EP0652891A1

EP0652891A1 - T-cell membrane inducing factors

Info

Publication number: EP0652891A1
Application number: EP93914341A
Authority: EP
Inventors: Jean-Michel Dayer; Dan T. Stinchcomb; Michael J. Milhausen
Original assignee: Synergen Inc
Current assignee: Amgen Boulder Inc
Priority date: 1992-06-05
Filing date: 1993-06-04
Publication date: 1995-05-17
Also published as: WO1993025572A1; CA2135397A1; JPH08501281A; AU4403993A

Abstract

Two novel proteins on the surface of activated T-cells are provided which induce monocytes to express cytokines and methods for purifying said T-cell surface proteins.

Description

T-CELL MEMBRANE INDUCING FACTORS

Field of the Invention

This invention relates to the field of immunology and immunological diseases. More specifically, the invention provides therapeutically useful proteins for immune deficiency disorders.

Background of the Invention and Information Disclosure Statement

Many diseases are characterized by the suppression, dysfunction or deficiency of T cells. Diseases which involve the suppression of T cells include HIV infection, diabetes mellitus, malaria, and cholera. Diseases which involve the dysfunction of T cells include ataxia-telangiectasia, Hodgkin's disease, hypogammaglobulinemia, and chronic lympnocytic leukemia. Diseases which are characterized by a deficiency of T cells include thymic aplasia, Di George's syndrome and lepromatous leprosy.

One of the actions of T cells is to induce monocytes to produce cytokines or perform other inflammatory functions (Amento, E. P. et al., Proc. Natl. Acad. Sci. USA 79:5307 (1982); Amento, E. P. et al., J. Immunol. 134:350 (1985);

Knight, E. Jr. et al., J. Immunol. 146:2280 (1991); Halpern, M.T. et al., J. Clin. Immunol. 11:1 (1991)). Several T cell surface molecules have been implicated in the induction of IL- lβ production by monocytes. These include membrane-associated IL-lα (Dinarello, C.A., et al., J. Immunol. 139:1902 (1987)), membrane-associated TNFα (Weaver, C.T., et al., J. Immunol. 142:3469-3476 (1989)), CD2 (Webb, D.S., et al.. Science 249:1295-1297 (1990)), and CD69 (Manie, S. et al., FASEB J. 5:A1455 (1991)). A need exists to control the ability of T cells to induce cytokine production by monocytes. The present invention satisfies this need and provides related advantages.

SUBSTITUTESHEET Summary of the Invention

The present invention relates to the identification, characterization and purification of the T cell surface proteins which induce monocytes to produce cytokines and methods for purifying said T cell surface proteins.

The purified T cell-derived proteins which are designated as TMIP-1 and TMIP-2 have the following properties. They a) are naturally located on the plasma membrane of T cells; b) induce monocytes to produce cytokines; c) are substantially present on activated but not substantially present on unactivated T cells; d) are sensitive to trypsin; e) are solubilized by 3-( (3-cholamidopropyl)- dimethylammonio)-l-propanesulfonate (CHAPS); f) bind to Q-Sepharose resin at approximately pH 8.5 and can be eluted from the Q-Sepharose with

1.) 0.12 to 0.18 M NaCl (TMIP-1), or 2.) 0.28 to 0.36 M NaCl (TMIP-2); and g) have a molecular weight of

1.) 12 to 13 kD by size exclusion chromatography

(TMIP-1) , or 2.) 36 to 37 kD by size exclusion chromatography (TMIP-2) . The present invention also provides methods for purifying the T cell-derived proteins.

Detailed Description of the Invention

When referred to herein, "substantially present" means that the factor is present in sufficient quantity on the surface of T-cells to induce cytokine production from monocytes. The cytokine measured, for example, is IL-13. When referred to herein, "activated T cells" means T cells that have been treated with any of a variety of agents that are known to mimic the physiological activation of T cells through contact with antigen presenting cells. Such agents may include, but are not to be restricted to, PMA

SUBSTITUTESHEET (phorbol-12-myristate 13-acetate) , PHA (phytohemagglutinin) , other plant lectins, antibodies to T cell surface molecules such as CD3, CD28 or T cells treated with antigen presenting cells and a specific antigen or a non-specific superantigen. These agents are collectively referred to as "T cell activating agents".

