which makes EIR _T , B cell hybridomas which produce EIR _β -.-L and EIR _B _ ₂ , respectively, are described. Also described is a T cell hybridoma which secretes this new species of SFA.

Table I summarizes the various IgE-induced Regulants described in the literature and those discovered by applicants.

TABLE I. EIR _B _! and E1R _B Cascade Products

Hybridoma Product Physical Characteristics Produced By Biological Function . ftTCC No*

EIR _B _ _χ Non-IgE-binding, U-20kd B Cells in Response to Induces Lyt-1 ⁺ Cells to B Cell IgE make SFA

Induces B Cell FcR. - »

Expression

EIR _B _ ₂ Non-IgE binding. 30-3Jkd B Cells in Response to Induces B Cell Fc ^ - ^~ Cell IgE Expression

Unknown nfluence on 0

IgE Synthesis

SFA _A Protein, Non-IgE- ^' Lyt-l* Cells in response Induces Lyt-l ⁺ Cells T Cell binding, 20-30 kd to EIR B, -l to make SEM *

SFA ₂ Non-IgE-Binding, 3J-40kd Lyt-l* Cells in response Induces Lyt-l* Cells ~ Cell to EIR _B to make SEM * SEM IgE-Binding. 10-l5kd Lyt-l* Cells in response Suppresses IgE Synthesis ~ Cell to SFA by B Cells

Inhibits EIR _B -induced » FcRt-expression by B cells

EIR _j . Protein. Non-IgE- Lyt-2 ⁺ Cells in response Induces Lyt-2* Cells to T Cell binding. 4J-60kd to the Enhanced Express¬ Express FcR^ ion of F Rf caused by Induces Lyt-2* Cells to 0

EIR _B make EIR _j

EIR _T IgE-B inding, 10-Ukd Lyt-2 Cells in response Potentiates IgE Synthesis to EIR _j , by suppressing continued ^EIR B-l production and thus suppresses endogenous SFA synthesis

EFA Non-IgE-Binding. 15-20kd T-Cells Induces Lyt-2* Cells to T Cell make EEM if IgE is added as co-stimulus

EEM Identical to EIR _T Lyt-2* Cells in response Identical to EIR _j to EFA plus IgE

EIR-Kβ Unknown Macrophages in response Needed as a co-stimulus to IgE for Induction of SEM

Production

• Cell lines on permanent deposit in the American Type Culture Collection in compliance with the Budapest Treaty on cell line deposit preservation and availability under stated accession number.

"The regu lants produced by IgE-st imul at ed lymphocytes , including those described herein , are described below:

I . B cells exposed to appropriate concentrations of

IgE produce B cell-derived IgE-induced regulants (EIR _B ) . Two species of EIR _B are described .

EIR _B _ι

— produced by B cells in response to stimulation with IgE

also produced by a recently identified B cell hybridoma

— induces other B cells to express Fc receptors for IgE (FcR _e )

induces Lyt-1 ⁺ T cells to make SFA (as defined by inhibition of FcR _e -expression)

— Inhibits IgE synthesis j-ri vivo, presumably by inducing SFA

— 15-20 kd in size

EI B-2

— produced by B cells in response to stimulation with IgE

— produced by a recently identified B cell hybridoma

— induces B cells to express FcR _e

— does not induce Lyt-1 ⁺ cells to make SFA (as

defined by inhibition for FcR _£ -expression)

appears to inhibit IgE synthesis jLn vivo (mechanism of action unknown)

30-35 kd in size

II. The enhanced expression of FcR _e induced in B cells exposed to EIR _B somehow stimulates Lyt-2 ⁺ T cells to make the T cell-selective EIR _T . ^' The net effect of ElRi production is the suppression of continued EIR _B release, which inhibits subsequent induction of SFA and facilitates IgE synthesis.

EIR _T

produced by Lyt-2 ⁺ T cells in response to interaction with EIR _B -activated FcR _£ ⁺ B cells

— produced by a T cell hybridoma line

induces Lyt-2 ⁺ T cells to express FcR _e

induces Lyt-2 ⁺ cells to release an IgE-binding molecule denoted enhancing effector molecule,

EEM (EEM has also been termed EIR _j )

enhances IgE synthesis in an isotype-specific manner through the action of EEM

EIRrp is 45-60 kd in size

EIRrp is not an IgE-binding factor

EEM

— produced by Lyt-2 ⁺ T cells in response to stimulation with EIR _T — produced by Lyt-2 ⁺ T cells in'Tresponse to stimulation with EFA plus IgE

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

— functions to inhibit IgE-induced EIR _B _ ₁ production, and thus inhibits production of

SFA synthesis triggered by elevated levels of IgE.

III. SFA acts on Lyt-1 ⁺ cells, in the presence of EIR- M , to induce the production of a suppressive effector molecule (SEM) . ' One of the functions of SEM is to inhibit EIR _B -induced FcR _e -expression by B cells. This SEM-mediated inhibition can, therefore, usurp induction of EIR _T and, thus, potentiates continued EIR _B production, facilitating continued SFA production. Such factors can directly inhibit B cells from producing IgE. SEM-containing fluids can also function in this manner. It remains to be determined if it is the same 10-15 kd molecule described below. SFA _j (described hereinafter)

— produced by Lyt-1 ⁺ cells in response to stimulation with EIR _B _ ₁

— acts on Lyt-1 ⁺ cells to induce production of SEM

— inhibits IgE-induced FcR _£ -expression by lymphocytes through the action of SEM

believed to inhibit IgE synthesis by B cells through the action of SEM

— 20-30 kd in size

S_FA ₂ (described hereinafter)

biologically identical to SFA_ ₁ , but is larger in size (i.e. , SFA_ ₂ is 35-40 kd, while SFA_ ₁ is 20-30 kd)

— produced by a recently identified T cell hybridoma

SEM

— produced by Lyt-1 ⁺ T cells and Lyt-1 ⁺ B cells stimulated with SFA plus EIR-M0

binds to IgE

inhibits EIR _B -induced FcR _e -expression by B cells

— appears to inhibit IgE synthesis by B cells

10-15 kd in size

EIR-M0 produced by macrophages in response to stimulation with IgE

needed as co-stimulus for induction of SEM by SFA

Abbreviations The following abbreviations are used herein: B cell - bone marrow-derived lymphocyte BSA - - bovine serum albumin C - complement

DMEN - Dulbecco's modified Eagles ^! s medium

DNP - 2,4-dinitrophenyl

EEM - enhancing effector molecule

EFA - enhancing factor of allergy EIR - IgE-induced regulant

EIR _B - B cell-selective IgE-induced regulant EIRp - inhibitory IgE-induced regulant EIRp - T cell-selective IgE-induced regulant EIR-M - EIR derived from macrophages cEIR - conventional lymphoid cell-derived EIR mcEIRrp - monoclonal T cell hybridoma-derived

EIR _T FcR - cell surface receptors for Fc portions of immuno-globulin molecules FcR ^{+ *} - indicative of exhibiting cell surface

FcR FcR _e - Fc receptor for IgE

FcR _e ⁺ - indicative of exhibiting cell surface

FcR _e FCS - fetal calf serum

IgE-BF - IgE-binding factor

MEM - minimum essential medium

PCA Reaction - passive cutaneous anaphylaxis

RAME - rabbit anti-mouse IgE antibodies SEM - suppressive effector molecule

SFA - suppressive factor of allergy

SRBC - sheep red blood cells T cell - thymus-derived lymphocyte TNP - trinitrophenyl

SUMMARY OF THE INVENTION Four new molecular isolates which are novel in isolated form and unpredictable from prior knowledge are disclosed as are hybridoma cell lines which produce each of the respective factors.

As a composition of matters, one facet of the invention ' consists essentially of the isolated EIR _B fraction having a molecular mass of 15-20 kd, or a molecular mass of 30-35 kd, or both fractions in combination, but unseparated from, for example, as found in natural fluids.

Further, the invention also comprises the regulant denoted SFA ₂ which in isolated form consists of a molecule exhibiting a molecular mass of 30-40 kd. SFA ₂ functions as a molecular ' intermediary in the suppressive aspect of IgE class-selective regulation.

Another aspect of the invention consists of the regulant EIRp, a molecular intermediary in the potentiating or enhancing component of IgE class- selective regulation. EIRp is previously unidentified molecule, which exhibits a molecular mass of 45-60 kd.

