WO1991012330A1

WO1991012330A1 - Production of immunosuppressive factor

Info

Publication number: WO1991012330A1
Application number: PCT/JP1991/000176
Authority: WO
Inventors: Shuichi Kaminogawa
Original assignee: Takeda Chemical Industries, Ltd.
Priority date: 1990-02-16
Filing date: 1991-02-14
Publication date: 1991-08-22

Abstract

The present invention provides a method for producing an ISF-T immunosuppressive factor. The method of the present invention comprises cultivating ISF-T producing cells under conditions suitable for ISF-T production and recovering ISF-T. One such ISF-T producing cell is the mouse T cell line, 13G2. ISF-T is useful for the treatment of autoimmune diseases or immunosuppression in organ transplantation.

Description

PRODUCTION OF IMMUNOSUPPRESSIVE FACTOR

BACKGROUND OF THE INVENTION

The present invention relates to a production method for an immunosuppressive factor. More specifically, the present invention relates to a production method for an immunosuppressive factor which serves well in the treatment of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus (SLE), immunosuppression in organ transplantation and the diagnosis of various diseases due to accentuation of immunoreaction.

Although much has been reported on immunosuppressive factors produced by animal cells, none of such factors has been obtained as a pure active substance. In addition, there has been no report on an antigen- nonspecific immunosuppressive factor which is produced by antigen-specific suppressor T cells and which suppresses the proliferation of antigen-specific helper T cells as does ISF-T, the immunosuppressive factor obtained by the production method of the present invention. For example, concerning the T cell-derived immunosuppressive factor reported by H. Cantor et al. [J. Exp. Med. 153, 1260 (1981)], the T hybridoma-derived immunosuppressive factor reported by C. J. Bellone et al. [J. Immunol. 142, 244 (1989)], the T cell- derived immunosuppressive factor^" reported by L. Adorini et al. [Eur. J. Immunol. 17, 575 (1987)] and the T hybridoma-derived immuno suppressive factor reported by T. Tada et al. [J. Immunol. 133, 1371 (1984)], all have an antigen binding site and suppress nothing more than immune responses to specific antigens. Furthermore, the soluble immune response suppressor (SIRS) reported by C. W. Pierce et al. [J. Immunol. 135, 3238 (1985)] differs from the immunosuppressive factor ISF-T, obtained by the production method of the present invention, in that hydrogen peroxide is necessary for the expression of its activity. The immunosuppressive factor reported by T. Tsunematsu et al. [J. Immunol. 136, 2904 (1986)] differs from the immunosuppressive factor ISF-T of the present invention in that it is produced by concanavalin A-activated T cells, like SIRS of C. W. Pierce; it suppresses the production of T cell-independent antibodies as well. Moreover, TGF- ?, a substance which transforms rat fibroblasts and which has recently been found to show immunosuppressive action, is a basic protein, but the mouse T cell line 13G2 (IFO 50220), a cell line which produces the immunosuppressive factor ISF-T of the present invention, does not produce TGF-β. TGF-jS is judged as differing from the immunosuppressive factor ISF-T of the present invention.

The currently available immunosuppressive agents used as antirheumatic drugs are mainly gold preparations, including sodium aurothiomalate and auranofin; however, the effects of these drugs are not always satisfactory. Drugs used to suppress immunological rejection in organ transplantation include azathioprine, steroid, cyclophospamide and cyclosporin A, but azathioprine, steroid and cyclophospamide all have severe side effects, which may cause serious adverse reaction in some cases. Thus, there has been demand for drugs free of such a drawback. On the other hand, cyclosporin A possesses the problem of weak action and renal toxicity.

As stated above, there are no practically satisfactory immunosuppressive agents. On the other hand, the immunosuppressive factor ISF-T of the present invention is expected to have relatively weak side effects because it is produced by living bodies. Furthermore, this immunosuppressive factor itself acts antigen-nonspecifically, but acts on nothing more than T cells activated by antigenic stimulation,* therefore, it is expected to specifically act on the immune system and also to suppress a broad range of immurioreactions. Thus, it has potential for use as a therapeutic drug for various diseases associated with accentuated immunoreaction. Also, since the immunosuppressive factor of the present invention has an action mechanism different from that of any conventional immunosuppressive agent, it is expected to show additional effect when used in combination with a conventional drug.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 shows the proliferation suppressive action of the immunosuppressive factor ISF-T on 3D20 in a culture supernatant of 13G2 (20-fold dilution) (see Example 2). Figure 2 shows the suppressive action of 13G2 on the antigen-specific proliferation of 3D20 (see Example 2).

Figure 3 shows the suppressive effect of the immunosuppressive factor ISF-T on the proliferation of mouse lymph node cells immunized with various antigens (see Example 3).

Figure 4 shows the suppressive effect of the immunosuppressive factor ISF-T on 3D20 proliferation induced by various stimulations (see Example 4).