When referred to herein, "purified" means that biochemical steps have been taken to separate the active proteins from other cellular constituents. "Substantially pure" means that a single peak of protein which is isolated from cell membranes is observed upon reverse phase HPLC analysis or a single band is observed upon SDS- polyacrylamide gel electrophoresis.

Monocytes are uninduced unless otherwise specified. In uninduced monocytes, cytokine production is low or nil (e.g., measured IL-lj8 production less than about 10 pg/ml at monocyte cell concentration of about 2.5 x 10⁵ cells/ml). In induced monocytes, cytokine production is significant (e.g., measured IL-1/3 production greater than about 10 pg/ml at a monocyte cell concentration of about 2.5 x 10⁵ cells/ml).

One of the actions of T cells is to induce monocytes to produce cytokines or perform other inflammatory functions (Amento, E. P. et al., Proc. Natl. Acad. Sci. USA 79:5307 (1982); Amento, E. P. et al., J. Immunol. 134:350 (1985); Knight, E. Jr. et al., J. Immunol. 146:2280 (1991); Halpern, M.T. et al., J. Clin. Immunol. 11:1 (1991)). However, prior to the instant invention, the mechanism by which T lymphocytes induce monocytes by direct contact in a non-antigen dependent manner was unknown. We first demonstrated that monocytes are induced to produce cytokines by direct cell-cell contact with activated T lymphocytes. We then identified, characterized and purified two T cell plasma membrane proteins (TMIPs) that induced monocytes to express cytokines.

The present inventors have identified, characterized and purified two novel proteins on the surface of activated T cells that induce monocytes to express the inflammatory cytokine, IL-lβ. We term these novel proteins TMIP for T

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SUBSTITUTESHEET cell-derived monocyte inducing p_roteins. These T cell-derived monocyte inducing proteins (or TMIPs) may prove to be useful therapeutics in patients suffering from dysfunctions or deficiencies of T cells, discussed above, for example. In addition, TMIPs may be useful in the .in vitro induction of tumor infiltrating monocytes for treatment of cancer. TMIP may also be useful in the treatment of listeriosis.

Therefore, the present invention solves the problems discussed above by providing proteins useful to treat diseases which are characterized by suppressed, dysfunctional or deficient T cell action or to induce tumor infiltrating monocytes to destroy cancer cells.

The effect of contact by T cells on monocyte cytokine production was investigated using double-chamber culture wells. Adherent monocytes were cultured in the lower chambers of Transwell plates. The lower and upper chambers were separated by a 3-μm membrane which prevented physical contact between the cells but allowed diffusion of soluble factors. T cells were seeded in either the upper or the lower chamber with or without the addition of a T cell activating substance. Cells were cultured and cytokine production was measured in the supernatants. When activated T cells and uninduced monocytes were co-cultured, separated by a 3μm membrane, cytokine concentrations were six- to eight-fold higher than when unactivated T cells and uninduced monocytes were co- cultured in the absence of a separating membrane. This result shows that soluble molecules made by activated T cells can induce cytokine production by monocytes. However, when activated T cells and uninduced monocytes were co-cultured without a separating membrane, production of IL-lα, IL-lβ and TNFα were enhanced three-fold over the levels observed when the cells were separated. This observation shows that cell- cell contact between activated human T cells and human monocytes induces or augments the expression of cytokines by the human monocytes.

To further assess the ability of T cell-monocyte contact to induce or augment monocyte cytokine expression, we fixed T

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SUBSTITUTE SHEET cells before contact with monocytes. Fixation was used to preserve the integrity of cell surface molecules while preventing the release of cell-derived lymphokines or other metabolic products of the T cells that could induce monocytes. In these examples, a monocyte cell line having a homogeneous population was used to facilitate the assay of monocyte cytokine induction. The monocyte cell line was chosen for its very low baseline release of IL-lβ, even when co-cultured with fixed unactivated T cells (Tsuchiya, S., et al. Int. J. Cancer 26:171 (1980)). Freshly isolated T cells were activated with PMA and PHA and were fixed with paraformaldehyde. The fixed PMA/PHA-activated T cells strongly induced IL-lβ production by monocytes.