The invention also comprises compositions consisting of the aforesaid 15-20 kd EIR _B fraction, the

30-35 kd EIR _B fraction, the 30-40 kd SFA ₂ fraction, or the 45-60 kd EIRp fraction in a physiologically compatible carrier.

Finally, the invention includes the respective hybridoma cell line from which each of the aforesaid regulants can be derived, which includes 1) the B cell hybridoma denoted 99E9 which secretes EIR _B _ _1/ 2) the B cell hybridoma denoted 2C7 which secretes EIR _B _ ₂ , 3) the T cell hybridoma denoted MBI-1 which makes SFA and 4) the T cell hybridoma denoted MBI-2 which produces EIRp. Also being included in the invention is a regulant produced by IgE-stimulated macrophages (EIR-

M ) , which is a required co-stimulus for SFA-induced SEM production.

Brief Description of the Drawing Figure 1 graphically depicts the results of experiments which demonstrate that both the cEIRp derived from heterogeneous lymphoid cells and the mcEIRp derived from the T cell hybridoma MBI-2 induce only Lyt-2 ⁺ cells to express FcR _e . Normal CAF-_ 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. Subsequently, the cells were washed and cultured in either medium alone, medium containing 50% mcEIRpi derived from T cell hybridoma MBI-2 or 50% cEIRp. derived from normal lymphocytes (which in this experiment is in the form of unfractionated lymphocyte- derived cEIR) . After 16 hr incubation, the cells were harvested, washed, and assayed for FcR _e ⁺ 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 bars indicate standard errors.

Figure 2 graphically depicts results of experiments which show that the mixture of mediators in cEIR inhibits, while T cell hybridoma-derived mcEIRp

^" enhances, IgE antibody responses of SJL mice. Groups of

SJL mice were either not irradiated (group I) or given

250 rads (groups II to IV) just prior to carrier preimmunization with 2 ;g of KLH in 4 mg alum on day -7. On days -1 and 0, 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 the supernatural fluid from cultures of T cell hybridoma MBI-2 (mcEIRp) . Each injection consisted of 0.1 ml of supernatant fluid (diluted to 0.5 ml in

saline) , given 4 times spaced at 8-16 hr intervals over a 48-hr period. On day 0, all mice were immunized with 2 ;g of DNP-KLH in 2 mg of alum. The day 10 primary anti-DNP IgE responses were measured by PCA reactions, and the anti-DNP IgG responses were determined by solid- phase radioimmunoassay. There were no statistically significant differences in IgG anti-DNP antibody responses among the various groups which were: 11.6 (1.20), 9.8- (1.60), 10.3 (1.30) and 8.5 (2.10) ;g/ l for groups I to IV, respectively.

Figure 3 graphically depicts data showing that both cEIRp and mcEIRp are protein structure. Normal CAF-L spleen cells were cultured in either medium alone, in medium containing 2% untreated EIRp, or 2% EIRp which had been treated with the indicated enzyme. After 8 hr incubation, the cells were harvested, washed and assayed for FcR _e ⁺ cells as described above respecting Figure 1.

Figure 4 graphically depicts molecular sieve chromatography data (AcA-54) of cEIR and mcEIRrp which demonstrates that EIRp. has a molecular mass of 45-60 kd — induction of FcR _e ⁺ lymphoid cells, and the isolation and separation of -15-20 kd and 30-35 kd EIR _B fractions which have been shown to possess allergy suppressive activity. Normal CAF _j spleen cells were either unfractionated (C only) , or depleted of T cells by anti-0-dependent complement-mediated cytotoxicity. The cells were subsequently 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 mcEIR _p (panel B) . After 8 hr the cells were harvested and assayed for FcR _e ⁺ cells as described above respecting Figure 1.

Figure 5 graphically depicts molecular sieve chromatography data (AcA-54) of cEIR and mcEIRp which

demonstrates, 1) the presence of SFA and SEM in cEIR, and 2) the absence of such contaminants in mcEIRp — inhibition of FcR _e ⁺ cell induction. Normal CAF _j spleen cells either unfractionated (C only) , or depleted of T cells with anti-0 plus C were cultured in either of the above stated 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 mcEIRp plus 10 ;g/ml IgE (panel B) . After-24 hr, the cells were harvested and assayed for FcR, ⁺ cells as described respecting Figure 1. The data are expressed as the percent inhibition of IgE-induced FcR _e - expression, with unfractionated and T cell-depleted lymphoid cells cultured in the absence of IgE.exhibiting 1.0 (1.41) and- 3.0 (1.11) percent FcR _£ ⁺ cells, respectively, and control IgE stimulated cultures exhibiting 18.4 (1.03) and 29.0 (1.05) percent FcR _e ⁺ cells, respectively. Figure 6 graphically presents data showing that both EIRp and hybridoma-derived mcEIRp can induce- Lyt-2 ⁺ cells to produce the FcR _e -inhibitory EEM, but do not induce Lyt-1 ⁺ cells to produce SEM. Normal CAF _j spleen cells were depleted of T lymphocytes with anti-ø plus C, and cultured in either medium alone, in medium containing 10 ;g/ml IgE plus 20% supernatant fluid from cultures of Lyt-1 ⁺ cell-depleted lymphoid cells stimulated with the indicated regulant (panel B) , or in medium containing 10 ;g/ml IgE plus 10% supernatant fluid from culture of Lyt-2 ⁺ cell-depleted lymphoid cells stimulated with the indicated regulant (panel C) . After 24 hr incubation, the cells were harvested, washed, and assayed for FcR _e ⁺ cells as described in the respecting Figure 1. The data are expressed as the percent inhibition of FcR _e -induction using the values

indicated in panel A as controls.

Figure 7 graphically depicts data showing that EIRp and mcEIR _p do not exhibit IgE-binding affinity, whereas the effector molecule they induce (EEM) is clearly an IgE-binding factor. Normal CAF _j spleen cells, either unfractionated (panels A and B) or T cell- depleted with anti-0 plus C (panels C and D) , were cultured in either medium alone, in medium containing the indicated dilution of either untreated or IgE-adsorbed cEIRp, or mcEIRp ^' (panels A and B) , in medium containing 10 ;g/ml 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 passage of 2 ml of EIR on a 2 ml IgE-Sepharose column constructed with 5.25 mg affinity-purified anti-DNP IgE per gram of Sepharose 4B. After 8 hr incubation (panels A and B) , or 24 hr incubation (panels C and D) , the cells were harvested, washed, and assayed for FcR _e ⁺ cells as described respecting Figure 1. The data in panels C and D are expressed as the percent inhibition of IgE-induced FcR _e -expression, with lymphoid cells cultured in the absence of IgE exhibiting 5.3 (1.18) percent FcR _e ⁺ cells and control IgE-stimulated cultures exhibiting 32.2 (1.04) percent FcR _e ⁺ cells.

Figure 8 graphically presents data demonstrating that the cell line MBI-1 produces SFA activity characteristic of SFA ₂ , and that this cell line can be induced to secrete enhanced levels of this latter regulant by stimulation with EIR _B . 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 (EIR _B ) . After 30 hrs incubation, the supernatant fluids from such cultures were harvested, and four ml from each culture

was separately passed over the ACA 54 molecular sieve column. Normal CAF-L spleen cells either depleted of Lyt-2 ⁺ . T cells (Figure 6) with anti-Lyt-2 antibodies plus C, or depleted of all T cells (not shown) were cultures in either medium alone, or in medium containing varied dilutions of the indicated column fraction plus 10 ;g/ml IgE. After 24 hr, the cells were harvested and assayed for FcR _e ⁺ cells as described respecting Figure 1. The data are expressed as the last dilution of each fraction which could exert at least 35% inhibition of IgE-induced FcR ₆ expression.

Description of the Preferred Embodiment

Methods Animals - SJL, BALB/C and (BALB/C X /J)F-L (CAF-J) mice were obtained from the Jackson Laboratories, Bar Harbor, ME, and maintained at the breeding colonies at the Medical Biology Institute, La Jolla, CA. Adult male Lewis rats were purchased from Holtzman Co., Madison, I.

Proteins and Chemicals

The reagents used were purchased or prepared as follows: Bovine serum albumin (BSA) , DNase, phospholipase A ₂ , 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) — ICN 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) ₈ -BSA was prepared as previously described (J. Immunol.. 124:2728) .

Monoclonal and Conventional Antibodies and Complement.

