EP0726913A1

EP0726913A1 - Cytokines from natural killer or t cells

Info

Publication number: EP0726913A1
Application number: EP95901809A
Authority: EP
Inventors: Clifford M. Snapper; James J. Mond
Original assignee: Henry M Jackson Foundation for Advancedment of Military Medicine Inc
Current assignee: Henry M Jackson Foundation for Advancedment of Military Medicine Inc
Priority date: 1993-11-10
Filing date: 1994-11-08
Publication date: 1996-08-21
Also published as: JPH09505285A; EP0726913A4; AU1090895A; WO1995013294A1; CA2176114A1

Abstract

Cell-free preparations of a cytokine produced by natural killer (NK) cells activated with interleukin 2 (IL-2) or produced by CD4<+> T cells activated with anti-CD3 monoclonal antibody are described. This cytokine induces antibody secretion by activated B cells. Methods for producing the cell-free supernatants containing the cytokine are also described.

Description

CYTOKINES FROM NATURAL KILLER OR T CELLS I. GOVERNMENT INTEREST

This invention described herein may be

manufactured, licensed and used by or for the United States Government without the payment of any royalties to us thereon. The Federal Government has a

nonexclusive nontransferable, irrevocable, paid-up license to practice or have practiced for or on behalf of the United States any subject invention throughout the world. II. TECHNICAL FIELD OF THE INVENTION

The invention is in the field of cytokines. More specifically it is in the field of cytokines which potently induce antibody secretion by activated animal B cells. III. BACKGROUND OF THE INVENTION

Cytokines are proteinaceous substances or protein factors whose activity is derived from T cells or Natural Killer (NK) cells. They serve as the

biological center piece or source of origin for a variety of biological activities because of their ability to potently induce antibody secretion by activated animal B cells. As a natural consequence of this ability, derivative products can be made from cytokines which further expand the scope of their enormous utility in the medical and research community.

The specificity of the cytokines has raised great hopes for their use and products derived therefrom in the prevention and therapy of abnormal health

conditions. They can be used individually or in combination with other cytokines to stimulate antibody production in order to modulate complex responses such as immune responses. Thus, there is a continuing need and interest in the medical arts for new members of the cytokine arsenal useful in the battle to prevent, treat and/or ameliorate the deleterious effects of infection and disease states which affect the world community. IV. SUMMARY OF THE INVENTION

It is an object of this invention to provide a novel cytokine. In accordance with this invention, and as broadly described herein, a second object of this invention is to provide a cytokine which can potently induce antibody secretion by activated animal B cells. These and other embodiments of the invention are fulfilled by the following embodiments, as illustrated in a general discussion of cytokines derived from T cells and NK cells. In one embodiment a cell-free preparation of a novel active substance whose activity is derived from T cells or NK cells is provided which potently induces antibody secretion by activated animal B cells; resists neutralization by monoclonal

antibodies against cytokines produced by NK cells activated by IL-2 or neutralization by monoclonal antibodies or receptor antagonists against cytokines produced by activated T cells; has Ig secretion

activity which is not mimicked by the addition of known T cell cytokines or NK cell cytokines when added to separate cell-free preparations in the absence of applicants' novel cell-free preparations of cytokines. Additionally, relative to the active substance whose activity is derived from T cells, it has been

discovered that it is a protein; can be produced by activation of the T cell through the T cell receptor signalling pathway which induces cytokine production by T cells; has activity which is inhibited by treatment with a proteolytic enzyme; has activity which is optimal within 3 to 5 hours after activation of the T cell; has detectable activity at dilution levels as low as 1:3000 and operates as a late acting factor required to induce proliferated B cells to mature into antibody secreting cells. It appears that the similarity between the cytokines discovered by applicants as being derived from NK cells or T cells range from

substantially the same to identical. In another embodiment of the invention a method of producing a cytokine preparation is provided,

comprising: coating the bottom of a flask with anti-CD3 antibody; adding T cells into the flask and allow the T cells to remain in the flask for a period of 24 hours; harvesting the cell-free supernatant; inducing the activation and proliferation of B cells in the cell-free supernatant through the B cell antigen receptor mediated signalling pathway utilizing dextran

conjugated anti-IgD antibodies in the presence of Interleukin-1 and Interleukin-2 wherein the B cells are at least 98.5 percent purified through fluorescence-activated cell sorting (FACS); adding activated T cell supernatant; harvesting the B cell culture supernatant 6 days after culture; and measuring the concentration of secreted IgM in the B cell cultured supernatant by an enzyme-linked Immunosorbent Assay (ELISA).

These and other embodiments will be apparent to those skilled in the art from the detailed disclosure which follows.

Cytokines prepared in accordance with this invention are derived from mammalian cells, including humans, and can be used generally to stimulate the immune system in immunocompromised hosts. Their utility extends to such areas as adjuvants for

boostering mammalian immune responses to vaccination, organ transplantation, chemotherapy, radiotherapy, genetic immunodeficiencies, and AIDS (autoimmune deficiency syndrome) or ARC (AIDS related complex.

Additionally, an antagonist or monoclonal antibody to neutralize the cytokine can be made or uniquely identifying and isolating its receptor on the surface of the B cell would be useful in the modulation of antibody production. Moreover, the identification of the receptor on the surface of the B cell can be utilized to create an antagonist (soluble receptor to prevent binding of the cytokine to the receptor.

Further, cytokines may be used alone or in combination with other cytokines (i.e., as pharmaceutical cocktail) to stimulate antibody production. V. BRIEF DESCRIPTION OF THE FTGURFS

Figure 1 depicts total, RBC-lysed, spleen cells that were stained with FITC-labelled anti-CD3 mAb (2C11) and phycoerythrin-labelled polyclonal goat anti-mouse IgM (left upper panel [unsorted]). B cells are IgM⁺CD3-, T cells are IgM-CD3⁺ , and non-B, non-T cells are IgM-CD3-. Re-analyses of sorted cells are displayed in right upper panel (B cells), left lower panel (T cells), and right lower panel (non-B, non-T cells). Figure 2 shows B^e and B^sp cells that were cultured at 1.25 × 10⁵ cells/ml with αδ-dex (3 ng/ml) and/or IL-2 (150 U/ml) in the absence or presence of 10% non-B, non-T cells (IgM-CD3-). six days after initiation of culture, SN was harvested for determination of secreted IgM concentrations by ELISA.