To obtain a homogeneous T cell population for further biochemical studies, we tested several T cell lines for the ability to induce monocyte cytokine expression. Fixed PMA/PHA-activated JURKAT or HuT-78 T cell lines induced monocytes to produce IL-lβ. These data demonstrate that activated T cells or activated T cell lines can induce monocytes by cell-cell contact alone.

To further localize the factors expressed by activated T cells that induce monocyte cytokine expression, we prepared subcellular fractions of PMA/PHA-activated and unactivated T cells. Each fraction was tested for its ability to induce monocytes to produce cytokines. Virtually all of the monocyte-inducing activity was present in the membrane fraction of PMA/PHA-activated T cells. The nuclear fraction had little effect and no inducing activity was found in the cytosolic fraction of activated T cells. Likewise, inducing activity was not found in any of the subcellular fractions of unactivated T cells. These data further support the concept that cytokine production by monocytes can be induced by cell- cell contact with activated T-cells and is not solely because of the release of soluble T cell factors. To develop an efficient and reproducible source of the above-described inducing factor, we adapted the plasma membrane preparation procedure of Maeda, et al. (Biochim.

SUBSTITUTESHEET Biophys. ACTA, 731, 115-120 (1983)) for use with activated T cell lines. The cell lines were unactivated or activated with PMA and PHA. Serial dilutions of the plasma membranes purified from T cells were added to monocytes, and IL-lβ was measured in the supernatants by ELISA. Plasma membranes isolated from unactivated T cells failed to induce measurable IL-lβ expression. In contrast, plasma membranes isolated from the activated T cells induced high levels of IL-lβ expression by monocytes. To determine whether the monocyte inducing factor present in the plasma membrane preparations was due to a protein, we treated the T cell membranes with the protease, trypsin. Trypsin treatment destroyed at least 70% of the inducing activity present in the plasma membranes isolated from induced T cells. Therefore, a protein component present on the plasma membranes of activated T cells is required for the induction of IL-lβ secretion by monocytes.

To begin biochemical characterization of the T cell plasma membrane-associated inducing protein, we solubilized the plasma membrane proteins with various detergents. Serial dilutions of the detergent solubilized plasma membrane proteins were added to monocytes and the secreted IL-lβ was measured. Of all the detergents tested, the CHAPS-solubilized protein preparation showed activity comparable to the intact membranes.

CHAPS-solubilized plasma membrane protein prepared from activated T cells was applied to a Q Sepharose (Pharmacia, Upsalla, Sweden) column. Bound proteins were eluted with a gradient of NaCl in the presence of CHAPS and individual fractions were tested for their ability to induce monocyte IL- lβ expression. The inducing activity eluted as two symmetric peaks; the first peak (peak I) eluted at 0.12 to 0.18 M NaCl and the second peak eluted at 0.28 to 0.36 M NaCl (peak II). Fractions representing each peak of activity were pooled separately, concentrated and applied to a Superdex 75 s ~ . exclusion column. Peak I eluted as a single, symmetric peak of 12 - 13 kilodaltons. Peak II eluted as a single, symmetric

SUBSTITUTESHEET peak of 36 - 37 kilodaltons. Sizes were determined by comparison to molecular weight size standards applied to the same column; the estimates have a range of error of about 2 to 5 kilodaltons. All molecular weights stated herein are presumed to include this range of error.

Several other T cell surface molecules have been implicated in the induction of IL-lβ production by monocytes. These include membrane-associated IL-lα, membrane-associated TNFα, CD2 and CD69. We have shown that a specific inhibitor of IL-lα and IL-lβ, an inhibitor of TNFα and TNFβ

(lymphotoxin) and antibodies directed against CD2 and CD69 fail to inhibit the induction of monocyte IL-lβ expression mediated by activated T cell plasma membrane proteins. Thus, the factors purified from activated T cells are novel proteins capable of inducing monocytes to synthesize IL-lβ.