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

Monoclonal anti-Thy 1.2 (anti-0) , derived from F7D5e7 hybridoma clone (Eur. J. Immunol. 9.:825) was obtained as an ascites fluid and clarified by centrifugation prior to use. Monoclonal anti-Lyt-1.2 and monoclonal anti-Lyt-2.2 were purchased from Accurate Chemical and Scientific Corp. , Westbury, NY, reconstituted as directed, and stored at -20°C until needed. Guinea pig complement (C) was obtained from Pel-Freeze Biologicals, Rogers, AR, and extensively adsorbed at 4°C with CAF- _j _ spleen cells prior to use.

Preparation of Lymphoid Cells, and Depletion of Specific Subpopulations Prior to Induction of FcR _e .

Spleens were dispersed gently in cold minimum essential medium (MEM) in a tissue grinder. Subsequently, 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 FcR _£ as described in the next section, or resuspended in MEM for depletion of Lymphoid subpopulations as described immediately below.

T cells were depleted prior to FcR _£ induction by two sequential treatments of lymphocytes with monoclonal anti-0 plus C (J. Exp. Med. 136:455) . Selective treatments with anti-Lyt-1.2 and anti-Lyt-2.2 monoclonal antibodies was conducted in the same manner. Control cells were treated with C only. The efficiency of lymphocyte subpopulation depletion by anti-ø treatment was greater than 95%, as determined by

susceptibility to lysis with monoclonal an i-ø plus C, and immunofluorescence (J. Immunol. 133:2821).

Normal peritoneal macrophages were obtained by washing peritoneal cavities of mice with PBS, and were isolated by adherence to tissue culture flasks. The peritoneal cells were plated at a density of 3 x 10 ^s cells/ml in DMEM, and non-adherent cells eliminated by washing after 3 hours incubation at 37°C. In Vitro System-for Induction of FcR _£ ⁺ Lymphocytes Unf actionated spleen cells or cells fractionated as described above were resuspended in DMEM plus 10% FCS and antibiotics. The lot of FCS employed was specifically selected for compatibility with EIR- induced FcR _e -expression. The cells were adjusted in concentration such that, unless otherwise stated, a final cell density of 10 ⁷ nucleated cells/ml was achieved after addition of monoclonal IgE, and/or EIR- contain'ing culture supernatant fluids or column fractions. The cells were plated in Linbro 24-well macroplates such that a final volume of 0.5 ml/well was achieved after addition of monoclonal IgE, SFA or EFA, or culture supernatant- fluids or additional medium. The cultures were then incubated in a 10% C0 ₂ atmosphere for the indicated time period, after which the cells were harvested and analyzed for frequencies of FcR _£ ⁺ cells, using the rosette assay described below.

Assay for Rosette Formation a) Preparation of Indicator Cells Sheep red blood cells (SRBC) were obtained from the blood of a single sheep maintained at the Medical Biology Institute animal facility. TNP- derivatized SRBC were prepared by reaction with TNBS as described previously (J. Immunol. 127:166) . The IgE- coated TNP-SRBC were prepared by incubating the red cell

suspension (1% in MEM) with subagglutinating amount of DNP-specific monoclonal antibodies (5-10 ;g/ml) , determined in pilot experiments to give optimal red cell sensitization for detection of maximum numbers of FcR _g ⁺ 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% suspension in MEM. b) Preparation of Rosettes Fifty ;1 of lymphocytes (2.5 x 10 ⁶ cell ^' s), prepared as described above, were added to 12 x 75-mm Falcon tubes that contained 100 ;1 of appropriate indicator cells-. The cell mixtures were then centrifuged at 500 rpm for 5 minutes. After unperturbed incubation overnight at 4°C, the cell pellets were gently resuspended either with a 100 ;1 Eppendorf or by vortexing. Trypan blue was added to each tube just before enumeration of rosettes, the viability of cultured cells was routinely 60% or more. As a rule, the number of rosettes in approximately 250-300 viable small lymphocytes in each sample pool was counted 3 or 4 times, and the geometric means and standard errors. were calculated from these values. However, in the case of screening multiple column fractions, only a single count of approximately 300 viable cells was made per sample pool. Since the regulant in question was contained in multiple and consecutive column fractions, changes noted in the level of FcR _e -expression in consecutive cultures were considered an accurate reflection of EIR content. A rosette-forming cell is defined as a small lymphocyte that is tightly surrounded by at least 3 indicator cells, although most rosettes displayed more than the minimal number of indicator cells. Preparation of IgE-induced Regulants (EIR) . The protocol for producing EIR has been

described (J. Immunol. 133:2821) . Briefly, normal spleen cells (10 ⁷ /ml) , either unfractionated or fractionated into various B or T lymphocyte subpopulations, or peritoneal macrophages, were cultured in the presence of 10 ;g/ml of monoclonal IgE (monomeric form) for 24 hr as described above for IgE-induced FcR,- expression. The supernatant fluids from these cultures were harvested 24 hr later, depleted of residual IgE by passing over RAME-Sepharose affinity columns, and ^' the (RAME-adsorbed) effluent fractions " from the affinity columns retained for testing for biological activity. 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, B 5147, with alloactivated T cell blasts from DBA/2 donors as described in detail previously (J. Exp. Med. 152:946, 1980) , and were maintained at -70°C at the Medical Biology Institute. B cell hybridomas were constructed by fusion of the SP2/0 B cell myeloma with in vivo antigen-primed 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 supernatant fluids from those wells showing growth within 10 to 14 days were screened for the presence of

EIR in the .in vitro FcR _e -induction assay as described below for screening of molecular sieve column fraction. The hybridoma clones used in the present study was found to be free of myccplasma by analysis with Vero cells and fluorescent DNA staining with Hoechst 33258.

Gel Filtration

Four ml of EIR-containing culture supernatant fluid was applied to a column of Ultrogel AcA-54 (2.6 x 97 cm) equilibrated with phosphate-buffered saline

containing 0.01% PEG. Elution was carried out with the same buffer at 4°C, and 2.5 ml fractions were collected for assay. 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 the gel filtration of whole spleen cell EIR or monoclonal EIR was assayed for the presence of the FcR _£ -inductive EIRp and EIR _B , and the FcR _£ -inhibiting SFA, EFA and IgE-binding factors SEM and EEM. In the former assay, 50 ;1 of each AcA-54 fraction (or buffer) was added to 0.45 ml culture media containing 5 x 10 ^δ of various types of lymphoid cells as indicated. After 8 hr incubation, the cells were harvested and assayed for FcR _£ ⁺ cells as described above. Significant induction of FcR _£ ⁺ cells in both unfractionated and T cell-depleted lymphoid cell cultures is indicative of the presence of EIR _B in that fraction. Induction of FcR _£ in only the unfractionated (T cell-containing) lymphoid cell culture is indicative of the presence of EIRp in the fraction being tested.

In the latter assay of inhibition of IgE-induced FcR _e - expression, 50 ;1 of each AcA-54 fraction (or buffer) was added to 0.45 culture media containing 5 ;g monoclonal IgE plus 5 x 10 ⁶ of the indicated lymphoid cells. In this assay, significant inhibition of IgE- induced FcR _£ -expression in unfractionated and Lyt-2 ⁺ cell-depleted , but not T cell-depleted indicator cells, is indicative of the presence of SFA. Inhibition of FcR _£ -induction only in the unfractionated lymphoid cell cultures, and not in the Lyt-2 ⁺ cell-depleted or total T cell-depleted cell cultures, indicates the presence of EFA. Finally, inhibition of IgE-induced FcR _£ -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 Regulants

The ability of EIR to selectively modulate in vivo IgE synthesis was ascertained using the irradiation-enhanced primary antibody response in SJL mice (J. Immunol. 120:205Q, J. Immunol..120:2060) . Briefly, on day -7 SJL mice were exposed to 250 rads and then carrier primed with 2 ;g KLH adsorbed on 4 mg aluminum hydroxide gel (alum) . On days -1 and 0, the - animals were injected intravenously with the indicated regulant, each injection consisting of 0.1 ml of supernatant fluid diluted to 0.5 ml with saline, given 4 times spaced at 8-16 hr intervals over a 48 hr period. Intervening these injections, on day 0, the mice were immunized with 2 ;g DNP-KLH in 2 mg alum. Mice were bled from the retroorbital plexus at indicated intervals thereafter and their sera assayed for IgE and IgG anti- DNP antibody contents as described previously (J. Immunol. 124:2727, J. Immunol. 117:1629).