Figure 5 shows a gel filtration pattern of the immunosuppressive factor ISF-T (see Example 6). Figure 6 shows results of flow cytometric analysis of the 13G2 cell surface antigen (see Example 7).

Figure 7 shows the suppressive action of 13G2 on anti-bovine αsl- casein antibody production (see Example 8).

Figure 8 shows the effects of 13G2 on cell proliferation of lymph node cells derived from C3H/He and BALB/c mice immunized with bovine αsl- casein (see Example 9).

Figure 9 shows the effects of 13G2 or 3D20 on the production of T cell- independent antibody (see Example 10).

Figure 10 shows the effects of 13G2 on the cell proliferation of 3D20 stimulated with a specific antigen determinant and I-Ab-positive L cells (see Example 11).

Figure 11 shows the cytotoxicity of 13G2 (see Example 12).

Figure 12 shows the ISF-T activities in 13G2 culture supernatant after stimulation with anti-T cell receptor antibody [ O — O denotes the results obtained with the culture supernatant at 1 day of cultivation, X — X the results obtained with the culture supernatant aϊδ days of cultivation and Δ the results obtained when 3D20 was not antigenically stimulated (control)] (see Example 5).

Figure 13 shows the 13G2 cell proliferation after anti-T cell receptor antibody stimulation (see Example 5).

Figure 14 shows a gel filtration pattern of the immunosuppressive factor ISF-T(see Example 6).

Figure 15 shows pH stability of the immunosuppressive factor ISF-T (see Example 6). Figure 16 shows the pi value of the immunosuppressive factor ISF-T

(see Example 6). DETAILED DESCRIPTION

The present inventor investigated the antigenicity of bovine σsl-casein and succeeded in establishing a cell line which proliferates in response to stimulation with bovine αsl-casein and IL-2, and discovered a suppressor T cell line (mouse T cell line 13G2) which shows immunosuppressive activity [Proc. Jap. Soc. Immunol., 18, 100 (1988)] and a helper T cell line (mouse T cell line 3D20) whose antigen-specific proliferation is suppressed by that activity [Journal of the Agricultural Chemical Society of Japan, 63, 252 (1989)]. The inventor made further investigations based on this finding and developed the present invention.

Accordingly, the present invention provides a method for producing an

ISF-T immunosuppressive factor. The method of the present invention comprises cultivating ISF-T producing cells under conditions suitable for ISF- T production and recovering ISF-T. One such ISF-T producing cell is the mouse T cell line, discussed above, 13G2.

Any cell line can be used to produce the immunosuppressive factor, ISF-T, as long as it is capable of producing the immunosuppressive factor, ISF-T as described below. Examples of such cell lines include T cells, particularly mouse T cells, specifically typical T cells of CD3(+), CD4(-), CD8(+) and TCR(+), noncytotoxic T cells and MHC-non-restricted T cells, more specifically the mouse T cell line 13G2 [IFO 50220, FERM BP-3088] disclosed in working examples given below.

Any culture medium can be used to cultivate cells capable of producing the ISF-T of the present invention, as long as it allows the cells to produce the

ISF-T of the present invention. Examples of useful media include animal cell culture media suitable for cultivation, such as commercially available RPMI

1640 medium [Journal of American Medical Association, vol. 199, p. 519

(1967)]. These media may be supplemented with an antibiotic such as canamycin, penicillin or streptomycin, preferably at about 0.01 to 1 mg/m .

Cultivation may be conducted in a medium prepared by adding an animal serum such as fetal bovine serum or calf serum at about 0.01 to 50 W/W , preferably about 0.1 to 20 W/W%, to the above-mentioned medium, but it is more advantageous to conduct the cultivation without adding an animal serum to the medium because ISF-T purification is easier in the absence of animal serum. Examples of the inducer added upon cultivation include lectins [e.g., concanavalin A (ConA), phytohemagglutinin A (PHA)], various antigens and phorbol esters [12-o-tetradecanoylphorbol-13-acetate (TPA)]. These substances may be used singly or in combination of two or three. Examples of the antigen described above include alloantigens [e.g., xenogenic or heterogenic B lymphocytes whose cell division has been suppressed by mitomycin C treatment (treatment normally carried out at 10 to 200 μg/r € for 30 minutes to 5 hours) or X-ray irradiation]. Also, a specific antigen (e.g., 1 to 500 μg/ € αsl-casein for 13G2) and antigen-presenting cells (e.g., 5 to 50 X 10⁶ cells/ m€ C57BL/6 mouse splenocytes for 13G2) may also be added.

Examples of their combinations include a combination of lectin, phorbol ester and alloantigen. Specifically, it is preferable to use ConA as the lectin, TPA as the phorbol ester and heterogenic splenocytes as the alloantigen, which are added, for example, at concentrations of about 5 to 80 μg/ €, about 1 to 1,500 ng/m€ and about 1 X 10⁴ to 5 X 10⁶ cells/ mi, respectively.