Figure 3 shows B^e cells that were treated for 30 min at 4°C with a polyclonal rabbit anti-AsGm-1 antisera (150 mg/ml) followed by a 30 min incubation at 37°C in a complement-containing solution in order to lyse AsGm-1⁺ cells. B^e cells with or without anti-AsGm-1 antibody treatment were cultured at 2 × 10⁵ cells/ml in the presence or absence of αδ-dex (3 ng/ml), and/or IL-1 (100 U/ml) plus IL-2 (150 U/ml). six days after

initiation of culture, SN was harvested for

determination of secreted IgM concentrations by ELISA.

Figure 4 depicts a pure population of activated NK cells were established by IL-2 stimulation, in vitro, of spleen cells derived from scid mice (see

methodologies). 7 days after initiation of scid spleen cell cultures, cells were harvested for flow cytometrio analysis. The following antibodies were utilized: (l) polyclonal rabbit anti-AsGm-1 antisera + FITC-labelled polyclonal goat anti-rabbit IgG, (2) FITC-labelled anti-CD3 mAb (2C11), (3) FITC-labelled anti-Thy-1 mAb (30H12), (4) FITC-labelled anti-MAC-1 mAb (M1/70), (5) FITC-labelled anti-Ia^d mAb (MKD6), and (6)

FITC-labelled anti-Ia^k mAb (Y3-P; control mAb-i.e. scid mutation established on BALB/c background [Ia^d]). Each flow cytometric tracing represents the fluorescence pattern of 10,000 viable cells. Figure 5 shows B^sp cells that were cultured at 1.25 × 10⁵ cells/ml with αδ-dex (3 ng/ml) and/or IL-1 (100 U/ml) plus IL-2 (150 U/ml) in the absence or presence of 10% sort-purified splenic non-B, non-T cells

(lgM-CD3-), 10% sort-purified splenic T cells (lgM-CD3⁺), or 3% in vitro-generated pure NK cells. Six days after initiation of culture, SN was harvested for

determination of secreted IgM concentrations by ELISA. Figure 6 shows B^sp cells that were cultured at 1.5 × 10⁵ cells/ml with αδ-dex (3 ng/ml), IL-1 (150 U/ml), plus IL-2 (150 U/ml) in the presence of increasing numbers of in vitro-generated pure NK cells (0-80% of B^sp cell numbers). 7 days after initiation of culture, SN was harvested for determination of secreted IgM

concentrations by ELISA. Figure 7 shows B^sp cells that were cultured at 1.5 × 10⁵ cells/ml with αδ-dex (3 ng/ml), IL-1 (100 U/ml) plus IL-2 (150 U/ml) in the absence or presence of varying concentrations of NK-BMF (10-50% v/v). Six days after initiation of culture, SN was harvested for

determination of secreted IgM concentrations by ELISA. Figure 8 shows B^5p cells that were cultured at 1.25 × 10⁵ cells/ml with αδ-dex (3 ng/ml), IL-1 (150 U/ml) plus IL-2 (150 U/ml) in the absence or presence of SN obtained 24 h after activation, with plate-bound anti-CD3 mAb (2C11), of the following CD4⁺ Th1 clones: (1) RA1 (1:0% SN v/v), (2) RA5 (1.0% SN v/v), (3) RA8 (1.0% SN v/v), (4) RC5 (1.0% SN v/v), and (5) RC9 (2.5% SN v/v). SN was harvested for determination of secreted IgM concentrations by ELISA.

Figure 9 shows B^sp cells that were cultured at 1.25 × 10⁵ cells/ml with αδ-dex (3 ng/ml), IL-1 (150 U/ml), plus IL-2 (150 U/ml) in the absence or presence of varying, final concentrations of SN (0.01-1.0% v/v) from anti-CD3-activated Th1 clones (see Fig 8). SN was harvested for determination of secreted IgM

concentrations by ELISA. Figure 10 shows a 24 h SN from anti-CD3-activated RA5 Th1 clone (T-BMF) was incubated at 37°C in the presence (RA5-SN-PK) or absence (RA5-SN-Ctrl) of proteinase K. B^sp cells were cultured at 1.25 × 10⁵ cells/ml with αδ-dex (3 ng/ml), IL-1 (150 U/ml) plus IL-2 (150 U/ml) in the absence or presence of either RA5-SN-PK (1.0% v/v) or RA5-SN-Ctrl (1.0% v/v). As an additional control, RA5-SN-PK (1.0% v/v) and RA5-SN-Ctrl (1.0% v/v) were added to B^sp cell cultures activated with αδ-dex (3 ng/ml) plus IL-5 (150 U/ml). SN was harvested for determination of secreted IgM concentrations by ELISA. Figure 11 shows a 24 h SN from the anti-CD3-activated RA5 Th1 clone (T-BMF) or SN from unactivated resting RA5 cells (Control SN) was tested in an IL-6-specific sandwich ELISA. Recombinant IL-6 was used to generate a standard curve. Figure 12 shows B^sp cells that were cultured at 1.25 × 10⁵ cells/ml with αδ-dex (3 ng/ml), IL-1 (150 U/ml) plus IL-2 (150 U/ml) in the absence or presence of varying concentrations of anti-CD3-activated RA5 SN (0.001-1.0% v/v) obtained at various time points after anti-CD3 activation (0-24h). SN was harvested for determination of secreted IgM concentrations by ELISA. Figure 13 shows B^sp cells that were cultured at 1.25 × 10⁵ cells/ml with αδ-dex (3 ng/ml), IL-1 (150 U/ml) Plus IL-2 (150 U/ml). T-BMF (1% v/v of RA5 SN) was additionally added to replicate cultures on different days (day 0-6). SN was harvested from all experimental groups 6 days after initiation of culture for

determination of secreted IgM concentrations by ELISA. VI. DETAILED DESCRIPTION OF THE INVENTION

A biological activity has been defined whose dominate function is to potently induce antibody secretion by activated animal B cells. More precisely, applicants have discovered:

1. A cell-free preparation of a novel active substance whose activity is derived from T cells or NK cells which potently induce antibody secretion by activated animal B cells, said substance having Ig secretion activity which is not mimicked by the

addition of cytokine in the absence of said cell-free preparation.