Enzymatic Digestion of EIRp

Enzymes were used at the following final concentrations for the digestion of EIR _T : trypsin, pepsin, pronase and phospholipase A ₂ were used at 1 mg/ml, RNase at 100 ;g/ml, DNase at 1 ;g/ml, and neuraminidase at O.lU/ml. 100 ;1 of a lOx enzyme solution was added to a tube containing 900 ;1 of serum- free EIR, or tissue culture medium only. The reaction mixture was incubated at 37°C for one hour, after which 100 ;1 FCS was added. The treated EIR _T preparations, and controls, were then tested for residual FcR _£ - inductive activity at a final dilution of 1:50.

Summary of the Molecular Mediators in the IgE Cascade Heterogeneous lymphoid cells of the normal

mouse spleen produce a mixture of IgE-selective regulatory mediators following exposure to IgE xxx vitro (J. Immunol. 133:2821, 133:2829, 133:2837, 133:2845) . Because of their contrasting, as well a ^' s synergistic effects, the presence of some of these mediators tends to obscure or modify the biological activities of the others, making it necessary to use selective subsets of lymphoid cells in order to produce and/or detect the activity of each respective mediator. We have termed this mixture of mediators conventional EIR (cEIR) . Such preparations have been shown to exhibit the following distinguishable biological -activities: (1) EIRp, an activity able to induce Lyt-2 ⁺ cells to express FcR _£ and produce EEM, (2) EEM, and IgE-BF which can suppress IgE-induced EIR _B production, an event manifest as an inhibition of B cell FcR _£ expression, (3) SFA, a molecule capable of inducing Lyt-1 ⁺ cells to produce SEM, and (4) SEM, an IgE-BF exhibiting the capacity to inhibit EIR _B -in'duced FcR _£ expression by B cells, again, like.EEM, detectable in its ability to inhibit IgE-induced FcR _£ expression by isolated B cells. As described herein, EIR _B , a mediator(s) able to induce B cell FcR _£ expression, and able to induce Lyt-1 ⁺ T cells to make SFA is also contained in cEIR. [Thus, cEIR = cEIRp + cEIR _B + cSFA + cSEM + cEEM] .

Constitutive Production of an EIR-Like Activity by T Cell Hybridoma MBI-2. As indicated above, supernatant fluid derived from cultures of IgE-stimulated lymphoid cells contains the full spectrum of EIR activities, including EIRp. For this reason we elected to exploit the limited heterogeneity of product secreted by hybridoma T cell lines to augment this analysis of EIRp.

Examination of 200 clones of T cell hybridomas for production of EIRp revealed 8 that constitutively secreted appreciable quantities of regulant capable of inducing the expression of FcR _£ by unfractionated lymphoid cells. The soluble product (mcEIRp.) of one such clone (MBI-2) was selected for further analysis and comparison with cEIRrp derived from heterogeneous lymphoid cell populations.

Both cEIRpi Derived From Heterogeneous Lymphoid Cells and the mcEIR<p Derived from T Cell Hybridoma MBI-2 Induce Only Lyt-2 ⁺ T Cells to Express FcR _£ .

The experiment summarized in Figure 1 illustrates that the cellular targets involved in the expression of FcRg resulting from exposure to either cEIRp. or mcEIRp are absolutely identical in both cases. Consistent with our previously reported results obtained with cEIRp. alone, both cEIRp. and mcEIRp induced FcRg expression in both unfractionated (Groups II and III) , but not in entire T cell-depleted (Groups V and VI) , lymphoid cells. When lymphoid cells selectively depleted of T cell subsets were examined, both cEIRp and mcEIRp. readily induced FcR, expression in Lyt-l ^"1" cell- depleted (Groups VIII and IX) , but not in Lyt-2 ⁺ cell- depleted (Groups XI and XII) , lymphoid cells. These results illustrate that both cEIRp. and mcEIRp induce Lyt-2 ⁺ T cells to express FcR _£ membrane markers.

The Mixture of Mediators in cEIR Inhibits, While T Cell Hybridoma-Derived mcEIRp Enhances, IgE antibody

Responses of SJL Mice.

EIRp. should have an enhancing influence on IgE antibody responses . However, when cEIR _p was administere ^' d j-n. vivo , we observed a significant suppression of IgE antibody production, this suppression

was shown to reflect the presence of appreciable quantities of SFA (as well as cEIR _B ) within the preparation of cEIRp. In order to document the selective existence of EIRp in supernatant fluids derived from the T cell hybridoma, MBI-2, we compared the .in. vivo effects of such presumed mcEIRp to those of cEIRp.

The experiment in Figure 2 illustrated the well-described conversion of low IgE-responder SJL mice (Group I) into high responder animals when low-dose irradiation precedes antigenic stimulation (Group II) . Administration of cEIR — which contains a mixture of cEIRp, cEIR _B and SFA — reversed this irradiation- enhanced IgE response (Group III) . In contrast, administration of mcEIRp derived from hybridoma MBI-2 not only failed to reverse the high responder state of such mice but, actually, further enhanced such responses 4-fold (Group IV) , such effects were selective for IgE responses, IgG responses being unaffected. . These results thus demonstrate that this preparation of mcEIR _p , in contrast to cEIR, contains no detectable SFA (or other inhibitory factors) which can exert inhibitory effects on xxx vivo IgE antibody responses, and thus displays the predicted enhancing effects of EIRp.

Preliminary Biochemical and Physicochemical Characterization of cEIRp and mcEIRp

In order to document the existence of comparable molecular properties of the active entities present in preparation of cEIRp and mcEIR _p , the following experimental procedures were performed:

(a) Both cEIRp and mcEIRp are Protein Structures.

Preparations of cEIRp were generated under serum-free conditions and tested, respectively, for

sensitivity to various forms of enzymatic digestion. As shown in Figure 3, the abilities of both cEIRp and mcEIR _p . to induce FcR _£ expression were inactivated by prior treatment with all three of the proteolytic enzymes employed — trypsin, pepsin and pronase — (Groups III, IV, V and XII, XIII, XIV) . In contrast, neither cEIRp nor mcEIRp were inactivated by RNase, DNase, -.neuraminidase or pholipase A ₂ (Groups VI, VII, VIII, IX and XV, XVI, XVII, XVIII) . Although not shown, none of these enzymes exhibited the capacity to directly induce expression of FcR _£ at any of the concentrations employed. b) Molecular Sieve Chromatography (AcA-54) of cEIR and mcEIRp Demonstrates That EIRp. has a Molecular Mass of 45-60 kd — Induction of FcR _e ⁺ Lymphoid Cells.

Serum-free preparations of cEIR (containing cEIRp, mcEIRp, SFA and other mediators) and of MBI-2 hybridoma-derived mcEIRp were subjected to AcA-54 molecular sieve chromatography. The resulting fractions of both cEIR and mcEIRp were tested for FcR _£ - inducing activity on unfractionated and T cell-depleted lymphoid cell populations, this allows us to distinguish the target cell specificities of such activities, in particular the presence of EIR _B (which induces FcR _£ 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 peaks of FcR _£ -inducing activity can be distinguished among the various AcA-54 fractions of cEIR when unfractionated lymphoid cells are used as indicator targets, one peak in the range of 45-60 kd (fractions 30-34) , another in the range of 30-35 kd (fractions 41-45) , and the last in the range of 15-20 kd (fractions 49-52) . On T cell- depleted indicator cells, fractions 30-34 (40-60 kd)

were inactive, indicating that the FcRg-inductive activities of molecules in this size range are dependent on the presence of T-cells, which is characteristic of EIR _p . This conclusion was confirmed by demonstrating that the FcR _£ -inductive activity of fractions 30-34 of cEIR was totally abolished by depletion of Lyt-2 ⁺ cells. This conclusion was further substantiated by the chromatogram of mcEIRp on the same AcA-54 column (panel B) . On unfractionated lymphoid cells, significant induction of FcR _£ was obtained with fractions 28-32, corresponding to a molecular mass of approximately 45-60 kd, the same precise size range depicted in Panel A for cEIR. Moreover, these same fractions (28-32) from mcEIR _p were capable of inducing FcR _e .expression on lymphoid cell populations depleted of Lyt-1 ⁺ , but not on Lyt-2 ⁺ (data not shown) or total T cell-depleted (panel B) populations.