Cultivation is achieved by standing culture, spinner culture, roller bottle culture or other methods known in the art. To obtain the ISF-T of the present invention by mass culture using a serum-containing medium, standing culture is preferred. Cells-are normally inoculated at a density of about 0 to 50 X 10⁶ cells/ m€, preferably about 1 to 5 X 10⁶ cells/ m€, and cultivated at about 30 to 40°C in the presence of about 1 to 20% CO₂.

During cultivation, culture medium pH is maintained normally at about 6 to 8, preferably about 7 to 7.4.

Cultivation time is determined based on the amount of the ISF-T of the present invention to be inductively produced, but it is preferable to separate and harvest the culture supernatant after cultivation for normally about 10 to 120 hours, preferably about 48 to 96 hours. To measure the ISF-T of the present invention accumulated in the medium, the αsl-casein specific helper T cell line 3D20 can be used.

To separate and purify the components of the ISF-T of the present invention from the culture supernatant, known methods of separation and purification can be used in combination. Example,? of such known methods of separation and purification include methods _'based on solubility, such as salting-out and solvent precipitation; methods based mainly on molecular weight difference, such as ultrafiltration, gel filtration and SDS- polyacrylamide gel electrophoresis; methods based on charge difference, such as ion exchange chromatography; methods based on specific affinity, such as affinity chromatography; methods based on hydrophobicity difference, such 5 as reversed phase high performance liquid chromatography and hydroxyapatite; and methods based on isoelectric point difference, such as isoelectric focusing.

The ISF-T of the present invention is separated and harvested from the culture broth of cells capable of ISF-T production described in the present 20 specification. In addition to this method, so-called gene manipulation technology permits us to obtain the ISF-T of the present invention in large amounts. It is also possible to obtain a monoclonal or polyclonal antibody against the ISF-T of the present invention by immunizing the mouse, rabbit or another animal with the ISF-T of the present invention used as - c immunogen. Obtaining such an antibody and preparing an antibody affinity column therewith permits separation and harvesting of the ISF-T of the present invention.

The immunosuppressive factor ISF-T obtained by the production method of the present invention is a new immunosuppressive factor having 20 the following biological, physical and chemical features, i) Specifically suppresses immunoreactions. ii) Antigen-nonspecifically suppresses antigen-specific immunoreactions. iii) Does not suppress immunoreactions due to T cell-independent antibody production. 25 iv) Shows immunosuppressive action by inhibiting the path of antigen presentation to T cells via antigen-presenting cells, v) Has a molecular weight of 10,000 to 100,000. vi) Inactivated by acid treatment (pH 2.0). vii) Suppresses immunoreactions caused by antigen-specific proliferation- 30 promoting action of helper T cell (e.g., mouse T cell line 3D20). viii)Stable atpH between 6-11. ix) Unstable with heat treatment, 56° C, 30 min. and 100° C, 3 min. x) Unstable under reducing coditions. xi) Has a pi value of around 6-8. 35 Since the ISF-T described above inhibits proliferation of the mouse T cell line 3D20 caused by stimulation with bovine αsl-casein, a specific antigen against 3D20, the mouse T cell line 3D20 can be used to determine ISF-T activity. For example, if we assume that an ISF-T concentration of 1 unit m is needed to obtain a 50% inhibition of mouse T cell line 3D20 proliferation, the mouse T cell line 13G2 [IFO 50220, FERM BP-3088] will produce about 100 unit m€ ISF-T.

The object of the present invention is to apply the immunosuppressive factor ISF-T, produced by an antigen-nonspecific suppressive T cell line, which suppresses antigen-specific immunoreactions, in the treatment of autoimmune diseases such ^"as rheumatism and SLE and in immunosuppression in organ transplantation, by purifying and isolating it via a highly sensitive assay system using a highly sensitive cell line and determinining its biological nature. '

Also, since conventional immunosuppressive agents are weak in immunosuppressive action in terms of drug effect, or are strong in side effects, they are not practically satisfactory, while the immunosuppressive factor ISF-T obtained by the production method of the present invention is expected to have potential for service as an icteal immunosuppressive agent.

The immunosuppressive factor ISF-T obtained by the production method of the present invention can be safely administered, orally or parenterally, to warm-blooded animals (e.g., humans, monkeys) directly or after being prepared as a pharmaceutical composition (e.g., injection) along with a pharmacologically acceptable carrier, excipient or diluent.

In preparing such a pharmaceutical preparation, monosaccharides such as glucose, amino acids, various salts, human serum albumin and other additives may be added. Isotonizing agents, pH regulators, analgesics, preservatives etc. may also be added to prepare a stable and effective immunosuppressive preparation.