2. The cell-free preparation of Claim 1 wherein the active substance is produced by NK cells.

3. The cell-free preparation of Claim 1 wherein the active substance is resistant to neutralization by monoclonal antibodies against

cytokines produced by NK cells activated by IL-2 and has Ig secretion activity which is not mimicked by the addition of NK cell cytokines in the absence of said cell-free preparation. 4. The cell-free preparation of Claim 1 wherein the active substance is a protein whose activity is derived from T cells which potently induce antibody secretion by activated animal B cells, said substance:

a. is resistant to neutralization by monoclonal antibodies or receptor antagonists against cytokines produced by activated T cells;

b. has Ig secretion activity which is not mimicked by the addition of T cell cytokines in the absence of said cell-free preparation; c. can be produced by activation of the T cell through the T cell receptor signalling pathway which induces cytokine production by T cells;

d. has activity which is inhibited by treatment with a proteolytic enzyme;

e. has activity which is optimal within 3 to 5 hours after activation of the T cell; f. has detectable activity at dilution levels as low as 1:3000; and

g. operates as a late acting factor required to induce proliferated B cells to mature into antibody secreting cells. 5. The cell-free preparation of Claim ₄ wherein the active proteinaceous substance is a cytokine.

6. The cell-free preparation of Claim ₅ which is produced by T cells. 7. The cell-free preparation of Claim ₁ which is free of mycoplasm.

8. The cell-free preparation of Claim 6 wherein the proteinaceous substance can increase the induction of antibody secretion 10 to 40 fold. 9. The cell-free preparation of Claim 3 wherein the active substance can increase the induction of antibody secretion 10 to 30 fold.

10. The cell-free preparation of Claim 1 wherein the active substance whose Ig secretion activity is not mimicked by the addition of T cell or UK cell cytokines comprising Interleukin-3;

Interleukin-4 ; Interleukin-6 ; Interleukin-10 ; TNF-α; IFN-γ; IFN-γ and TNF-α; IFN-α; IFN-α and IFN-γ; IFN-α and TNF-α; IFN-α, IFN-γ and TNF-α; GM-CSF; and TGF-β1. 11. The cell-free preparation of Claim 8 wherein the proteinaceous substance is resistant to neutralization by monoclonal antibodies or receptor antagonists against cytokines comprising anti-IFN-γ; TNFRIg; anti-GM-CSF; anti-IL-3; anti-IL-4; anti-IL-5; anti-IL-6; anti-IL-10; and huCD40Fc. 12. The cell-free preparation of Claim 8 wherein the proteinaceous substance is produced by activation of the T cell through the T cell receptor pathway which induces cytokine production by T cells as shown in Figure 12. 13. The cell-free preparation of Claim 9 wherein the active substance is resistant to

neutralization by monoclonal antibodies against cytokines comprising anti-TNF-α; Anti-IFN-γ; anti IFNα/γ; anti-IL-4; anti-IL-5; anti-lL-6; and anti-IL-10. 14. A method of producing a cell-free preparation containing the active proteinaceous substance of Claim 4, comprising:

a. coating the bottom of a flask with anti-CD3 antibody;

b. adding T cells into the flask and allow said T cells to remain in the flask for a period of 24 hours;

c. harvesting the cell-free

supernatant;

d. inducing the activation and

proliferation of B cells in the cell-free supernatant through the B cell antigen receptor mediated signalling pathway utilizing dextran conjugated anti-IgD

antibodies in the presence of Interleukin-1 and

Interleukin-2 wherein said B cells are at least 98.5 percent purified through fluorescence-activated cell sorting (FACS);

e. adding activated T cell supernatant; f. harvesting the B cell culture supernatant 6 days after culture; and

g. measuring the concentration of secreted IgM in the B cell cultured supernatant by an enzyme-linked Immunosorbent Assay (ELISA). 15. The product produced in accordance with the method of Claim 14. In sum, we have established a novel in vitro assay which has allowed us to screen for potentially novel factors which induce immunoglobulin (Ig) secretion by murine B lymphocytes. We refer to such factors as _B cell maturation factors (BMF). Utilizing this assay we have identified two such activities which we have shown to be distinct from the well-described and

characterized cytokines which are known to play a role in B cell activation and differentiation. The first activity was identified in culture supernatants from pure populations of in vitro-generated IL-2-activated murine natural killer (NK) cells. The second activity was identified in the culture supernatants of a panel of anti-CD3-activated murine CD4⁺ T lymphocyte clones. While these two activities are functionally similar, they could represent distinct molecules, studies to identify the murine BMF from one of the CD4⁺ T cell clones are currently underway. A cytokine with the ability to induce high rate Ig secretion, or a

selective antagonist of such a molecule, could be of potential value as a therapeutic in a diverse number of clinical settings as well as an in vitro tool to generate antibody-forming cells. VII. Methods

Mice. Female DBA/2 mice were obtained from the National Cancer Institute (Frederick, MD) and were used at 7-10 weeks of age. The experiments were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Institute of Animal Resources, National Research Council, Department of Health, Education, and Welfare. Publ No. (National Institutes of Health) 78-23. Culture medium. RPMI 1640 (Biofluids, Rockville, MD) supplemented with 10% fetal bovine serum (Gibco Laboratories, Grand Island, N.Y.), L-glutamine (2 mM), 2-mercaptoethanol (0.05 mM), penicillin (50 mg/ml), streptomycin (50 μg/ml), and gentamicin (50 mg/ml) were used for culturing cells. Reagents. αδ-dex was prepared by conjugation of Hδ^a/1 (monoclonal mouse IgG2b (b allotype) anti-mouse IgD (a allotype) to a high molecular weight dextran ₍₂ × 10⁶ M.W.). Approximately 6 Hδ^a/1 were conjugated to each dextran molecule. LPS W, extracted from

Escherichia coli 0111:B4, was obtained from Difco Laboratories, Inc. (Detroit, MI) and was used at 20 mg/ml in all experiments. Affinity-purified,

phycoerythrin-labelled polyclonal goat anti-mouse IgM was obtained from Southern Biotechnology Associates (Birmingham, AL). FITC-labelled monoclonal hamster IgG anti-CD3e (2C11) monoclonal antibody (mAb) was obtained from Pharmingen (San Diego, CA). Polyclonal rabbit anti-AsGm-l was obtained from Wako (Osaka, Japan).

Murine recombinant (r)IL-1 was a gift from Dr.