The other pertinent observation in Figure 4 relates to the biological activities of certain fractions retrieved from the AcA-54 column on T cell- depleted target cells. Thus, as shown in panel A, two peaks of such activity were obtained on the chromatogram of cEIR — one corresponding to fractions 40-45, (30-35 kd) and the second corresponding to fractions 49-56, (15-20 kd) — thereby indicating the presence of EIR _B in such fractions. This nicely illustrates how it is possible to clarify a heterogeneous molecular population into fractions which display activity (e.g. , EIR _B ) not detectable in the starting unfractionated population (e.g. , cEIR, see Fig 1) . Further, the smaller molecular mass species of EIR _B (i.e. , 15-20 kd EIR _B ) was shown to induce T cells _. in vitro to secrete SFA, the activity of which was confirmed using the selective .in vitro FcR _£ assay system (not graphically presented) . In contrast, the chromatographic profile

obtained from mcEIRp. (panel B) lacked any FcR _£ -inductive activity on T cell-depleted populations, thereby further confirming the absence of EIR _B . Additional studies confirm that EIR _B produced in conventional lymphoid cell populations and by EIR _B -secreting B cell hybridomas, fall into the same size categories as those depicted in panel A of Figure 4. Thus, we conclude that EIR _T falls in the size range of 45-60 kd and that EIR _B falls in the size range of 30-35 kd (fractions 40-45) and 15-20 kd (fractions 49-56) .

The discovery of these EIR _B fractions is of great importance in the present application. These isolated fractions have been shown to have allergy suppressive activity (i.e., such molecules suppress IgE antibody synthesis when used together) and are considered to be valuable in therapy.

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

As shown above, either or both of the isolated fractions possess an allergy suppressive activity not possessed by the beginning fluids, and which never before existed in any composition either natural or synthetic. Such isolated EIR _B fractions possess therapeutically activity and utility not heretofore known or suggested by prior work.

These isolated fractions are normally used in the form of the EIR _B fraction in physiologically active amounts of, for example, from a few nanograms to 100 micrograms per dose in a biologically compatible carrier, such as normal saline, and may be injected or may be prepared in ingestible form. Once isolated from the natural fluid, or

prepared through hybridoma expression or synthetically, for example using Merrifield's method, the compositions of matter, which are completely novel separate from interfering fluid constituents, described herein may be formulated for therapy using conventional pharmaceutic practice.

As used herein, a- composition consisting essentially of one or both of the above described EIR _B fractions contains a physiologically active amount of either the 15-20 kd or 30-35 kd fractions (or a mixture thereof) free of interfering 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, adjuvents, carriers, preservatives, etc. c) Molecular Sieve Chromatography (AcA-54) of cEIR and mcEIRp Demonstrates (1) the Presence of SFA and SEM in cEIR, and (2) the Absence of Such Contaminants in mcEIRp — Inhibition of FcR _£ ⁺ Cell Induction The same AcA-54 fractions of cEIR and mcEIRp were also assayed for presence of inhibitory activity on IgE-induced FcR _£ ⁺ cells, such activity would be associated with the presence of SFA, EFA, SEM or EEM. As shown in Figure 5, panel A, three peaks of inhibitory activity were observed with fractionated cEIR, in the ranges 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-depleted indicator cells were used, indicating that EFA is not solely responsible for the activity present in any of these fractions. However, when total T cell-depleted indicator cells were employed, only the cEIR fractions in the range of 10-15 kd (fractions 57-60) exhibited inhibitory activity (panel A) . More importantly the FcRg-inhibitory

activity of fractions 57-60 was specifically removed by adsorption with igE-Sepharose, indicating that such activity is due to the presence of the IgE-binding factors, SEM and/or EEM. These data also substantiate that SFA-like mediators are present in preparations of cEIR and migrate as molecules of the size of 20-25 and 35-40 kd, respectively.

In contrast, none of the mcEIRp fractions exerted inhibitory activity on IgE-induced FcR _fc expression by either unfractionated or T cell-depleted lymphoid cell cultures (Fig. 5, panel B) . This permits us to conclude that only mcEIRp. activity is present in the supernatant fluid derived from hybridoma MBI-2 and that neither SFA, EFA nor EIR _B is produced by these cells.

Both cEIRp and Hybridoma-Derived ^' mcEIRp Can ^* Induce Lyt-2 ⁺ Cells to Produce the. FcR _£ -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 abilities of cEIRp. and mcEIRp. — as well as appropriate fractions of corresponding activities obtained from the chromatograms illustrated in Figures 4 and 5 — to induce the production of final effector molecules in the cascade, specifically EEM and/or SEM. It should be recalled that EIRp. 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 FcR _e expression by B cells, albeit at different points in the cellular cascade, and so can be differentiated in this respect.

Thus, Lyt-1 ⁺ and Lyt-2 ⁺ cell-depleted primary cultures were exposed to various preparations of cEIRp.,

mcEIRp and SFA, and the supernatant fluids from these primary cultures were harvested and tested for their abilities to directly inhibit IgE-induced FcR _£ expression by B cells. As illustrated in panel A of Figure 6, IgE-stimulated B cells exhibited enhanced expression of FcR _£ . However, the data presented in panel B illustrates that EEM was produced by Lyt-2 ⁺ T cells exposed to cEIRp (Group I) or mcEIRp (Group IV) . Similarly, fraction 31 of both cEIRp and mcEIRp induced the production of EEM as indicated by the approximately 50% inhibition of FcR _£ expression (Groups II and V) . In contrast, the EIRp-depleted fraction 41 of cEIR and mcEIRp was unable to induce Lyt-2 ⁺ cells to make EEM (Groups III and VI) . It should also be noted that, consistent with the data presented in the next experiment (Figure 7) , the EEM-like activities induced by cEIRp or mcEIRp-enriched fraction 31 were totally depleted by adsorption on IgE affinity columns.

The reciprocal experiment in panel C demonstrates that cEIR clearly induces Lyt-2 ⁺ cell- depleted lymphoid cells to produce SEM that can inhibit FcR _£ -induction in B cells, as could the SFA-enriched (and the EIRp-depleted) fraction 41 (Groups VII and IX) . However, neither unfractionated mcEIRp (Group X) nor the EIRp-enriched fraction 31 of cEIRp (Group VIII) or of mcEIRp (Group XI) could induce Lyt-2 ⁺ cell-depleted lymphoid cells to release SEM. Moreover, fraction 41 of mcEIRp (Group XII) , which corresponds to the SFA-enriched fraction from cEIR (Group IX) , also lacks the ability to induce production of SEM by Lyt-l cells.

We conclude that the EIRp-enriched fraction 31 is free of detectable SFA-like activity and that the sole EIR- like activity present in these two EIRp-enriched preparations, as well as the unfractionated material from hybridoma MBI-2, is that of EIRp.

cEIRp. and mcEIR _p Do Not Exhibit IgE-Binding Affinity, Whereas the Effector Molecule 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 enhancing molecule which acts, in part, by (1) inhibiting the development of FcR _£ ⁺ B cells, (2) blocking the production of EIR _B which, in turn, (3) diminishes 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 EIRp. is significantly involved in the process of generating EEM, it is important to determine whether the relevant EIRp active entity is an IgE-BF — a question that can now be addressed unambiguously now that mcEIRp is available.

The experiment summarized in Figure 7 addresses these questions in a comparative manner. Panels A and B of Figure 7 illustrate the effects of IgE-Sepharose adsorption on the FcR _£ -inductive activity of cEIRp and mcEIRp, respectively. Significant induction of FcR _e expression was observed with as little as a 10 ^~2 dilution of either untreated cEIRp or mcEIR _p . Stimulation of lymphoid cells with IgE-adsorbed preparations of either regulant yielded identical titration curves. We conclude from these data that cEIRp and mcEIRp, like EFA, do not possess affinity for IgE and are not IgE-binding factors.

Panels C and D of Figure 7 clearly establish that preparations of cEIRp and mcEIRp can induce the production of an effector molecule which can inhibit IgE-induced FcR _£ expression in B cells and, like EEM, is an IgE-BF. In This instance, Lyt-l cell-depleted lymphoid cells were cultured for 48 hrs in the presence of either 10% cEIRp or mcEIRp. The resulting

supernatant fluids were split into two portions, one of which was subjected to IgE-adsorption. The various preparations were then tested for the ability to directly inhibit FcR _£ expression by B cells. Unabsorbed regulant, induced by either cEIRp

(panel C) or mcEIRp (panel D) clearly was inhibitory to IgE-induced FcRg express-ion, causing a 40-45% suppression of this response at dilutions of as little as 1:50. However, the parallel preparations subjected to IgE-adsorption, irrespective of whether induced with cEIR _p or mcEIRp were totally devoid of FcR _£ -inhibitory activity. Additionally, supernatant fluid from unstimulated Lyt-l cell-depleted lymphoid cell cultures, and the initial EIRp preparations as well, were similarly devoid of FcR _£ -inhibitory activity. We conclude that the regulant induced by EIRp is an IgE-BF, and is probably homologous to that induced by EFA (i.e. , EEM) .