The immunosuppressive factor ISF-T of the present invention serves well for the improvement and treatment of autoimmune diseases such as rheumatism and systemic lupus erythematosus, immunosuppression in organ transplantation and diagnosis*t>f various diseases occurring due to accentuation of immunoreactions. For example, when using the ISF-T described above to improve or treat articular rheumatism, the dose is selected as desired according to the route of administration (e.g., intravenous, intra- articular), symptom severity etc^ but intravenous administration is appropriate, normally in the range of about 0.01 to 100 _/*g/kg/day. It should be noted that since optimal dose conditions vary depending on the method of administration, duration of administration etc., this range is not an absolute limitation.

EXAMPLES

The present invention is hereinafter described in more detail by means of the following examples, but the invention is not by any means limited by them.

The mouse T cell line 13G2 used in the following working examples has been deposited under accession number IFO 50220 at the Institute for Fermentation, Osaka since January 9, 1990, and has also been deposited under accession number FERM BP-3088 at the Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan since September 10, 1990.

Example 1: Establishment of immunosuppressive T cell line and immunopotentiating T cell line After C57BL/6 mice were immunized repeatedly with bovine αsl- casein, a major causative substance of milk allergy, lymph node cells were taken out and cultured. Lymph node cells were stimulated repeatedly by X- ray irradiated C57BL/6 spleencells (3000R) and 100 μg/m bovine αsl-casein once weekly. Also added was 10% culture supernatant of splenocytes of the same strain stimulated with concanavalin A, to produce T cell growth factor. At 2 weeks of cultivation, cloning was conducted by the limited dilution method to establish a mouse suppressive T cell line 13G2 having immunosuppressive activity [IFO 50220, FERM BP-3088] (hereinafter also simply referred to as 13G2). Also established was a mouse immunopotentiating T cell line.3D20 (hereinafter simply referred to as 3D20) which antigen-specifically proliferates by the same method as above.

Example 2: Determination of immunosuppressive activity

Using 3D20, the activity of the immunosuppressive factor ISF-T was determined as follows: A 20 μ€ dilution, obtained by properly diluting a measuring sample containing ISF-T (a fraction with a molecular weight above 10,000 obtained by 20-fold-concentrating a 13G2 culture supernatant by ultrafiltration) with an RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 5 X 10^"5 M 2-mercaptoethanol, 100 jug/ m£ penicillin and 100 unit m streptomycin, 30 μβ of culture medium, 50 μ-S of a 4 X 10⁵ cells/ m 3D20 suspension, 50 yΛ of a 4 X 10⁶ cells/ m£ C57BL/6 mouse splenocyte suspension and 50 μi of a 400 jig/ m€ solution of bovine αsl-casein (antigen), were transferred to each well of a 96- well microculture plate and mixed therein. Cultivation was conducted at 37°C in the presence of 5% CO2 for 3 days. Then, 1 Ci ³H-thymidine was added, and cultivation was continued for 16 hours; the ³H-thymidine incorporated in the cells was then measured using a liquid scintillation counter. As shown in Figure 1, determination of ISF-T activity (suppression of proliferation of 3D20) in the 20-fold-concentrated sample obtained from an ultrafiltrative fraction with a molecular weight of more than 10,000 revealed the presence of about 2,000 unit/ m£ ISF-T. In the absence of bovine αsl-casein, ISF-T had no effects (in Figure 1, - O - represents the antigen stimulated group, - D - the unstimulated group). The same applied when cultivation was performed in the presence of X-ray-irradiated 13G2 cells in place of the sample containing ISF-T: cell proliferation due to various antigen stimulations was suppressed (Figure 2) (in Figure 2, - O - represents the antigen stimulated group, - Δ - the unstimulated group).

Example 3: Antigen-specificity of immunosuppressive factor

Lymph node cells of C57BL/6 mice immunized with bovine αsl-casein (αsl-CN), keyhole limpet hemocyanine (KLH) or jS-lactoglobulin Q3-LG) were stimulated with respective corresponding antigens, and the effects of ISF-T on their cell proliferation were assessed.

A 50 μg sample of each antigen, along with Freund's complete adjuvant (FCA), was given to C57BL/6 mice, at the paw, for immunization. Seven days later, lymph node cells were collected and transferred to a 96-welI microculture plate at 4 X 10⁵ cells per well. Secondary antigenic stimulation was performed by adding 100 μgl m£ of each antigen to each well. Cultivation was conducted in the presence of a 1/200 to 3/20 portion of the culture supernatant of 13G2 for 4 days; proliferation inhibitory action on lymph node cells was then examined. Suppressive activity is 'expressed by ³H-thymidine incorporation inhibitory ratio (Figure 3). In all antigenic stimulation systems, ISF-T inhibited cell proliferation

(in Figure 3, - Δ -, - O - and - D - respectively represent the KLH, αsl-CN and J5-LG stimulation groups). Although there were differences in the degree of inhibition, it cannot be said that the effect of ISF-T varies among antigen types, since the state of cell immunization differed among the systems.

