JPS62123126A - Cell dna synthesis inhibitory factor - Google Patents

Abstract

PURPOSE:The titled factor, produced from human lymphocytic cells and useful for inhibiting transplantation rejection of transplanted patients or treating autoimmune disease, allergy, abnormal lymphocytosis and cancer. CONSTITUTION:A cell DNA synthesis inhibitory factor, separated and purified from a cultivation supernant of T cell hybridoma prepared by fusing human lymphotic cells or human pripheral blood lymphotic cells, e.g. T cell groups separated as human peripheral blood lymphocytes oor human lymphocytes, etc., with human leukemic cell strains, etc., and having the following properties; (i) Molecular weight; 45,000-70,000 (measured by gel filtration chromatography). (ii) Unadsorbed on immobilized concanavalin A-sepharose. (iii) Isoelectric point; 4.5-5.5. (iv) Eluted with FPLC-Mono Q anion exchange chromatography at 0.5-0.7M common salt concentration. (v) Physico-chemical properties shown in the table. The above-mentioned factor is capable of inhibiting cell DNA synthesis of a wide range beyond animal species, e.g. humans, rats, mice, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel human-derived cellular DNA synthesis inhibitor.

[Conventional technology]

DNA of cells released from lymphocytes, especially Ta1 cells, activated by stimulation with various lectins and alloantigens, etc.
Many physiologically active substances that have the effect of inhibiting synthesis, division, and proliferation have been widely praised. For example, Nam
ba et al. extracted biologically active factors (inhibitors of DNA 5ynthes) that have the effect of inhibiting DNA synthesis from rat tile cells stimulated with concanavalin A1 or ovalbumin, mouse cells, or rat lymph node cells activated with phytohemagglutinin.
is, IDS) was found to be released, and by purification, molecules 1i80,000, with an isoelectric point of 2.
reported that it is a glycoprotein of 7 to 2.9 (In
flammatron, IS, p. 5).

In addition, Aune et al. detected a physiologically active factor (soluble immunostimulant) that suppresses antibody production against sheep red blood cells from mouse tile cells stimulated with concanavalin A.
ne response 5uppressor,
It was discovered that S[R3) is released, and the factor is a protein with a molecular weight of 48,000 to 67,000 and
reported that it is produced from suppressor T cells positive for the 23 antigen and becomes activated by hydrogen peroxide produced by macrophages (J. Immunol, 1).
27, 1828%), and 5chnaper et al. reported that when human Tomo 1 cells were stimulated with concanavalin A, etc., a bioactive factor with 5IR3-like activity of 1,110,000 to 150,000 molecules was produced. (J, Immunoj, 132 e, 2
429 pages).

In addition, Greene et al. reported that human peripheral blood lymphocytes stimulated with concanavalin 8 were found to contain a bioactive factor (5oluble immunesu
ppressor 5uppernatant of T
Cell proliferation.

5ISS-T) was found to be released. 5I
It has been reported that SS-T has a molecular weight of 30,000 to 40,000 and its activity is inhibited by N-acetyl-D-glucosamine (J, Immunol,
, 126, p. 1185) 6 On the other hand, the present inventors have obtained a physiologically active factor (s
timed Tcell-derived 1
nhibitory factor for cell
ularDNA 5ynthesis + 5TIF
) was found to be produced. Rat 5TIF has a molecular weight of 45,000-50,000 and an isoelectric point of 5.1-5.
．． 6. Concanavalin'A is a non-cephalinose adsorbing protein that has been reported to have the activity of inhibiting DNA synthesis in various cells of rat, mouse, or human origin (J, Immunol.

134　S　page 1019).

[Problem that the invention seeks to solve]

As mentioned above, the existence of various activated lymphocyte-derived physiologically active factors has been reported many times, but in most cases, sufficient knowledge of their molecular properties has not been obtained.
Moreover, at present, the elucidation of instant release purification and its mechanism of action is not sufficient.

[Means to solve the problem]

In view of these circumstances, the present inventors have conducted extensive research and have discovered that a novel cellular DNA synthesis inhibitor is produced from human lymphocytes, leading to the completion of the present invention.

That is, the present invention relates to a cellular DNA synthesis inhibitor derived from human lymphocytes and characterized by the following properties.

(a) Molecular weight is 45,000 to 70,000 (gel i
I! (b) non-adsorbent to 1 Ml regulated concanavalin A-Sepharose; (C) has an isoelectric point of 4.5-5.5; (d) FP
(e) Insensitive to deoxyribonuclease, ribonuclease, periodic acid treatment, trypsin, α-chymo-1-lipsin, eluted at a salt concentration of 0.5-0.7M by LC-Mono Q anion exchange chromatography. , sensitive to proase, (f) stable in the pH range of 2 to 7, (g) inactivated by treatment at 90°C for 30 minutes.