In summary, these data demonstrate that PMA/PHA activated human T cells are able to induce, by cell-cell contact alone, monocytic cells to produce and release inflammatory cytokines. We have identified, characterized and purified novel activated T cell-derived monocyte inducing proteins (or TMIPs) , one of which is 12 - 13 kDa in size (TMIP-1) , the other is 36 - 37 kDa in size (TMIP-2) . These monocyte inducing proteins may be of therapeutic use in the treatment of immunodeficiency diseases or for the induction of tumor-infiltrating monocytes.

The method for purifying the T-cell derived proteins is as follows:

Activated T cells are disrupted. This can be accomplished by using a homogenizer, sonicator, or a decavitation device. Plasma membranes are prepared from the homogenized, activated T cells. The plasma membranes may be isolated by differential centrifugation or on a density or velocity gradient. Density gradients which can be used include: sucrose, dextrose or dextran sulfate. Plasma membrane proteins are solubilized in a zwitterionic detergent such as CHAPS ((3-[ (3-cholamidopropyl)-dimethylammonio]-l- propanesulfonate) ) , bound to an anionic exchange resin such

SUBSTITUTESHEET as Q-sepharose resin (Pharmacia, Uppsala, Sweden) and are eluted with a 0.0 to 0.5 linear gradient of NaCl. Other zwitterionic detergents which may be used for this purpose include: CHAPSO (3-[ (3-cholamidopropyl)-dimethylammonio]-2- hydroxy-1-propanesulfonate) , N-decyl-N,N-dimethyl-3-ammonio-l- propanesulfonate, N-dodecyl-N,N-dimethyl-3-ammonio-l- propanesulfonate, N-hexadecyl-N,N-dimethyl-3-ammonio-l- propanesulfonate, N-octadecyl-N,N-dimethyl-3-ammonio-l- propanesulfonate, N-octyl-N,N-dimethyl-3-ammonio-l- propanesulfonate, N-tetradecyl-N,N-dimethyl-3-ammonio-l- propanesulfonate. Other anionic exchange resins which may used for this purpose include: DEAE (diethylamino)-sepharose, diethylaminoethyl cellulose, diethylamino sephadex, diethyl[2- hydroxypropy1]aminoethyl-sephadex, epichlorohydrin triethanolamine cellulose, polyethyleneimine cellulose, diethyl-[2-hydroxypropyl]aminoethyl cellulose. Active fractions eluting at 0.12 to 0.18 M NaCl (TMIP-1) and at 0.28 to 0.36 M NaCl (TMIP-2) from the Q-sepharose column are separately pooled, concentrated and applied to a Superdex 75 size exclusion column (Pharmacia, Uppsala, Sweden) . Other size exclusion resins include Bio-Gel A (Biorad, Richmond, CA) , Bio-Gel P (Biorad, Richmond, CA) , Superose (Pharmacia) , and Sepharose (Pharmacia) . TMIP-1 activity elutes as a peak with a mobility indicating a molecular weight of 12 - 13 kD. Active TMIP-1 fractions can be pooled and applied to reverse phase HPLC. The active peak may be collected and may be substantially pure at this stage. TMIP-2 activity elutes from Superdex 75 as a peak of 36 - 37 kD. Active TMIP-2 fractions may be pooled and applied to a reverse phase HPLC column. Because TMIP-2 is inactivated by the reverse phase solvents, TMIP-2 will be identified by correlating an isotopically labelled protein with the TIMP-2 activity (e.g., Hannum, C. H., et al.. Nature 343:336-340 (1990)). Once labelled, the protein can be collected from reverse phase HPLC as a peak of radioactivity. TMIP-2 may be substantially pure at this stage.