Stephanie Vogel (USUHS, Bethesda, MD). Murine rIL-2 was a gift from-Dr. Maurice Gately (Hoffman LaRoche,

Nutley, NJ). Murine rIL-5 was a gift from Dr. Richard Hodes (NIH, Bethesda, MD). Preparation and culture of B cells. Enriched populations of B cells were obtained from spleen cells from which T cells were eliminated by treatment with monoclonal rat IgM anti-Thy-1 (H013-4), rat IgG2b anti-CD4 (GK1.5), and rat IgG2b anti-CD8 (2.43), followed by mouse anti-rat Igk (MAR 18.5) and complement. Cells were then fractionated into high and low density populations by centrifugation over a

Percoll gradient (Pharmacia, Piscataway, NJ) consisting of 70, 65, 60, and 50% Percoll solutions (with

densities of 1.086, 1.081, 1.074, and 1.062 g/ml, respectively). The high density cells were collected from the 70 to 66% interface and were used in all experiments. The average percentage of Ig⁺ cells was 85-90% in the 70 to 66% fraction. B cells obtained in this way were referred to as "B cell-enriched" (B^e) cells. This cell population is also known to contain small numbers of NK cells, macrophages, mast cells, and cells of the granulocytic series. To remove NK cells from the B^e cell preparation we further treated B^e cells with polyclonal rabbit anti-AsGm-1 antibodies (Waco, Osaka, Japan) plus complement as follows: B^e cells (10⁷ cells/ml) were incubated in "cytotoxicity medium" (CM) (RPMI + 2.5 mM Hepes + 0.3% BSA) with anti-AsGm-1 antibody (150 mg/ml final concentration) at 4°C for 1 h. Cells were then washed once in cold CM and

resuspended in CM containing a 1/10 dilution of rabbit complement (Pel-Freeze, Brown Deer, Wisconsin) at 37°C for 1 h. Cells were then washed and resuspended in medium for further use. Functional assays were carried out in 96-well flat-bottom Costar plates (Costar, Cambridge, MA). Cultured cells were incubated at 37°C in a humidified atmosphere containing 6% CO₂. Establishment of NK cell cultures. Spleen cells from CB-17 SCID mice, obtained from NCI (Frederick, MD), were cultured in medium at 1 × 10⁷/ml in the presence of 500 U/ml of rhIL-2. Spleen cells were first treated with anti-Thy-1, anti-CD4, and anti-CD8 + complement as a precaution against the possible presence of small numbers of T cells resulting from "leakiness" in the SCID mutation. NK cells were maintained by splitting them 1:2 into fresh medium + 500 U/ml of IL-2 every 2-3 days. Cells were used for experiments beginning -7-10 days after establishment of culture. Such cells were monitored by flow cytometry to confirm the absence of CD3⁺ (T) cells. NK cell cultures were re-established every month, using fresh spleen cells from SCID mice, since longer culture periods were associated with deterioration of the NK cell line.

Cells derived from CB-17 mice, like those from DBA/2, express H-2^d MHC class I molecules. Preparation of supernatants (SN) from IL-2 activated, in-vitro-generated NK cell cultures

(NK-BMF). NK-BMF was prepared in two basic ways: (1) In vitro-maintained, IL-2-activated NK cells were washed 3x and recultured at 1.0 × 10⁵ cells/ml in the presence of IL-2 (500 U/ml). 24 h later cell-free culture SN was harvested, aliquotted, and stored at -20ºC until used (2) In vitro-maintained, IL-2-activated NK cells were washed 3x and recultured at 1.0 × 10⁵ cells/ml in the presence of 5 × 10⁵ B^e cells/ml, but in the absence of other exogenous stimuli. 24 h later cell-free culture SN was harvested, aliquotted, end stored at -20ºC until used

Preparation of supernatants (SN) from activated

CD4+ T cell clone (T-BMF). Antigen-specific, MHC class

II-restricted, CD4⁺ T cell clones were established elsewhere by standard methodologies. The following CD4⁺ T cell clones were assigned to the Th1 subset on the basis of their secretion of ιL-2 and IFN-g, but not IL-4: The rabbit gamma globulin-specific,

Ia^d-restricted Th1 clone, D1.6, was established in the laboratory of Dr. Abul Abbas (Harvard Medical School, Boston, MA) whereas the KLH-specific Th1 clones, RA1, RA5, RA8, RC5, and RC9 were established at Immunex

Corporation, Seattle, WA. These T cell clones were maintained by weekly stimulation with antigen, spleen cells (as a source of antigen-presenting cells [APCs]), and exogenous IL-2. Cytokine-containing SN were obtained from cultures of these CD4⁺ T cell clones in the following manner: Tissue culture wells were incubated with anti-CD3 mAb (2C11) at 10 mg/ml in PBS for 3h at 37°C and then washed 3x in fresh PBS. T cell clones which were allowed to return to their resting state after

stimulation with antigen, APCs, and IL-2 by maintaining them for ~7 days in the absence of any further

exogenous stimuli were added to anti-CD3-coated plates at 1×10⁶/ml for various times, upon which cell-free SN were obtained and either stored at -20°C or 4°C. In the latter case, SN was used in cellular assays within 1-2 weeks of having been harvested. Cytofluorometric analysis and cell sorting. Spleen cells were stained for 30 min with FITC-labelled anti-CD3 mAb + PE-labelled anti-IgM antibodies (final concentration of 10 mg/ml each in the presence of a 5-fold excess of anti-FcγRII mAb to prevent cytophilic antibody binding) at 10⁷ cells/ml in cold clear HBSS containing 3% FBS and 50 mg/ml each of penicillin, streptomycin, and gentamicin. Cells were then washed and resuspended in staining buffer at 10⁷ cells/ml in preparation for fluorescence analysis and/or cell sorting. For analysis, a FACStar Plus or FACSCAN (y Becton Dickinson, Mountainview, CA) was used and 15,000 cells were collected using logarithmic amplification. Only viable cells were analyzed on the basis of their characteristic forward and side scatter profiles, cell sorting was similarly carried out on a FACStar Plus, as well as on an Epics Elite (Coulter Corp., Hialeah, FL), and sorted cells were immediately reanalyzed to confirm their staining profile. Only sorting purities of >98% were acceptable for subsequent study. Sort-purified B cells (mIgM⁺CD^3-) were referred to as B^sp cells. Non-B, non-T cells (mIgM-CD3-) and T cells (mIgM-CD3⁺) were also collected and macrophages were routinely eliminated, during sorting, on the basis of their characteristic forward and side scatter profile

Quantitation of secreted IgM. IgM concentrations were measured by ELISA, with Immulon 4, 96-well

flat-bottomed ELISA plates (Dynatech Laboratories,

Alexandria, VA). Briefly, ELISA plates were coated with polyclonal goat anti-mouse IgM antibodies (Southern Biotechnology Associates), followed by addition of serial dilutions of samples and standards. Alkaline phosphatase-conjugated polyclonal goat anti-mouse lgM antibodies were then added, upon which a fluorescent product was generated from cleavage of

4-methylumbilliferyl phosphate (Sigma) by specifically-bound alkaline phosphatase-conjugated antibodies. Fluorescence was measured on a 3M 96 fluorometer (Mountainview, CA) and fluorescence units were converted to Ig concentrations by extrapolation from standard curves determined in each assay by using purified myeloma IgM of known concentration. IgM measurements showed no significant cross-reactivity or interference from the presence of other isotypes (IgD, IgG3, IgG1, IgG2b, IgG2a, IgE, and IgA). Ouantitation of IL-6. Flat-bottomed, 96-well microtiter plates (Nunc, Roskilde, Denmark) were coated with 6 mg/ml of anti-mouse IL-6 mAb (MP5-20F3) in 0.1 M carbonate buffer (ph 9.6) for 3h at room temperature, blocked with PBS-1% BSA for 1h at room temperature, then extensively washed with PBS-Tween. Supernatant or recombinant.purified lymphokine (Pharmingen) diluted in PBS-1% BSA was incubated on these. plates for 1h.