Example 4: Effects of immunosuppressive factor on 3D20 proliferation in response to various stimulations The effects of ISF-T on the cell proliferation, induced by stimulating 3D20 with IL-2 (culture supernatant of ConA stimulated splenocytes) or anti- CD3 antibody, were studied. To 20,000 cells of 3D20 were added i) 20,000 X-ray-irradiated 13G2 cells or ii) 10% culture supernatant of 13G2, followed by daily determination of the cell proliferation inhibitory action on 3D20. Figure 4 A shows the results obtained when antigenic stimulation was conducted in the same manner as in Example 2. Figure 4B shows the results obtained with IL-2 stimulation by adding 10% of a culture supernatant of ConA-stimulated splenocytes having an IL-2 activity of 40 unit m€. Figure 4C shows the results obtained when anti-CD3 antibody stimulation was conducted by adding 10% of a culture supernatant of 145-2C11 hybridoma (kindly supplied by Dr. O. Leo at NIH). In Figure 4, - O - represents the group to which o neither 13G2 nor ISF-T was added, - D - the 13G2 addition group and - Δ - the ISF-T addition group. In the case of bovine αsl-casein stimulation, ISF-T suppressed 3D20 cell proliferation to the same extent as in mixed culture with 13G2 at 3 to 4 days of cultivation (Figure 4A). On the other hand, ISF-T did not suppress 3D20 cell proliferation induced by antigenically nonspecific 5 stimulation such as with IL-2 or anti-CD3 antibody (Figure 4B and Figure 4C).

Example 5 : 13G2 cell proliferation and ISF-T production by antigenic stimulation 0 I X 10⁶ cells/ m€ 13G2 was stimulated with a solidified anti-mouse

CD3 antibody, anti-T cell receptor (TCR) antibody or IL-2, followed by determination of ISF-T production by the assay method described in Example 2. In all systems, the ISF-T activity in the medium reached maximum within 1 day of cultivation, thereafter showing almost no increase until 5 days 5 (Figure 12). In Figure 12, X — X represents the group of 5 days cultivation and O - O represents the group of day cultivation. On the other hand, cell proliferation of 13G2 stimulated with anti-TCR antibody reached maximum at 2 days of cultivation, as' determined on the basis of ³H-thymidine incorporation (Figure 13).

Example 6: Molecular weight of immunosuppressive factor

A culture supernatant of 13G2 was subjected to gel filtration using Sephadex G-100, and the molecular weight of ISF-T was deduced.

A 15 m portion of a 20-fold-concentrated culture supernatant of 13G2 was charged to a 100 m Sephadex G-100 column. Figure 5 shows the results of determination of the absorbance at 280 nm of each obtained fraction ( — ) and determination of the cell proliferation-suppressive activity of 10% of each fraction in the measuring system described in Example 2 ( — ). In Figure 5, BSA, OVA and α-LA represent bovine serum albumin, egg white albumin and α-lactoglobulin, respectively. From Figure 5, άt is evident that antigen- specific 3D20 proliferation suppressive activity is present in the fraction with a molecular weight of 10,000 to 100,000.

Physical and chemical features of ISF-T

I- Molecular weight

The 13G2 supernatant was concentrated using a Diaflo ^® cell with a PM10 ultrafiltration membrane (molecular weight-cut off, 10, 000) (Amicon Corporation, Danver, MA). The concentrated solution was applied to a Sephadex G-100 (Pharmacia LKB Biotechnologoy, Uppsala, Sweden) column (2.6 x 100 cm) equilibrated with PBS and gel filtration was performed at a flow rate of 23.0 ml hr. The ISF-T activity of each fraction of 4.6 ml was assayed according to the measuring system described in Example 2. In Figure 14, - represents the absorbance at 280 mm of each fraction and • - • represents uptake of 3H - thymidine of each fraction.

2. Stability

(1) pH stability

One volume of the 13G2 supernatant was mixed with 9 volumes of buffers of various pHs. The mixture was kept at 4° C for 24 hr and then dialyzed against PBS at 4° C overnight. The ISF-T activity of the neutralized mixture was assayed according to the measuring system described in Example 2. The buffer compositions used were 0.1 M glycine -HCl (pH 2 - 3), 0.1 M sodium citrate (pH 4 - 5), 0.1 M sodium acetate (pH5 - 6), 0.1 M sodium phosphate (pH 6 - 7), 0.1 M Tris-HCl (pH 7 - 8), 0.1 M glycine-NaOH (pH 9 - 10.5), 0.1 M Na2HP0 Na-NaOH(pH 10.5 - 12). Figure 15 shows that ISF-T is stable at pH between 6 - 11 and that unstable both at acidic pH below 6 and alkaline pH above 11.

(2) Heat stability

As shown in Table 1, ISF-T was unstable with heat treatment, 56° C, 30 min and 100° C, 3 min.