SUBSTITUTE SHEET The purified protein of the present invention (TMIP) can be used in a pharmaceutical composition comprising a pharmaceutically effective amount of the purified protein and a pharmaceutically acceptable carrier. A pharmaceutically effective amount is any amount of protein which is effective in treating an animal in need of treatment. Pharmaceutically acceptable carriers include, but are not limited to physiological saline, phosphate buffered saline, water and emulsions, such as oil and water emulsions. With the purified protein of the present invention

(TMIP) , the amino acid sequence of the prominent peptide can be determined. Antibodies that react with TMIP can be used for screening expression libraries in order to obtain the gene which encodes TMIP. Synthetic peptides can be synthesized which correspond to regions of the sequence of TMIP using an Applied Biosystems automated protein synthesizer. Such peptides can be used to prepare antibodies according to procedures known in the art. Such antibodies would be useful for diagnosing autoimmune diseases, or inflammatory diseases involving activated T cells by means well known to those skilled in the art.

EXAMPLE 1 Effect of co-culture of activated T cells and monocytes on cytokine production. The effect of cell contact between activated T cells and monocytes on cytokine production was investigated using double chamber culture wells. Buffy coats were prepared from healthy donors, diluted (1:4) with Hank's Buffered Salt Solution (HBSS) (Gibco, Paisley, Scotland) and the mononuclear cells isolated by centrifugation on Ficoll- Paque (Pharmacia, Uppsala, Sweden) . The cells were washed three times with Phosphate Buffered Saline (PBS) (Dulbecco's, Gibco) and then resuspended in culture medium. Monocytes were purified by plating mononuclear cells at 4 x 10⁶/ml in the lower chambers of Transwell plates (Costar, MA) at 37°C for 1 h. After attachment, the wells were washed vigorously with warm (37°C) PBS. Adherent cells were 90-95% peroxidase-

SUBSTITUTESHEET positive. The upper and lower chambers were separated by a 3 μm membrane which prevented physical contact between the cells, but allowed diffusion of soluble factors. T cells were isolated from non-adherent mononuclear cells by passing the cells through a nylon wool column two times. In some experiments, cells were further treated with L-Leucine methyl ester to eliminate any remaining monocytes. Purified unactivated T cells were 94-98% CD2 positive, 83-94% CD3 positive and less than 2% CD14 positive, as measured by indirect immunofluorescence on an EPICS V flow cytometer (Coulter, FL) . T cells were seeded in the upper or lower chamber as shown. The cells then were cultured for 72 h with or without the addition of T cell activating agent, phytohemagglutinin (PHA) (1 μg/ml, Wellcome Diagnostic (Dartford, England) ) , and monocyte cytokine production was measured in the supernatants. IL-1 alpha and IL-1 beta were measured using an ELISA described previously (J. Grassi et al. J. Immunol. Methods 123:193 (1989)) or an IL-lβ ELISA kit (R & D Systems, Minneapolis, MN) . The sensitivity of the assays for both IL-1 alpha and IL-1 beta is 10 pg/ml. TNF alpha was measured by a TNF alpha IRMA kit (Medgenix, Belgium) . The sensitivity of this assay is 15 pg/ml. The results are shown in Table 1.

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SUBSTITUTESHEET TABLE 1

Effect of co-cultures of activated T cells and monocytes on IL-lα, IL-1B and TNFα production.

Legend to Table 1: T cells (T) and monocytes (Mo) were co- cultured in two-chamber plates as described. After incubation, IL-lα, IL-lβ and TNFα released in the supernatants were measured by ELISA. Values of IL-lα and IL-lβ represent mean ± SD (n = 3) . Values of TNFα are monoplicates;

- : absence of cell or stimulus. ND: not detectable.

EXAMPLE 2 Fixed activated T cells induce THP-1 cells to produce IL-lβ.

To further assess the importance of cell-cell contact in monocyte induction, we measured the ability of paraformaldehyde-fixed T cells to induce THP-1 monocytic cells. The fixed T cells do not release measurable cytokines; thus, this experiment shows that cell-cell contact is sufficient to induce monocyte cytokine expression. T cells, isolated as described in Example 1, were activated at 4 X 10^s cells per ml with PMA (5ng/ml, Sigma Chemical (St. Louis, MO)) and PHA (1 μg/ml) for 72 hours at 37° and 5% C0₂. The T cells were washed three times with PBS, then fixed with freshly

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SUBSTITUTESHEET The fixed cells were washed three times with PBS, resuspended in PBS and allowed to set overnight at 4°C. The cells were washed again before use and resuspended at 4 x 10⁶/ml in medium. To find a ready source of the activity, we also tested various T cell lines for the ability to induce IL-lβ production by THP-1 cells. HUT-78, a human cutaneous T cell lymphoma line, and JURKAT, a human leukemic T cell line, were obtained from the American Type Culture Collection (Rockville, MD) . The T cell lines were activated and fixed as described above for peripheral blood-derived T cells. Unactivated and fixed T cells or T cell lines were used as controls.