Biotinylated anti-IL-6 mAb (MP5-32c11) was used at 1 mg/ml and avidin-conjugated alkaline phosph atase at a dilution of 1/2,000. Preliminary experiments showed this assay to be sensitive to 0.22 ng/ml of rIL-6.

Specificity was documented by the absence of binding by 1,000-fold higher concentrations of IL-4, IL-5, and IFN-γ. VIII. EXAMPLES

The following working examples are not intended to limit the invention which is defined by the claims appended below. A. Dextran-conjugated anti-IgD antibodies

(αδ-dex) potently induce proliferation of murine B cells in vitro. -We synthesized a novel construct which potently activates murine B cells through the membrane (m)Ig (antigen receptor) -mediated signal transduction pathway. This construct consisted of covalent linkage of multiple anti-IgD monoclonal antibodies (mAbs) to a high molecular weight dextran backbone. We henceforth refer to this construct as αδ-dex (Ref 1). The high valency of anti-IgD antibodies linked to dextran resulted in extensive B cell mlg crosslinkage at extremely low concentrations of anti-IgD. This resulted in minimal modulation of mlgD from the B cell surface and hence allowed for continuous B cell signalling. Given the small numbers of antigen-specific B cells in the unimmunized mouse, the advantages of αδ-dex lied in its ability to polyclonally activate the vast majority of mature B cells through the mlg signal transduction pathway. Further, αδ-dex mimicked the repeating epitope nature of polysaccharides, such as those found within bacterial cell walls, and could be used as an in vitro model to study immune responses to that class of antigens. αδ-dex induced resting murine splenic B cells to proliferate but did not, by itself, stimulate Ig secretion. Compared to unconjugated anti-IgD, αδ-dex induced substantially higher maximal levels of

proliferation at 1000-fold lower concentrations of anti-IgD (Ref 1). B. Interleukin (IL) -2 or IL-5 induce Iα secretion in αδ-dex-activated B cell-enriched, T cell-depleted spleen cell cultures-Addition of the cytokines IL-2 or IL-5 to αδ-dex-activated B cell cultures resulted in the secretion of large amounts of IgM (Ref 2). By contrast, unconjugated anti-IgD was ineffective at stimulating cytokine-mediated Ig secretion. Thus, αδ-dex represented the first efficient in vitro system for inducing cytokine-dependent polyclonal Ig secretion through the mlg signal transduction pathway. The population of small, resting B cells utilized in these studies were established by depleting T lymphocytes from spleen cells with a cocktail of anti-T cell antibodies and complement followed by fractionation, according to density, by centrifugation on a

discontinuous Percoll gradient. Cells were obtained from the high density Percoll fraction and represented cells in the resting or G_o state of the cell cycle. This population of small T cell-depleted spleen cells consisted of 85-90% B cells with the remainder of cells consisting mostly of NK cells, macrophages, and granulocytes. We henceforth refer to this B

cell-enriched spleen cell population as B^e cells. IL-2 stimulated Ig secretion by αδ-dex-activated B^c cells but not by highly purified B cells. To

determine whether IL-2 or IL-5 could act directly on the αδ-dex-activated B cell to induce Ig secretion we obtained a highly purified population of small B cells through the use of a florescence activated electronic cell sorter (FACS) (Fig 1). Thus, small B^e cells were stained with phycoerythrin (PE)-labelled anti-IgM antibody which selectively binds to mIgM⁺ B cells, plus FITC-anti-CD3 to identify and eliminate any residual T cells and/or cells binding anti-IgM non-specifically. Sort-purified mIgM⁺ B cells were obtained at >99% purity and are henceforth referred to as B^sp cells.

Whereas LPS or αδ-dex plus IL-5 stimulation resulted in a comparable level of Ig secretion by small B^e and B^sp cells, activation with αδ-dex plus IL-2 led to Ig secretion only by B^e cells (Table 1). This suggested that a non-B, non-T cell removed during the FACS procedure was critical for induction of Ig secretion by B^sp cells in response to αδ-dex plus IL-2. Bcell populations which were less than 98.5% purity typically contained sufficient non-B, non-T cells to allow for induction of Ig secretion in response to αδ-dex plus IL-2. Hence FACS was required to obtain sufficiently pure B cells to abrogate the αδ-dex plus IL-2-induced Ig secretory response. We demonstrated that the Ig secretory response of small B^sp cells in response to αδ-dex plus IL-2 could be restored by adding back the splenic non-B, non-T cells which were removed by FACS (Fig 2). The splenic non-B. non-T cells responsible for induction of Ig secretion in αδ-dex plus IL-2-activated B^sp cells are AsGm-1⁺-Depletion of macrophages from the B^e cell population, by exploiting the property of macrophages to selectively adhere to plastic, had no effect on Ig secretion in αδ-dex plus IL-2 activated B^e cells (data not shown). Thus, we postulated that the non-B, non-T cell necessary for induction of Ig secretion in this system was an NK cell. Since the vast majority of splenic NK cells, as well as some

macrophages, selectively express the marker Asialo Gm-1 (AsGm-1) on their surface, we eliminated NK cells by incubating small B^e cells with anti-AsGm-1 antibody plus complement. This procedure abrogated the Ig secretory response of B^e cells to αδ-dex plus IL-2 strongly suggesting that NK cells were responsible for induction of Ig secretion in this system (Fig 3).

Additional experiments demonstrated that depletion of AsGm-1⁺ cells from the small B^c cell population had no effect on the Ig secretory response to αδ-dex plus IL-5 (data not shown), consistent with the ability of these latter stimuli to act directly at the level of the B cell A pure population of activated NK cells release an Ig-inducing activity. To better define the role of NK cells in inducing Ig secretion by activated B^sp cells we established a pure population of NK cells in vitro.