(3) Stability under reduing conditions

ISF-T was unstable under reducing conditions in the presence of dithiothreitol (DTT). Reduction with 0.1 M DTT at 37° C for 1 hr markedly decreased the activity (Table 1).

3. Isoelectric point (pi)

Isoelectric focusing was performed with a Rotofore cell ^® (Bio-Rad Laboratories, Richmond, CA) using 2% Bio-Lyte 3/10 (Bio-Rad Laboratories). o After running at 12 W, at 4° C for 4 hr, pH of the fraction was measured. The fractions were dialyzed against PBS and then the activity was determined according to the measuring system described in Example 2. As shown in Figure 16 the pi value of ISF-T was around 6 - 8. In Figure 16, Δ - - - Δ represents the absorbance at 280 nm of each fraction, • - • represents 5 uptake of 3H-thymidine of each fraction, and O - O represents pH of each fraction.

0

5 Table 1. Stability of ISF-T

aMean ± S.D. (n = 3). Uptake of [3H]thymidine of negative control (antigen- unstimulated proliferation), 1,069 ± 171 cpm; positive control (antigen- stimulated proliferation), 31,695 ± 968 cpm. bISF-T was incubated at 37°C for 1 hr with or without 0.1 M DTT..

Example 7: Cell surface antigen of 13G2 cells

13G2 cells were analyzed for cell surface antigen by flow cytometry. When using an anti-CD4 antibody [culture supernatant of hybridoma GK1.5 (kindly supplied by Dr. F. Fitch at Chicago University)] and anti-CD8 antibody [culture supernatant of hybridoma 53-6.72 (kindly supplied by Dr. L. A. Herzenberg at Stanford University)] to stain 13G2, 13G2 was treated with these antibodies and then treated with FITC-labeled goat anti-rat immunoglobulin antibody. When using an anti-CD3 antibody (culture supernatant of hybridoma 145-2C11), 13G2 was treated with this antibody and then stained with FITC-labeled goat anti-hamster immunoglobulin antibody. When using an anti-T cell receptor (V ?8) antibody [culture supernatant of hybridoma F23.1 (kindly supplied by Dr. U. D. Starerz at Scripps Clinic and Research Foundation)], 13G2 was treated with this antibody and then stained with FITC-labeled goat anti-mouse immunoglobulin antibody. 13G2 stained with secondary antibody alone was used as control. Cell surface antigen positive cell population was determined using FACS. Results are shown in Panels A and B of Figure 6 (in Figure 6A, — represents the control, — the anti-CD4 antibody and — the anti-CD8 antibody; in Figure 6B, — represents the control, — the anti-CD3 antibody and — the anti-V_.08 antibody).

As seen in Panels A and B of Figure 6, 13G2 became stained with anti- CD3 antibody, anti-CD8 antibody and anti-TCR(V 8) antibody (F23.1), while it did not become stained with anti-CD4 antibody. 13G2 was thus found to be a typical suppressor T cell line.

Example 8: Effects on antibody production

Effects of X-ray irradiated 13G2 on the anti-bovine αsl-casein antibody production induced by antigenically stimulating mouse lymph node cells immunized with bovine αsl-casein were assessed. Specifically, C57BL/6 mice were immunized with 50 μg of bovine αsl-casein, along with Freund's complete adjuvant, by injection at the paw. At 14 days, lymph node cells were collected. To each well of a 48-well culture plate, 2 X 10⁶ lymph node cells and 100 to 3,000 X-ray-irradiated 13G2 or 3D20 cells were transferred, followed by secondary stimulation with 10 μgl mi bovine αsl-casein. After 11 days of cultivation, the culture supernatant was assayed for anti-bovine αsl- casein antibody level by the EIISA method, performed by adding the culture supernatant to a microculture plate coated with bovine αsl-casein and determining the amount of bound antibody using an alkaline phosphatase- labeled anti-mouse immunoglobulin antibody. Results are shown in Figure 7, where - O - represents the 13G2 addition group and - Δ - the 3D20 addition group. As seen in Figure 7, when 300 cells of 13G2 were added to 2 X 10^€ lymph node cells, anti-bovine αsl-casein antibody production decreased to about one-fifth level. When 1,000 cells of 13G2 were added, antibody production was almost completely suppressed. Such suppressive activity on antibody production was not noted in 3D20. On the other hand, as is evident from Figure 2, generated in Example 2, to completely suppress the antigen- specific cell proliferation of 3D20, 10,000 cells of 13G2 were necessary for 20,000 cells of 3D20.