THP-1, a leukemic cell line derived from a patient with acute monocytic leukemia was obtained from the American Type Culture Collection (Rockville, MD) . All cells were cultured in RPMI 1640 medium (Gibco, Scotland or Mediatech, Washington, D.C.) supplemented with 10% heat induced fetal calf serum (Gibco, Scotland) or 10% defined fetal bovine serum (Hyclone Laboratories, Inc., Logan, Utah), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, Scotland or Irvine Scientific, Santa Ana, CA) . Some isolates grew faster with the addition of 5 x 10^"5 M β-mercaptoethanol (Sigma Chemical, St. Louis, MO).

Fixed activated T cells prepared above (4 x 10⁵ eelis/ml) were plated with THP-1 cells (5 x 10⁴ cells/ml) . After 48 hours of co-culture at 37°C. IL-lβ production was measured in the supernatants by ELISA. Data representative of five experiments are shown in Table 2. The data demonstrate that activated T cells and T cell lines may induce the induction of monocytic THP-1 cells by cell-cell contact alone.

12 SUBSTITUTESHEET TABLE 2

EXAMPLE 3 Subcellular fractionation of the monocyte inducing factors. Subcellular fractions of 5 x 10° unactivated or activated T lymphocytes were prepared as described by A. A. Aderem et al. (1986) Proc. Nat . Acad. Sci. USA 83. 5817. Briefly, cells were suspended at 5 x 10⁷/ml in calcium and magnesium-free PBS containing 0.25 M sucrose, 0.1 M MgCl₂, 10 mM Tris-HCl pH 7.4, 1 mM phenylmethylsulfonylfluoride, 250 Units of aprotinin and 15 mM EDTA (ethylenediaminetetraacetic acid, Sigma, St. Louis, MO) . The cell suspension was sonicated with three 5-second bursts of 90 watts power. The homogenate was centrifuged at 1,000 x g for 15 min. to isolate unbroken cells and nuclei. The supernatant was centrifuged at 100,000 x g for 30 min. in a Beckman Airfuge to separate cytosol and membranes. All manipulations were done at 4°C. Each fraction was cultured for 48 hours with 5 x 10⁴ THP-1 cells. IL-lβ in the supernatants was measured by ELISA. The results are presented in Table 3, below. As can be seen, the inducing factors were found primarily in the membrane fraction of activated T cells.

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SUBSTITUTESHEET TABLE 3

EXAMPLE 4 The monocyte inducing factors present on T cell plasma membranes are sensitive to trypsin. The HUT 78 or JURKAT cell lines were activated with 5 ng/ l PMA and 1 pg/ml PHA, at 1 x 10⁶ cells/ml for 24 hours at 37°C. The activated cells were harvested by centrifugation and washed three times in phosphate buffered saline (PBS) (Dulbeσco's, with Ca⁺² and Mg⁺²) . For breakage, the cells were resuspended at 3 x 10⁶ to 1 x 10⁷ cells/ml in buffer A (20 mM Tris; pH7.5, 10 mM NaCl, 0.1 mM MgCl₂) with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.5 μg/ml Dnase I added prior to resuspension. Cells were disrupted by three ten-second bursts with a Polytron homogenizer (Brinkman) at setting five (12,000 rpm) . Efficient breakage was assessed by microscopic examination. Nine ml of 41% sucrose (v/v) in buffer A with 1 mM PMSF was applied under 28 ml of the broken cell suspension in a 1" by 3 1/2" polyallomar ultracentrifuge tube (Beckman) . After spinning at 26,000 rpm for one hour, two to three mis of plasma membranes were harvested at the sucrose interface. The plasma membranes were diluted to eight ml with either culture media (RPMI 1640, 2mM 1-glutamine, 100 U/ml penicillin, 10° μg/ml streptomycin and 1 mM PMSF) or PBS (without Ca⁺² and g⁺² and with 1 mM PMSF) and then spun at 100,000 x g. Pellets were washed two times as described above and then resuspended as desired for bioassays or further biochemical analysis. Plasma membranes were isolated from PMA/PHA induced HuT 78