Thus, we isolated spleen cells from mice which were homozygous for the severe combined immunodeficiency (scid) mutation. Such mice genetically lack both B and T cells but contain functional NK cells. Culture of scid spleen cells in relatively high concentrations of IL-2 resulted, within 6 days, in a pure population of activated NK cells as demonstrated by flow cytometric analysis (Fig 4). Thus, virtually all cells expressed the NK cell marker AsGm-1 as well as Thy-1, and lacked markers for T cells (CD3), B cells (la, and macrophages (MAC-1). The use of scid spleen cells were essential for obtaining this population since high doses of IL-2 would also expand even small numbers of T cells which might contaminate the B^e cell population. Addition of in vitro-generated pure NK cells to αδ-dex plus

IL-2-activated B^sp cells resulted in induction of Ig secretion which was comparable to that observed when sort-purified splenic non-B, non-T cells were added (Fig 5). Sort-purified small naive splenic T cells, by contrast, were ineffective at inducing Ig secretion in this system. NK cells induced optimal Ig secretion when present at 5-10% of the B cell population (Fig 6).

Addition of conditioned medium from these

IL-2-activated NK cell cultures also induced Ig secretion in αδ-dex plus IL-2-activated B^sp cells in the absence of NK cells indicating that NK cells released a BMF (Table 2). We henceforth refer to this BMF as NK-BMF. Optimal Ig induction by NK-BMF occured at 50% final volume of NK-BMF with little if any activity observed at 10% (Fig 7). NK-BMF is not among the cytokines known to induce B cell activation and maturation. To better

characterize the nature of NK-BMF we performed two types of experiments to determine whether NK-BMF was among the cytokines known to induce B cell activation and differentiation. In the first set of experiments we added known cytokines, either singly or in combination, at various concentrations, to cultures of B^sp cells stimulated with αδ-dex plus IL-2. Neither IL-3 , IL-4, IL-10, TNF-α, IFN-γ, lFN-α/β, GM-CSF, or TGF-β was capable of inducing Ig secretion in this system. In the second set of experiments we demonstrated that

neutralizing antibodies to IL-4, IL-5, IL-10, TNF-α, IFN-γ, and IFN-α/β failed to abrogate the Ig secretary response (Table 3). In separate experiments, using various concentrations of recombinant IL-12 and a polyclonal goat anti-mouse IL-12 antisera, we also demonstrated that NK-BMF was not IL-12 (data not shown). As indicated below, several distinct

experiments were further carried out which indicated that neither NK-BMF nor T-BMF was IL-6 (see Table 4, Table 5, and Figure 11)

Anti-CD3-activated murine CD4⁺ T cell clones also release a BMF. Studies with NK-BMF indicated that this activity was present at relatively low titers as

indicated by a requirement for its presence at 50% final volume to induce optimal Ig secretion. This has made the further characterization and identification of this activity technically difficult. Since no one has yet been successful at establishing long-term murine NK cell clones we were further hindered by an inability to select for a high BMF-producing NK cell clone. We thus determined whether other cell types were capable of releasing an activity which could similarly induce Ig secretion by αδ-dex plus IL-2-activated B^sp cells. Many antigen-specific murine in vitro CD4⁺ T cell clones have been established in numerous laboratories. It has been well-established that such clones secrete large amounts of cytokines when cultured in tissue culture wells that have been pre-coated with anti-CD3 mAb. CD3 is a component of the T cell antigen-receptor and its crosslinkage induces T cell activation. Further, CD4⁺ T cell clones have been subdivided into two broad

categories dependent upon the profile of cytokines they release upon activation. Thus Th1 clones exclusively release IL-2, IFN-γ and lymphotoxin upon activation whereas Th2 clones exclusively release IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13. Both types of Th clones share the ability to release TNF-α, IL-3, and GM-CSF. Since we demonstrated that IL-5, present

exclusively in SN from anti-CD3-activated Th2 clones, could induce Ig secretion by αδ-dex or αδ-dex plus IL-2-activated B^sp cells we evaluated Th1 clones for their potential release of a novel BMF.

Thus, SN from a single anti-CD3-activated Th1 clone was added to cultures of αδ-dex plus IL-2-activated B^sp cells and was shown to stimulate large amounts of secreted Ig (Table 2). We similarly tested 5 additional distinct Th1 clones for their ability to release a BMF upon anti-CD3 activation. All Th1 clones were found to release BMF (Fig 8). This BMF is henceforth referred to as T-BMF. In contrast to NK-BMF which was required at a 25-50% final volume to induce Ig secretion, T-BMF induced near-optimal Ig secretion at a final volume of 0.1% and continued to have Ig-inducing activity at 0.01% (Fig 9). Thus, BMF-containing T cell supernatants appeared to be at least 250-fold more potent in

Ig-inducing activity than supernatants derived from activated NK cell cultures. T-BMF, in the absence of αδ-dex+IL-1+IL-2, had no Ig-inducing effect on small B^sp cells (data not shown). T-BMF is a protein and is not among the cytokines known to induce B cell activation and differentiation. That T-BMF is a protein was demonstrated by the ability of proteinase K treatment to abrogate the Ig-inducing activity of T-BMF (Fig 10). This effect was not due to an inhibitory effect of residual, active proteinase K upon addition of proteinase K-treated T-BMF to the activated B^sp cell cultures since addition of proteinase K-treated T-BMF to αδ-dex plus IL-5-activated B^sp cultures did not reduce Ig secretion. To determine whether T-BMF was among the cytokines known to stimulate B cell activation and/or

differentiation, we stimulated Bsp cells with αδ-dex, IL-1, IL-2, and 0.1% T-BMF and added neutralizing antibodies or antagonists to IL-3, IL-4, IL-5, IL-6, IL-10, GM-CSF, TNF-α, and IFN-γ. none of the

antibodies tested inhibited Ig secretion (Table 4).