Example 9 : Major histocompatibility complex (MHC) dependency of immunosuppression

C3H/He or BALB/c mice were immunized with bovine αsl-casein, and it was examined whether the antigen-specific proliferation of their lymph node cells is inhibited by mixed culture in the presence of X-ray-irradiated 13G2. Specifically, C3H/He or BALB/c mice were immunized with 100 μg of bovine αsl-casein along with Freund's complete adjuvant by injection at the paw. At 7 days, lymph node cells were collected. To each well of a 96-well microculture plate, 4 X 10^δ lymph node cells and 3 X 10⁴ X-ray-irradiated 13G2 cells were transferred, and cultivation was conducted for 4 days in the presence of 100 μgl m€ antigen, and the proliferation suppressive effect on the lymph node cells was assessed. Suppressive activity was determined on the basis of suppression of ³H-thymidine incorporation. Results are shown in Figure 8. Figure 8 shows that, as in C57BL/6 mice, antigen-specific proliferation of lymph node cell was suppressed in C3H/He or BALB/c mice. In other words, the immunosuppressive action of 13G2 had no restriction of ^MRC- Example 10: Effects on production of T cell-independent antibody

Effects of X-ray-irradiated 13G2 on the IgM antibody production induced by stimulating C57BL/6 mouse splenocytes with lipopolysaccharide (LPS), a B cell mitogen, were assessed. Specifically, effects of X-ray- irradiated 13G2 or 3D20 (100 to 3,000 cells/well) on the IgM antibody production induced by adding 25 μgl mi LPS to C57BL/6 mouse splenocytes (4 X 10^δ cells/well) and carrying out cultivation for 4 days were assessed. The amount of IgM antibody produced was determined by ELISA. ELISA was performed by adding the culture supernatant to a microculture plate coated with goat anti-mouse IgM antibody and determining the amount of bound IgM antibody using an alkaline phosphatase-labeled goat anti-mouse IgM antibody. Results are shown in Figure 9 (in Figure 9, - O - represents the 13G2 addition group, - Δ - the 3D20 addition group and the LPS-free group). Figure 9 shows that 13G2, like helper T cell line 3D20, had no effect on LPS-induced IgM production.

Example 11: Effects on antigen-presenting cells

The gene of I-Ab, an H-2 class II antigen, was introduced into mouse L cells to prepare L cells wherein I-Ab antigen is expressed on the cell surface (kindly supplied by Dr. R. Germain at NIH). These cells were fixed with 1% p- formaldehyde. Using these cells as antigen-presenting cells (20,000 cells/well), 3D20 cells (20,000 cells/well) were stimulated with 100 μgl mi of bovine αsl-casein or the 3D20-recognizing bovine αsl-casein antigen site (amino acid residues at 136 through 155 positions) as antigen, and cultivation was carried out in the presence of X-ray-irradiated 13G2 (10,000 cells/well) for 4 days; the effects on 3D20 cell proliferation were then assessed. Results are shown in Figure 10. As seen in Figure 10, when 10,000 X-ray-irradiated 13G2 cells were added to 20,000 3D20 cells, 3D20 cell proliferation was suppressed to about a one-third level only when the bovine αsl-casein antigen determinant fragment was used as stimulant. This finding suggests that the immunosuppressive action of 13G2 is not mediated by antigen-processing by macrophages or dendritic cells and that the I-Ab antigen on L cells and bovine αsl-casein itself are not mutually interactive.

To further clarify the action mechanism of ISF-T, it was investigated whether ISF-T acts on antigen-presenting cells or helper T cells, on the basis of ³H-thymidine incorporation by 3D20. X-ray-irradiated antigen-presenting cells were cultivated in the presence of ISF-T for 16 hours. After ISF-T removal by washing, the X-ray-irradiated antigen-presenting cells were cultivated along with 3D20 and an antigen (αsl-casein). 3D20 cell proliferation was suppressed to abput one-third level in comparison with the ISF-T-free group. By contrast, 3D20 cell proliferation remained unchanged even when 3D20 was cultivated along with antigen-presenting cells and an antigen after they were treated with ISF-T (Table 2).

Table 2. Action mechanism of ISF-T

Precultivation ³H-thymidine incorporation by 3D20 in 4-day cultivation

Cells ISF-T (CPM)

Antigen-presenting cells - 8876 ± 1636

Antigen-presenting cells + 2675 ± 300

3D20 - 15269 ± 876

3D20 + 22462 ± 3044

Example 12: Cytotoxicity of 13G2

Using allokiller cells against H-2b as control, the cytotoxicity of 13G2 on 3D20 and MLPBβ, a C57BL/mousf dβrived B cell line, was assessed.

Specifically, the cytotoxicity of the,H-2b specific allokiller cells, obtained by stimulating C3H He mouse splenocytes with X-ray-irradiated C57BL/6 mouse splenocytes for 8 days and 13G2, was assessed in a ⁵¹Cr release assay for 4 hours using 31⁾20 or MLPBβ, as target. Results are shown in Panels A and B of Figure 11 (Figures 11A and B show the cytotoxicity on 3D20 and MLPBβ, respectively; - • - and - O - denote the cytotoxicity of allokiller cells and that of 13G2, respectively). As is evident from Figures, 11A and B, 13G2, like normal splenocytes, had almost no cytotoxicity on 3D20 or MLPBβ. In conclusion, it can be considered that the cell proliferation suppressive action of 13G2 is not mediated by its cytotoxicity but is exhibited by cell inactivation of helper T cells.