Finally, the untreated and IgE-adsorbed EIR _T preparations described in panel A and B of Figure 6 were also compared with respect to ability to induce Lyt-2 cells to produce EEM. Adsorption of either cEIRp or mcEIRp with IgE-Sepharose did not alter subsequent induction of EEM by either regulant. In the course of analyzing the various fractions obtained by passing cEIR over a molecular sieve column two peaks of SFA-like activity were quite unexpectedly observed. The fact that SFA was present in cEIR was not too surprising since we have previously demonstrated that supernatant fluids derived from cultures of IgE-stimulated lymphocytes contain this activity (J. Immunol. 133:2837), but there was no reason to predict two separate SFA-like functions. Initial observations were confirmed by xxx vivo suppression of IgE antibody responses (Fig. 2) , and xxx vitro by the

Lyt-1 ⁺ cell-dependent inhibition of IgE-induced FcR _c -- expression (Figure 5) and induction of Lyt-1 ⁺ cells to produce the IgE-BF, SEM (Figure 6) . However, the fact that two SFA species of distinct molecular mass were present in such supernatant fluids was unexpected.

As shown in Figure 8, a T cell hybridoma (denoted MBI-1) has been identified- which secretes SFA molecules exhibiting a molecular mass characteristic of SFA ₂ , and that such secretion can be dramatically enhanced upon stimulation of such cells with EIR _B . Thus it can be noted in Figure 8 that unstimulated MBI-1 cells produce SFA molecules of 35-40 kd, but in low quantity. However, EIR _B -stimulated MBI-1 cells produce this same molecular entity, but in very much enhanced levels.

Also observed upon molecular - sieve fractionation of cEIR were fractions which could inhibit IgE-induced FcR _£ expression by isolated B cells. The peak of this activity exhibited an elution pattern typical of molecules of between 10-15 kd. Moreover, the mediator present in these fractions could be depleted by passage on an IgE-Sepharose column indicating that it is an IgE-BF. It is likely that both SEM and EEM were contained in this peak of activity. The lack of detectable B cell FcR _£ -inductive activity (EIR _B ) in unfractionated lymphoid cell-derived EIR observed in Figure 1 is due to the concomitant presence of SEM and SFA in these same preparations. Indeed, from the data illustrated in panel A of Figure 4, EIR _B exists as molecules of 15-20 kd and as molecules of 30-35 kd. Reconstitution experiments showed that the SFA and IgE-BF containing fractions illustrated in Figure 5 could suppress expression of FcR _e induced with the EIR _B -containing fractions illustrated in Figure 4. Moreover, the influence of IgE-BF and SFA present in

cEIR on the activity o.f EIR _B also present in these preparations, can be discerned upon analysis of the various fractions in Figures >4 and 5. Thus, the shoulder in FcR _£ induction on unfractionated lymphoid cells by fractions 40-42 illustrated in panel A of Figure 4 corresponds to the presence of SFA-like activity in these same fractions as illustrated in panel A of Figure 5. Moreover, it can be seen that when T cells are depleted from the indicator cell population in panel A of Figure 4, a single large peak of EIR _B activity now incorporates the previous shoulder, remember, that for SFA to inhibit FcR _£ -expression, Lyt-1 ₊ T cells must be present to produce the final effector molecule, SEM. A similar relationship between the activities of SFA and EIR _B illustrated in fractions 49-51 of Figures 4 and 5 is also apparent. Thus, we conclude that EIR _B activity is present in unfractionated cEIR, but that this activity is obscured by the concomitant presence of SFA-like mediators and IgE-BF. Since the activity of EIRp is not altered by the presence of these FcR _£ -inhibitory mediators, only EIRp- induced FcR _£ expression is observed with whole spleen cell-derived cEIR (Figure 1) .

Table II presents a summary of experiments concerning the characterization of the secreted product derived from selected B cell hybridomas. Basically, B cell hybridomas which secrete EIR _B produce such regulants with biological activity characteristic of either EIR _B _p or EIR _B _ ₂ . Thus, the B cell hybridoma 99E9 which was selected on the basis of inducing B cell FcRg expression can be shown to also stimulate Ly-1 ⁺ T cells to produce SFA in vitro. 99E9 is biologically indistinguishable at this point from the EIR _B _-j_ produced by polyclonal B lymphocytes upon stimulation with IgE. The B cell hybridoma 2C7, which was also selected on the

basis of the ability to induce B cell FcR _£ expression, does not induce T cell secretion of SFA in vitro. EIR _B derived from cell line 2C7 exhibits a molecular mass of 30-35 kd and is thus presently indistinguishable from the EIR.α..—»-_. derived from IgE-stimulated B cells.

TABLE II

COMPARISON OF EIR _B PREPARATIONS

Induction of T

Induction of B Cell Cell SFA

Inductive Stimulus FcRE Expression Secretion cEIR _B + +

15-20 kd cEIR _β .! + +

99E9-Derived mcEIR _β .! + +

30-35 kd cEIR _B _ ₂ + -

2C7-Derived mcEIR _B . -2 + —

M Maaccrroopphhaaggeess PPllaayy aa CCeennttrraall rroollee iinn tthhee EEIIRR CCaassccaaddee..

We have reported that SFA acts on Ly-1 ⁺ T cells to induce production of the suppressive Ige-BF termed SEM (J. Immunol. 133 :2845) . These initial studies were conducted with SFA derived from CFA-induced ascites, a preparation expected to exhibit considerable molecular heterogeneity. However, when we attempted to induce production of SEM with SFA derived from in vitro cultures of MBI-1 cells such attempts uniformly failed. Since MBI-1 cell-derived SFA could inhibit IgE-induced FcR _£ expression, presumably through induction of SEM in situ, this suggested that IgE might be acting on another cellular population to trigger production of an EIR able to act as co-stimulus with SFA for induction of SEM secretion. Further, since ascites-derived SFA (a preparation able to induce secretion of SEM) was obtained from an environment rich in macrophages, EIR derived from macrophages was tested for its ability to act with monoclonal SFA to induce secretion of SEM. Thus, purified T cells (G-10 passed and panned on anti-Ig-coated plates) were cultured in the presence

or absence of SFA, with and without EIR derived from macrophages, and subsequent secretion of SEM monitored using inhibition of IgE-induced FcR _£ expression by B cells as for Figure 6. As shown in Table III, unstimulated T cells could not produce appreciable quantities of SEM, whereas T cells stimulated with ascites-derived SFA could (Group I vs. Group II) . However, monoclonal SFA (mcSFA) could not directly mediate SEM production (Group III) , nor could EIR derived from macrophages (Group IV) . However, when mcSFA was used in conjunction with EIR-M0, SEM production was noted. We conclude that SFA is necessary, but insufficient, to induce SEM production, EIR-M0 is needed as co-stimulus. TABLE III

BOTH SFA AND EIR DERIVED FROM MACROPHAGES (EIR-M0) ARE NEEDED FOR INDUCTION OF T CELL SEM SECRETION

% Inhibition of B Cell FcR _£ Group Lymphocytes Inductive Stimulus Expression

I None 0

II Ascites-Derived SFA 42

III T Cells mcSFA 3

IV EIR-M0 0

V mcSFA + EIR-M0 43

BALB/c spleen cells were fractionated to enrich for T lymphocytes by sequential G-10 Sepharose passage and panning on anti-Ig-coated plates. Subsequently, the cells were cultured at 10 ⁷ cells/ ml for 24 hours in either medium alone, in medium containing 10% ascites-derived SFA, 10% supernatant

fluid from cultures of MBI-1 cells (i.e. , SFA) . 10% EIR derived from macrophages [generated by culturing normal peritoneal macrophages for 48 hours in the presence of 10 ;g/ml IgE] , or in medium containing 10% mcSFA and 10% EIR-M0. The supernatant fluids derived from such cultures of stimulated T cells were then tested for SEM content as described in the Legend for Figure 6.