Since IL-6 has been shown to be a cytokine capable of inducing B cell maturation to Ig secretion, applicants further tested whether T-BMF was IL-6. T-BMF was not IL-6 as indicated in two distinct ways: (1) direct measurement of IL-6 in T-BMF, utilizing a highly sensitive ELISA, indicated undetectable amounts of IL-6 (<200 pg/ml) in undiluted T-BMF (Fig 11). since T-BMF induces Ig at 0.01% final volume, IL-6 if present would be <0.02 pg/ml. Since IL-6 has been reported to exhibit B cell maturation effects only at concentrations in the ng/ml range, it is highly unlikely that T-BMF was IL-6. (2) Addition of IL-6 from 2-20,000 pg/ml to cultures of αδ-dex plus IL-2-activated B^sp cells failed to induce Ig secretion (Table 5). Finally, since TNF-α and

lymphotoxin bind to the same receptor and exhibit nearly overlapping functional effects, it is highly unlikely that T-BMF is lymphotoxin, since TNF-α is not active as a BMF in this system. T-BMF is released early after anti-CD3-activation of a Th1 clone-Kinetic studies were performed to determine the time during which T-BMF was induced upon activation with anti-CD3 mAb. Approximately 90% of total T-BMF activity was induced between 2-3 h after anti-CD3 activation (Fig 12). m the absence of anti-CD3 no detectable BMF was induced in the Th1 clone,

T-BMF acts late in culture to stimulate Ig secretion by αδ-dex plus IL-1+IL-2-activated B cells. To determine the time in culture when T-BMF was required to induce Ig secretion by B cells activated with αδ-dex plus IL-1+IL-2, we established replicate cultures and added T-BMD on different days after initiation of culture. Culture SN was harvested from all experimental groups 6 days after culture

initiation. T-BMF strongly induced IG secretion, even when it was added as late as day 3 of the 6 day culture and the level of T-BMF-induced Ig secretion was

comparable to that observed when T-BMF was added at initiation of culture (Fig 13). Kinetic studies in our lab have indicated that the majority of Ig secretion in this system occurs between days 3 to 5 (data not shown). Addition of T-BMF 1 day after culture

initiation induced a substantially higher Ig secretory response relative to cultures in which T-BMF was added at culture initation (Fig 13). Peak Ig secretion, however, occured when T-BMF was added at day 2. Thus, T-BMF acts late in culture to induce Ig secretion, supporting the view that it functions as a true B cell maturation factor. The observation that T-BMF induced opitmal Ig secretion when its addition to culture was delayed by 2 days suggests that either T-BMF or a separate component in the T cell SN in inhibitory for Ig secretion when present early in culture, or that T-BMF activity progressively declines during the culture period and is not available in optimal amounts, when added at initiation of culture, for inducing Ig secretion late in culture.

Table 1

IgM secretion (ng/ml

B^sp B^e

Medium

< 50 < 50 αδ-dex

600 525

IL-2

< 50 70 αδ-dex+IL-2

2,250 16,900 αδ-dex+IL-5 7,500 7,500

LPS

119,000 90,600

Table 1. B^e and B^sp cells were cultured at 1.25 X 10₅ cells/ml in the presence of αδ-dex (3 ng/ml), IL-2 (150 U/ml), IL-5 (150 U/ml) and/or LPS (20 μg/ml). Six days after initiation of culture, SN was harvested for determination of secreted IgM concentrations by ELISA.

Table 2

IgM secretion (ng/ml) B^e B^sp

Med 26 < 5

αδ-dex 160 120

IL-l/IL-2 54 < 5

αδ-dex +

IL-l/IL-2 7,500 220

aδ-dex + IL-5 23,100 15,000

NK-BMF 16

αδ-dex +

IL-l/IL-2 + NK-BMF 8,750

T-BMF < 5

αδ-dex + T-BMF 150

αδ-dex + IL-1/IL-2+T-BMF 4,500 Table 2. B^e and B^sp cells were cultured at 1.25 X 10⁵ cells/ml in the presence of αδ-dex (3 ng/ml), IL-1 (150 U/ml), IL-2 (150 U/ml), IL-5 (150 U/ml), NK-BMF (25% v/v), and/or T-BMF (25% v/v). Six days after initiation of culture, SN was harvested for determination of secreted IgM concentrations by ELISA. Table 3

August 1992 IgM secretion (ng/ml)

B^e cells

Medium < 125 αδ-dex+IL-2 13,700 αδ-dex+IL-2+anti-TNF-α 11,500 αδ-dex+IL-2+anti-IFNγ 15,000 αδ-dex+IL-2+anti-IFNα/β 10,250 αδ-dex+IL-2+anti-IL-4 11,000 αδ-dex+IL-2+anti-IL-5 12,500 αδ-dex+IL-2+anti-IL-6 11,250 αδ-dex+IL-2+anti-IL-10 13,100 September 1992

B^sp cells IgM secretion (ng/ml)

Medium < 35 αδ-dex+IL-2 1,250 αδ-dex+IL-2+NK-BMF 35,000 αδ-dex+IL-2+IL-3 2,000 αδ-dex+IL-2+IL-4 975 αδ-dex+IL-2+IL-10 800 αδ-dex+IL-2+TNF-α 1,250 αδ-dex+IL-2+IFN-γ 2,000 αδ-dex+IL-2+IFNγ+TNFα 1,250 αδ-dex+IL-2+IFNa 1,875

αδ-dex+IL-2+IFNa+IFNγ 1,250

αδ-dex+IL-2+IFNa+TNFα 1,375

αδ-dex+IL-2+IFNa+IFNγ+TNFα 1,000

αδ-dex+IL-2+GM-CSF 1,313

αδ-dex+IL-2+TGFβ1 1,125 Table 3. (August 1992). B^e cells were cultured at 1.0 × 10⁵ cells/ml with αδ-dex (3 ng/ml), IL-1 (100 U/ml and IL-2 (150 U/ml) in the absence or presence of the following antibodies: (1) anti-TNF-α mAb (XT22; 50 g/ml), (2) anti-IFNγ mAb (XMG-6; 50 μg/ml), (3) polyclonal rabbit anti-IFNa/B anti-serum (1/50 v/v), (4) anti-IL-4 mAb (11b11; 50 μg/ml), (5) anti-IL-5 mAb (TRFK-5; 50 μg/ml), (6) anti-IL-6 mAb (P7; 50 μg/ml), and (7) anti-IL-10 mAb (SXC; 20 μg/ml). Six days after initiation of culture, SN was harvested for

determination of secreted IgM concentrations by ELISA. (September 1992). B^sp cells were cultured at 1.5 × 10⁵ cells/ml with αδ-dex (3 ng/ml), IL-1 (100 U/ml) and IL-2 (150 U/ml) in the absence or presence of the.

following cytokines: (1) IL-3 (WEHI 3 SN 25% v/v), (2) rIL-4 (1000 U/ml), (3) rIL-10 (10 U/ml), 4) rTNFα (100 U/ml), (5) rIFNα (10 U/ml), (6) rIFNa (100 U/ml), (7) rGM-CSF (100 U/ml), and/or (8) purified natural TGF-β1 (1.0 ng/ml). Six days after initiation of culture, SN was harvested for determination of secreted IgM concentrations by ELISA. (IL=interleukin, TNF=tumor necrosis factor, IFN=interferon, GM-CSF=granulocyte-macrophage colony stimulating factor, TGF=transforming growth factor)

TABLE 4

A. IgM secretion (ng/ml)

αδ-dex + IL-1 + IL-2 82αδ-dex + IL-1 + IL-2 + RA5-SN 4,000αδ-dex + IL-1 + IL-2 + RA5-SN + antiIFN-γ 3,250

+ TNFRIg 3,620 + anti-GM-CSF 2,400

+ anti-IL-3 2,300 + anti-IL-4 4,750 + anti-IL-5 4,000 + anti-IL-10 2,750

+ huCD40Fc 4,000 B.