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SUBSTITUTESHEET cells by modification of the method of Maeda, et al. and were resuspended in serum-free growth medium. The plasma membranes then were incubated with 33 μg/ml trypsin for 30 minutes at 37°C. After incubation, 66 μg/ml soybean trypsin inhibitor and 10% (v/v) fetal bovine serum (FBS) were added. Control plasma membranes were incubated first with the soybean trypsin inhibitor; trypsin and FBS were added after incubation. The results shown in Table 4 indicate that much of the T cell plasma membrane-bound inducing factors are sensitive to trypsin.

TABLE 4

* Control membranes were treated first with soybean trypsin inhibitor, then trypsin.

EXAMPLE 5 The monocyte inducing proteins can be solubilized by 1% CHAPS. The following procedure efficiently solubilized the inducing protein(s) without apparent loss of activity: Plasma membranes prepared as described above were spun at 100,000 x g for 30 minutes. The resulting pellet was resuspended in the desired volume of medium or buffer containing 0.25 - 1% 3-((3- cholamidopropyl)-dimethylammonio)-1-propanesulfonate (CHAPS) and 1 mM fresh PMSF. Vigorous resuspension was required to evenly distribute the plasma membrane pellet. Plasma membrane protein concentrations up to 1 mg/ml were used. The detergent-treated membranes were incubated on ice for one hour

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SUBSTITUTE SHEET and then spun at 100,000 x g for 30 minutes. After dialysis against 4 changes of media, the solubilized proteins were filtered through 0.2 μm filters, and fetal bovine serum (FBS) was added to 10% (v/v) . Plasma membranes were isolated as described from PMA/PHA-induced HuT78 cells. The membranes were resuspended in PBS, were diluted with 1 % CHAPS (w/v) , and were incubated at 4°C for one hour. The membrane/detergent mixture was centrifuged at 100,000 x g in an SW50.1 rotor (Beckman Instruments, Palo Alto, CA) . The remaining pellet was resuspended in the original volume of

PBS. Both pellets and supernatants then were dialyzed against one liter of serum-free growth media in a multi-chamber dialysis unit (Bethesda Research Laboratories, Gaithersburg, MD) overnight at 4°C. The pellets and supernatants were added to THP-1 cells and IL-1 beta was measured in the culture media. Activated control membranes were treated as above, except PBS was substituted for the CHAPS. The results are shown in Table 5. The addition of CHAPS caused all of the inducing proteins to be solubilized.

TABLE 5

EXAMPLE 6 T-cell derived monocyte inducing proteins bind Q Sepharose and can be eluted as distinct peaks. Three liters of HUT-78 cells were grown and induced (as described above) in a 10-chamber Cell Factory (Nunc; Roshilde, Denmark) . Cells were disrupted using a sucrose gradient, although this could also be accomplished by the use of a homogenizer. Plasma membranes were prepared and were solubilized by treatment with 20 mM

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SUBSTITUTESHEET Tris, pH 8.5; 0.25% CHAPS and 1 mM PMSF. Although CHAPS was selected for use in this experiment, it is expected that any zwitterionic detergent would be effective for solubilization. After incubation at 4°C for one hour, the insoluble material was pelleted by centrifugation (100,000 x g, 30 minutes, 4°C) . The soluble supernatant was filtered through a 0.2μ Acrodisc filter (Gelman Sciences, Ann Arbor, MI) . Approximately 1 - 2 mg of solubilized plasma membrane protein was applied to an anion exchange resin column. An 8 ml Q Sepharose column equilibrated with the above buffer was used herein. Bound proteins were eluted with a 0 to 0.4 M NaCl gradient. Monocyte inducing activity was measured as described in Example 2, diluting each fraction at least eight-fold in the assay to prevent any effect of the buffers on cell viability or IL-lβ production.