The compositions of this invention, in suitable carrier compositions and adjuvants, such as are commonly known in immunology and pharmacology are administered to the subject, typically by injection. Adjuvents of several classes which may be suitable for use in connection with the present invention are described in many publications, see, for example, Klein, J., IMMUNOLOGY, pp. 361 et.seq.

The following examples illustrate general compositional criteria which may be used by those skilled in formulating diagnostic and therapeutic compositions in preparing suitable compositions. Composition No. 1. An isolated EIR _B fraction having a molecular mass of 15-20 kd in a solution comprised of normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 - to 25 ;g/ml, of the isolated fraction and optionally including physiologically acceptable preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis. Composition No. 2. An isolated EIR _B fraction having a molecular mass of 30-35 kd. in a solution comprised of normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of the- isolated fraction and optionally including physiologically acceptable preservatives and/or known,

physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis. Composition No. 3. An isolated SFA ₂ fraction having a molecular mass of 35-40 kd. in a solution comprised of normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of the isolated fraction and optionally including physiologically ^' acceptable preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis. Compostion No. 4. An isolated EIRp fraction having a molecular mass of 45-60 kd. in a solution comprised of normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to " 25 ;g/ml, of the isolated fraction and optionally including physiologically acceptable preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis. Composition No. 5. An isolated EIR-Mø fraction, able to synergize with SFA to induce secretion of SEM in a solution comprised of normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of the isolated fraction and optionally including physiologically acceptable preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis. Composition No. 6. An allergic reaction

suppressive composition of matter comprising biologically compatible carrier and diluents and a physiologically effective amount of a composition consisting essentially of an EIR _B fraction having a molecular mass of 15-20 kd. the composition comprising normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of the isolated fraction and optionally including physiologically acceptable preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis.

Composition No. 7. An allergic reaction suppressive composition of matter comprising biologically compatible carriers and diluents and a physiologically effective amount of a composition consisting essentially of an EIR _B fraction having a molecular mass of 30-35 kd. the composition comprising normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of- the isolated fraction and optionally including physiologically acceptable preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis.

Composition No. 8. An allergic reaction suppressive composition of matter comprising biologically compatible carriers and diluents and a physiologically effective amount of a composition consistently essentially of an EIR-M0 fraction the composition comprising normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of the isolated fraction and optionally

including physiologically acceptable preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis.

Composition No. 9. An allergic reaction suppressive composition of matter comprising biologically compatible carriers and diluents and a physiologically effective amount of a composition consisting essentially of a SFA ₂ ^" fraction having a molecular mass of 35-40 kd. the composition comprising normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of the isolated fraction and optionally including physiologically acceptable preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis. Composition No. 10. An IgE class-selective potentially composition of matter, which could suppress allergic reactions by virtue of polyclonal stimulation of IgE secretion and, thus, saturating mast cell FcR, with IgE which does not recognize the allergen in question, comprising biologically compatible carriers and diluents and a physiologically effective amount of a composition consisting essentially of an EIR _p fraction having a molecular mass of 45-60 kd. the composition comprising normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of the isolated fraction and optionally including physiologically acceptable preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response

control therapy or diagnosis.

Composition No. 11. A composition consisting essentially of a physiologically compatible carrier and on allergy suppressive EIR _B factor having a molecular mass of 15-20 kd and biological characteristics equivalent to non-IgE binding EIR _B _ _j produced by B cells in response to IgE stimulus and which induces Lyt-1 ⁺ cells to make SFA and induces B cell FcR _£ expression, the composition comprising normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of the isolated fraction and optionally including physiologically acceptable preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis.

Composition No. 12. A composition consisting essentially of a physiologically compatible carrier and an allergy suppressive ΕIR _B factor having a molecular mass of 30-35 kd and biological characteristics equivalent to non-IgE binding EIR _B2 produced by B cells in response to IgE stimulus and which induces B cell FcR _£ expression, the composition comprising normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of the isolated fraction and optionally including physiologically acceptable preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis.

Composition No. 13. A composition consisting essentially of a physiologically compatible carrier and an allergic suppressive SFA factor having a molecular mass of 35-40 kd and biological characteristics

equivalent to the non-IgE-binding SFA ₂ produced by T cells in response to EIR _B _2. stimulus, and which suppresses FcR _£ expression in vitro and suppresses IgE antibody responses in vivo,the composition comprising- normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of the isolated fraction and optionally including physiologically acceptable preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml o 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis.

Composition No. 14. A composition consisting essentially of EIR _B fraction having a molecular mass of 15-20 kd. the composition comprising normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of the isolated fraction and optionally including physiologically acceptable preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis.

Composition No. 15. A composition consisting essentially of EIR _B fraction having a molecular mass of 30-35 kd., the composition comprising normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of the isolated fraction and optionally including physiologically acceptable preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis.

Composition No. 16. A composition consisting essentially of SFA ₂ - fraction having a molecular mass of 35-40 kd. , the composition comprising normal saline

containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of the isolated fraction and optionally including physiologically acceptable preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis.

Composition No. 17. A composition consisting essentially of EIRp fraction having a molecular mass of 45-60 kd., the composition comprising normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of the isolated fraction and optionally including physiologically acceptable preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis.

Composition No. 18. A lymphocyte derived composition of matter which exhibits SFA-like activity consisting essentially of EIR _B having a molecular mass of 15-20 kd. , the composition comprising normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of the isolated fraction and optionally including physiologically acceptable preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis.

Composition No. 19. A lymphocyte derived composition of matter which exhibits SFA-like activity consisting essentially of EIR _B having a molecular mass of 30-35 kd. , the composition comprising normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of the isolated fraction and optionally including physiologically acceptable

preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis Composition No. 20. A macrophage-derived composition of matter which exhibits EIR-M0 activity in the induction of SEM, the composition comprising normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of the isolated fraction and optionally including physiologically acceptable preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis.

Composition No. 21. A lymphocyte-derived or T cell hybridoma-derived composition of matter which exhibits IgE class-selective suppressive activity consisting essentially of SFA ₂ having a molecular mass of 35-40 kd. , the composition comprising normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of the isolated fraction and optionally including physiologically acceptable preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis.

Composition No. 22. A lymphocyte-derived or T cell-hybridoma-derived composition of matter which exhibits IgE class-selective enhancing activity consisting essentially of EIRp. having a molecular mass of 45-60 kd. , the composition comprising normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of the isolated fraction and optionally including physiologically acceptable

preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis. The methods of the invention may be carried out in accordance with good medical and immunological practices using the various diluents, carrier materials, adjuvants, etc. The following methods may be used by those skilled in the art as general guidelines to prepare the compositions and use the compositions effectively.

Method No. 1. The method of suppressing allergic reactions comprising administering a composition of matter selected from the group consisting of EIR _B _ _1; EIR _B _ ₂ , SFA ₂ , or EIR-M0 in a diluent composition comprising normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of the isolated fraction and optionally including physiologically acceptable preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis.

Method No. 2. The method of enhancing IgE antibody responses comprising administering a composition of matter consisting of EIR _T in a diluent composition comprising normal saline containing from about 0.01 ;g/ml to about 100 ;g/ml, preferrably from 1 to 25 ;g/ml, of the isolated fraction and optionally including physiologically acceptable preservatives and/or known, physiologically compatable immunological adjuvents is injected in amounts from 0.1 ml to 5 ml or more intraveneously into the subject in allergy response control therapy or diagnosis. In general, the compositions of this invention

which are suitable for therapeutic administration will consist essential of an effective amount of the active ingredient disclosed herein in the general range of from 0.001 ;g/ml to 1000 ;g/ml, or more, typically from one to one hundred micrograms per milliliter of carrier or diluent compositions such as normal saline, distilled water, etc., optionally, an effective amount from .0001% to .1% of adjuvent such as Freund ^r s adjuvent, alum, etc. Generally, the administration of the compositions of this invention may be carried by any of the methods described in the scientific and clinical literature in which the solution containing the active isolated fraction is inject into the subject. Method such as those described by: Humphrey, J.H &-White, R.G., IMMUNOLOGY, For Medical Students, Blackwell, Oxford (1970) : Natelson, S., Pesce, 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, Philadephia (1967) : Katz, D.H., Benacerraf, Baruj , IMMUNOLOGICAL TOLERANCE, Mechanisms and Potential Therapeutic Applications, Academic Press, New York (1974) : Klein, J., IMMUNOLOGY, John Wiley, New York, (1982) , and Alving, B.M. , Finlayson, J.S., Editors, IMMUNOGLOBULINS: CHARACTERISTICS AND USES OF INTRAVENEOUS PREPARATIONS, U.S. Dept. H&HS, Food and Drug Administration, DHHS Publication No. (FDA) -80- 9005, may be used as a basis for particular methods within the present invention. The isolated compositions of this invention, as shown in Table 1 for example, are useful in producing polyclonal antibodies according to conventional immunological practice and to produce monoclonal antibodies according the method of Kohler and Milstein, Nature (256:495) (see also Kennett, R.H., McKearn, T.J.