αδ-dex + IL-1 + IL-2 350 αδ-dex + IL-1 + IL-2 + RA5-SN 15,000 αδ-dex + IL-1 + IL-2 + RA5-SN + anti-IL-6 16,200 Table 4. B^sp cells were cultured at 1.5 × 10⁵ cells/ml with αδ-dex (3 ng/ml), IL-1 (150 U/ml), IL-2 (150 U/ml) and/or T-BMF (0.1% v/v [24 hr SN from anti-CD3-activated RA5 Th1 clone]) in the absence or presence of the following antibodies/antagonists: [A] (1) anti-IFN-γ mAb (XMG-6; 10 μg/ml), (2) TNFRIg (10 μg/ml;

[TNFRIg=tumor necrosis factor receptor-Ig hybrid molecule which binds and neutralizes free TNF-α]), (3) anti-GM-CSF mAb (10 μg/ml/ purchased from Genzyme), (4) anti-IL-3 mAb (8F8.1, 10 μg/ml), (5) anti-IL-4 mAb (11B11; 10 μg/ml), (6) anti-IL-5 mAb (TRFK-5; 10 g/ml), (7) anti-IL-10 mAb (2A5; 10 μg/ml), (8) huCD40Fc (10 μg/ml; [human recombinant CD40-lg hybrid with binds and neutralizes free CD40 ligand). [B] anti-IL-6 mAG (MP 520 Pc; 10 μg/ml). Six days after initiation of culture, SN was harvested for determination of secreted IgM concentrations by ELISA.

TABLE 5

IgM secretion (ng/ml)

Medium < 100

αδ-dex < 100

αδ-dex+IL-1+IL-2 450

αδ-dex+IL-1+IL-2+T-BMF 4000

αδ-dex+IL-1+IL-2+IL-6 lU/ml 380

10 U/ml 320

100 U/ml 550

1000 U/ml 400

10,000 U/ml 430

Table 5. B^sp cells were cultured at 1.5 × 10⁵ cells/ml with αδ-dex (3 ng/ml), IL-1 (150 U/ml), in the absence or presence of various concentrations of rIL-6 (1-10,000 U/ml) and/or T-BMF (1.0% v/v [24 hr SN from anti-CD3-activated RA5 Th1 clone]). Sic days after initiation of culture, SN was harvested for

determination of secreted IgM concentrations by ELISA.

Claims

WHAT WE CLAIM IS :

1. A cell-free preparation of a novel active substance whose activity is derived from T cells or NK cells which potently induce antibody secretion by activated animal B cells, said substance having Ig secretion activity which is not mimicked by the addition of cytokine in the absence of said cell-free preparation. 2. The cell-free preparation of Claim 1 wherein the active substance is produced by NK cells. 3. The cell-free preparation of Claim 1 wherein the active substance is resistant to

neutralization by monoclonal antibodies against cytokines produced by NK cells activated by IL-2 and has Ig secretion activity which is not mimicked by the addition of NK cell cytokines in the absence of said cell-free preparation. 4. The cell-free preparation of Claim 1 wherein the active substance is a protein whose activity is derived from T cells which potently induce antibody secretion by activated animal B cells, said substance: a. is resistant to neutralization by monoclonal antibodies or receptor antagonists against cytokines produced by activated T cells;

b. has Ig secretion activity which is not mimicked by the addition of T cell cytokines in the absence of said cell-free preparation; c can be produced by activation of the T cell through the T cell receptor signalling pathway which induces cytokine production by T cells;

d. has activity which is inhibited by treatment with a proteolytic enzyme;

e. has activity which is optimal within 3 to 5 hours after activation of the T cell;

f. has detectable activity at dilution levels as low as 1:3000; and

g. operates as a late acting factor required to induce proliferated B cells to mature into antibody secreting cells.

The cell-free preparation of Claim 4 wherein the active proteinaceous substance is a

cytokine.

6. The cell-free preparation of Claim 5 which is produced by T cells.

7. The cell-free preparation of claim 1 which is free of mycoplasm.

8. The cell-free preparation of Claim 6 wherein the proteinaceous substance can increase the induction of antibody secretion 10 to 40 fold. 9. The cell-free preparation of Claim 3 wherein the active substance can increase the induction of antibody secretion 10 to 30 fold. 10. The cell-free preparation of Claim ₁ wherein the active substance whose Ig secretion activity is not mimicked by the addition of T cell or NK cell cytokines comprising Interleukin-3;

Interleukin-4; Interleukin-6; Interleukin-10; TNF-α; IFN-γ; IFN-γ and TNF-α; IFN-α; IFN-α and IFN-γ; IFN-α and TNF-α; IFN-α, IFN-γ and TNF-α; GM-CSF; and TGF-β1. 11. The cell-free preparation of Claim 8 wherein the proteinaceous substance is resistant to neutralization by monoclonal antibodies or receptor antagonists against cytokines comprising anti-IFN-γ; TNFRIg; anti-GM-CSF; anti-IL-3; anti-IL-4; anti-IL-5; anti-IL-6; anti-IL-10; and huCD40Fc. 12. The cell-free preparation of Claim 8 wherein the proteinaceous substance is produced by activation of the T cell through the T cell receptor pathway which induces cytokine production by T cells as shown in Figure 12. 13. The cell-free preparation of Claim 9 wherein the active substance is resistant to

neutralization by monoclonal antibodies against cytokines comprising anti-TNF-α; Anti-IFN-γ; anti IFNα/γ; anti-IL-4; anti-IL-5; anti-IL-6; and anti-IL-10. 14. A method of producing a cell-free preparation containing the active proteinaceous substance of Claim 4, comprising:

a. coating the bottom of a flask with anti-CD3 antibody;

c. harvesting the cell-free

supernatant;

d. inducing the activation and

antibodies in the presence of Interleukin-1 and

Interleukin-2 wherein said B cells are at least 98.5