Two peaks of monocyte inducing activity eluted from the Q Sepharose column. The first peak, termed TMIP-1, elutes from the Q Sepharose resin at 0.12 to 0.18 M NaCl while the second peak, termed TMIP-2, elutes from the Q Sepharose resin at 0.28 to 0.36 M NaCl. When added to the THP-1 cells, TMIP-1 induces IL-lβ expression without any detectable change in morphology. In contrast, TMIP-2 induces both IL-lβ expression and striking morphological differentiation when added to THP-1 cells.

EXAMPLE 7 Size exclusion chromatography of TMIP-1 and TMIP-2. Active fractions that eluted from the Q Sepharose column were pooled and concentrated 3- to 8-fold using Centricon 10 filter devices (Amicon; Beverly, MA) . The material containing TMIP-1 was applied to a Superdex 75 size exclusion column (Pharmacia LKB Biotechnology, Uppsala, Sweden) and was eluted with PBS + 1% CHAPS + l mM PMSF equilibrated with the same buffer. A portion of each fraction was then assayed for the ability to induce THP-1 cells to produce IL-lβ. The inducing protein elutes as a single, symmetric peak. The TMIP-1 protein is about 12 - 13 kilodaltons by comparison with molecular weight

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SUBSTITUTESHEET size standards applied to the same column under the same conditions.

Similarly, the fractions containing TMIP-2 from the Q Sepharose column were concentrated and applied to a Superdex 75 column. Fractions were assayed as described above. TMIP-2 elutes as a single symmetric peak of approximately 36 - 37 kilodaltons.

Although this invention has been described with respect to specific embodiments, it is intended not be limited thereto and may be modified by those skilled in the art without departing from the spirit and scope thereof.

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SUBSTITUTESHEET

Claims

We claim:

1. A purified T cell-derived monocyte inducing protein (TMIP) .

2. A purified T cell-derived monocyte inducing protein (TMIP) according to claim 1, TMIP-1.

3. A purified T cell-derived monocyte inducing protein (TMIP) according to claim 2 characterized as follows: a) naturally located on the plasma membrane of T cells; b) induces monocytes; c) substantially present on activated but not substantially present on unactivated T cells; d) sensitive to trypsin; e) solubilized by 3-((3-cholamidopropyl)- dimethylammonio)-l-propanesulfonate (CHAPS) ; f) a molecular weight of 12 - 13 kD by size exclusion chromatography; g) binds to Q-sepharose at about pH 8.5; and h) elutes from Q-sepharose at 0.12-0.18 M NaCl.

4. A purified T cell-derived monocyte inducing protein (TMIP) according to claim 1, TMIP-2.

5. A purified T cell-derived monocyte inducing protein (TMIP) according to claim 4 characterized as follows: a) naturally located on the plasma membrane of T cells; b) induces monocytes; c) substantially present on activated but not substantially present on unactivated T cells; d) sensitive to trypsin; e) solubilized by 3-((3-cholamidopropyl)- dimethylammonio)-l-propanesulfonate (CHAPS) ; f) a molecular weight of 36 - 37 kD by size exclusion chromatography; g) binds to Q-sepharose at about pH 8.5; and h) elutes from Q-sepharose at 0.28-0.36 M NaCl.

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SUBSTITUTE SHEET

6. A process for the purification of T cell-derived monocyte inducing protein (TMIP) comprising the steps of: a) activating T cells; b) disrupting said T-cells; c) solubilizing said protein from the plasma membrane of said T-cells using a zwitterionic detergent; d) binding said protein to an anion exchange resin column; e) eluting said protein on a salt gradient; and f) applying said protein to a column containing a size exclusion resin.

7. A purified protein produced according to the process of claim 6.

8. A purified protein produced according to the process of claim 6, TMIP-1.

9. A purified protein produced according to the process of claim 6, TMIP-2.

10. An antibody having reactivity with the purified TMIP of claim 1.

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SUBSTITUTESHEET