& Bechtol, K.B., MONOCLONAL ANTIBODIES, Plenum Press, New York, 1980) which have both therapeutic and diagnostic application, according to principles set forth in the above cited references. These antibodies may, for example, be used to purify the compositions of this invention as well as in diagnosis.

The compositions of this invention are described in rather specific detail in order that the best mode for carrying out and using the invention may be fully disclosed, and the specific criteria-- and methods are to be considered in this light and not as limiting. For example, active fragments of these compositions are known or expected to exist and are encompassed within the respective definitions of the inventive compositions.

Industrial Application The present invention is useful in manufacturing pharmaceutical and diagnostic preparation.

REFERENCES

The following references disclose background methods and procedures and underlying principles.

1. Hoover, R. , and R. Lynch. 1983. Isotype- specific suppression of IgA: Suppression of IgA responses in BALB/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. Neauport-Sautes, J. Theze, and W. H. Fridman. 1983. Isotype regulation of antibody production: T cell hybrids can be selectively induced to produce IgGp and IgG ₂ subclass-specific suppressive immunoglobulin-binding factors. Proc. Natl .

Acad. Sci. 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: Fee receptors regulate. T and B cell collaboration for IgA responses. J. Immunol. 133:1087. *

5. Adachi, M. , J. Yodoi, N. Noro, T. Masuda, and H. Ochino. 1984. Murine IgA binding factors produced by FccR ⁺ T cells: 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. Marcelletti, J. F. , and D. H. Katz. 1984. FcR _e ⁺ lymphocytes and regulation of the IgE antibody system. I. A new class of molecules termed IgE-induced regulants (EIR) , which modulate FcR _£ expression by lymphocytes. J. Immunol. 133:2821. *

7. Marcelletti, J. F. , and D. H. Katz. 1984. FcR _e ⁺ lymphocytes and regulation of the IgE antibody system. II. FcR _£ ⁺ B cells initiate a cascade of

cellular and molecular interactions which control FcR _£ expression and IgE production. J. Immunol. 133:2829. *

8. Marcelletti, J. F., and D. H. Katz. 1984. FcR _£ ⁺ lymphocytes and regulation of the IgE antibody 5 system. III. Suppressive factor of allergy (SFA) is produced during the xxx vitro FcR _£ expression cascade and displays corollary physiological activity .in. vivo. J. Immunol. 133:2837. *

9. Marcelletti, J. F., and D. H. Katz. 1984. 0 FcR _£ ⁺ lymphocytes and regulation of the IgE antibody system. IV. Delineation of target cells and mechanisms of action of SFA and EFA in inhibiting _m vitro induction of FcR _£ expression. J. Immunol. 133:2845.

10. Ishizaka, K. , J. Yodoi, M. Suemure, and M. 5 Hirashima. 1983. Isotype-specific regulation of the

IgE response by IgE-binding factors. Immunol. Today ±:192. *

11. Yodoi, J., M. Hirashima, and- K. Ishizaka. 1980. Regulatory role of IgE-binding factors from rat T 0 lymphocytes. II. Glycoprotein nature and source of IgE-potentiating 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 5 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 0 IgE-binding activity. J. Immunol. 125:1442.

14. Yodoi, J. , M. Suemura, and T. Kishimoto. 1983. Fc _£ R and FciR expressed on murine IgE-specific suppressor T hybridomas: Dissociation between suppressor activity and Fc _£ R expression. Microbiol. Immunol. S 27:877.

15. Katz, D. H. , C. A. Bogowitz, and L. R. Katz. 1984. The IgE antibody system: Mature, peripheral B. lymphocytes exert regulatory influences on the IgE systems of self-reconstituting, sublethally irradiated mice. J. Mol. Cell Immunol. JL:83.

16. Spiegelberg, H. , G. Boltz-Nitulescu, J. Plummer, and F. Melewicz. 1983. Characterization of IgE Fc receptors on monocytes and macrophages. Fed. Proc. 42:126. 17. Spiegelberg, H. L. , R. D. O'Conner, R. A. ^"

Simon, and D. A. Mathison. 1979. Lymphocytes with immunoglobulin E Fc receptors in patients with atopic disorders. J. Clin. Invest. jS4_: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 Fc _e -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 (FcR _£ ) . I. Conditions for inducing

FcR _£ ⁺ 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. Regulation of IgE antibody production by serum

molecules. I. Serum from complete Freund's adjuvant- immune donors suppresses irradiation-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. Rev. 41:77.

26. Katz, D. H. 1979. Regulation of IgE antibody production by serum molecules. III. Induction of suppressive activity by allogeneic lymphoid cell interact ^■ ons 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 mice. 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. O'Hair, and D. H. Katz. 1981. Human IgE antibody synthesis xx

vitro: Stimulation of IgE responses by pokeweed mitogen and selective inhibition of such responses by human suppressive factor of allergy (SFA) . J. Immunol. 127:1169. 31. Katz, D. H. , M. Nonaka, B. L. Zuraw, P. A.

Cohen, and C. H. O'Hair. 1982. Regulation of human IgE antibody synthesis xxx vitro. In Human B-Lymphocyte Function: Activation and Immunoregulation. A. S. Fauci and R. E. Ballieux, editors. Raven Press, New York, NY. p. 181.

32. Katz, D. H. 1982. IgE antibody responses in vitro: From rodents to man. In Progress in Allergy. K. Ishizaka, editor. S. Karger AG, Basel, Switzerland. 32:105. 33. Katz, D. H., S.-S Chen, F.-T. Liu, C. A.

Bogowitz, and L. R. Katz. 1984. Biologically-active molecules regulating the IgE antibody system: Biochemical and biological comparisons of suppressive factor of allergy (SFA) and enhancing factor of allergy (EFA). J. Mol. Cell. Immunol. 1:157.

34. Uede, T. , T. F. Huff, and K. Kishizaka. 1984. Suppression of IgE synthesis in mouse plasma cells and B cells by rat IgE-suppressive factor. J. Immunol. 133:803.* 35. Liu, F.-T, J. W. Bonn, E. L. Ferry, H.

Yamamoto, C. A. Molinaro, L. A. Sherman, N. R. Klinman, and D. H. Katz. 1980. Monoclonal dinitrophenyl- specific murine IgE antibody: Preparation, isolation and characterization. J. Immunol. 124:2728.* 36. Lake, P., E. Clark, M. Khorshidi, and G.

Sunshine. 1979. Production and characterization of cytotoxic Thy-1 antibody-secreting hybrid cell lines.

Detection of T cell subsets. Eur. J. Immunol. 9.:875. *

37. Katz, D. H. and D. P. Osborne, Jr. 1972. The allogeneic effect in inbred mice. II. Establishment of

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. Construction 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. ₁ 976. Hapten-specific IgE antibody responses in mice. VI. 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 K. Ishizaka. 1983. Modulation of the biologic activities of IgE-suppressive factor to the formation of IgE- potentiating 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. Segal, and F. D. Finkelman. 1984. Characterization of murine lymphocyte IgE receptors by flow microfluorometry. J. Immunol. 133:1521.

45. Vender-Mailie, R. , T. Ishizaka, and K. Ishizaka. 1982. Lymphocytes bearing Fc receptors for IgE. VIII. Affinity of mouse IgE for FceR 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, Philadephia (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. Alving, B.M., Finlayson, J.Ξ., Editors, IMMUNOGLOBULINS : CHARACTERISTICS AND USES OF

INTRAVENEOUS PREPARATIONS, U.S. Dept. H&HS, Food and Drug Administration, DHHS Publication No. (FDA)-80- 9005.*

52. Ke.mett, R.H., McKearn, T.J. & Bechtol, K.B. , MONOCLONAL 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.