Claims

Claims:

1. A method for producing an ISF-T immunosuppressive factor, comprising cultivating ISF-T producing cells under conditions suitable for ISF-T production and recovering ISF-T.

2. A method according to claim 1, wherein the cells are T cells.

3. A method according to claim 1 , wherein the cells are mouse T cells.

4. A method according to claim 1, wherein the cells are selected from the group consisting of T cells of CD3( + ), CD4(-), CD8( + ) and TCR( + ), noncytotoxic T cells and MHC-non-restricted T cells.

5. A method according to claim 1, wherein the cell has the properties of the mouse T cell line 13G2 [IFO 50220, FERM BP-3088].

6. A method according to claim 1, wherein the cells are cultivated in an animal cell culture medium.

7. A method according to claim 6, wherein the medium is RPMI 1640 medium.

8. A method according to claim 6, wherein the medium contains an antibiotic.

9. A method according to claim 6, wherein the medium contains an inducer.

10. A method according to claim 9, wherein the inducer is a lection, antigen or phorbol ester used, singly or in combination of two or three.

11. A method according to claim 10, wherein the lectin is about 5 to 80 μg/ml of concanavalin A, the antigen is about 1 x 10⁴ to 5 x 10⁶ cells/ml of heterogenic splenocytes whose cell division has been inhibited, and the phorbol ester is about 1 to 1,500 ng/ml of 12-O-tetradexanoylphorbol.

12. A method according to claim 1, wherein about 0.1 to 50 x 10⁶ cells/m€ of the cells are cultivated at about 30 to 40°C in the presence of about 1 to 20% CO2 for about 10 to 120 hours in a culture medium of about pH 6 to 8.

13. A method according to claim 1, wherein the cells are cultivated by roller bottle culture using a serum-containing medium.

14. A method according to claim 1, wherein the ISF-T is separated and purified by a method based on solubility, molecular weight difference, charge difference, specific affinity, hydrophobicity or isoelectric point difference, or a combination thereof.

15. A substantially pure ISF-T made according to the method of claim 1.

16. The ISF-T immunosuppressive factor made by the method of claim 1, wherein the ISF-T has the following biological, physical and chemical features: i) Specifically suppresses immunoreactions. ii) Antigen-nonspecifically suppresses antigen-specific immunoreactions. iii) Does not suppress T cell-independent antibody production. iv) Shows immunosuppressive action by inhibiting the path of antigen presentation to T cells via antigen-presenting cells, v) Has a molecular weight of 10,000 to 100,000. vi) Inactivated by acid treatment (pH 2.0). vii) Suppresses immunoreactions caused by antigen-specific helper T cell, viii) Stable at pH between 6-11. ix) Unstable with heat treatment, 56°C, 30 min. and 100°C, 3 min. x) Unstable under reducing conditions, xi) Has a pi value of around 6-8.

International Application No: PCT/JP 91 / 00176

MICROORGANISMS

Optional Sheet In connection with the microorganism referred to on page 2 . line 5_ of the description ■

A. IDENTIFICATION OF DEPOSIT2

Further deposits are identified on an additional sheet D ³

Name of depositary Institution⁴ IFO: Institute for Fermentation, Osaka

FRI: Fermentation Research Institute, Agency of Industrial Science and Technology Ministry of International Trade and Industry

Address of depositary Institution (Including postal code and country)⁴

IFO: 17-85, Juso-honmachi 2-chome, Yodogawa-ku, Osaka-shi, Osaka 532 Japan

FRI: 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki 305 Japan

Date of deposit s Accession Number 6

IFO: 19 . 01. 90 IFO- 50220 FRI: 07 . 09. 90 FERMBP- 308f

B. ADDITIONAL INDICATIONS⁷ (leave blank if not applicable). This information is continued on a separate attached sheetD

In respect of those designations in which a European patent is sought, a sample of the deposited microorganism will be made available until the publication of the mention of the grant of the European patent or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a sample to an expert nominated by the person requesting the sample. (Ruel 28(4) EPC)

C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE 3 (if the indicationsare notfor all designated States)

States members of the European Patent Convention which have been designated for the purpose of a European Patent.

D. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indication listed below will be submitted to the International Bureau later⁹(Specify the general nature of the indications e.g., 'Accession Number of Deposit")

D This sheet was received with the international application when filed (to be checked by the receiving Office)

(Authorized Officer) !_>_. The date of receipt (from the applicant) by the International Bureau¹⁰

Form PCT/RO/134 (January 1981)