Human peripheral blood lymphocytes are used as the human lymphocytes used in the present invention, and these cells can be left unstimulated or stimulated with various stimulants. Further, as human lymphocytes, specific cell groups such as a differentiated T cell population or a T8 antigen- or Leu 2 antigen-positive subrenal T cell-cytotoxic T cell subgroup can also be used.

Any stimulant that can induce the cellular DNA synthesis factor of the present invention may be used, such as concanavalin A (ConA), phytohemagglutinin (PIIA), and bork wheat mitogen (I'WM). Examples include lectins such as ribopolysaccharide (LPS), various antigens, and phorbol esters (e.g., 4β-phorbol 12-myristate 13-acetate-1-: PMA), and these can be used in Chinese or in combination. can do.

Furthermore, human hybrid cell culture lines (TS [B-cell hybridomas]) obtained by fusing human peripheral blood lymphocytes with human leukemia cell lines, etc., and clones thereof, which produce the cellular DNA synthesis inhibitor of the present invention, are also available. can be used.

As a method for producing a human T cell hybridoma that produces the cellular DNA synthesis inhibitor of the present invention, a method normally used for cell fusion can be used. isolated human peripheral blood lymphocytes and isolated T cell populations, or
8 antigen or Leu2 antigen positive suppressor T cells-
Cytotoxic cell subpopulations and the like can be used. On the other hand, the parent cell line used for cell fusion is CCRF-CE, which is resistant to various drugs such as 8-azaguanine, 6-thioguanine, or 5-bromodeoxyuridine, and does not produce other physiologically active substances. I, C.C.
RF-[l5B2. Human Tl1l cell leukemia cell lines such as MOLT-3 and MOLT-4, as well as various human T-cell leukemia cell lines treated with various protein synthesis inhibitors, nucleic acid synthesis inhibitors, or both, can be used. can. Here, as the protein synthesis inhibitor, known eukaryotic protein synthesis inhibitors can be used, but irreversible protein synthesis inhibitors such as emetine hydrochloride are preferred. In addition, examples of nucleic acid synthesis inhibitors include DNA synthesis inhibitors, and specific examples include actinomycin D5 adriamycin, α-amantine, ethidium bromide, and rifampicin. In addition to various viruses such as Sendai virus, polyethylene glycol (PEG), polypropylene glycol (PPG), and polyvinyl alcohol (PPG) can be used to induce cell fusion reactions.
A cell fusion inducer such as VA'') can be used.

For selection of fused hybridomas, when various drug-resistant strains are used as parent strains, it is desirable to use a selective medium in which the parent strain cannot grow, such as a selective medium containing hypoxanthine, aminopterin, or thymidine. Furthermore, when a drug-treated human T-cell leukemia cell line is used as a parent strain, selection can be carried out in a normal medium.

The human T cell hybridoma producing the cellular DNA synthesis inhibitor of the present invention can be cloned by conventional methods such as limiting dilution method, semi-solid medium culture method (soft agar method), etc. For the clones, the activity was measured and the human 1-Tf that produced this factor was determined.
[Select cell hybridoma clones.

As an activity measurement method for selecting human T cell hybridoma clones that produce this factor, lymphoid cells such as rat and mouse bone marrow cells, thymocytes, lung cells, or human peripheral blood lymphocytes are cultured unstimulated or treated with concanavalin. A (Con A), phytohemagglutinin (PII
A), mitogens such as pork lyde mitogen (PWM), interleukin-1 (IL-1), interleukin-2 (IL-2), colony stimulating factor (
The inhibitory effect on thymidine uptake of cells induced by stimulation with various stimulants such as C3F) is measured as the DNA synthesis inhibitory effect of those cells.At this time, the active fraction of the cellular DNA synthesis inhibitor If thymidine is present in the cell culture fluid used as a cell culture medium, the activity should be evaluated based on the inhibitory effect on the increase in the number of the various cells mentioned above, or the inhibitory effect on the production of specific substances produced by those cells. This is desirable.

The selected clones can be cultured permanently after examining optimal conditions such as whether or not to add serum, cell concentration, culture time, stimulating substances, and culture scale.

In this way, the cellular DNA synthesis inhibitor of the present invention can be obtained in virtually unlimited quantities from the culture supernatant of these cells.

Furthermore, the cellular DNA synthesis inhibitor of the present invention can also be produced in large intestine seedlings or human cultured cell lines by genetic engineering means.

The cell DNA synthesis inhibitor of the present invention obtained by the above-mentioned cells or production methods can be separated and purified using conventional methods generally used for protein separation, such as salting out, centrifugation, dialysis, and gel filtration chromatography. It can be carried out by a combination of methods such as , ion exchange chromatography, affinity chromatography, ultrafiltration and lyophilization.

That is, after concentrating the culture supernatant obtained in the present invention by salting out ammonium sulfate, the molecular weight is io, ooo ~ 200.000.
Perform gel filtration using a carrier suitable for separating substances,
Furthermore, by subjecting the active fraction to affinity chromatography using immobilized concanavalin A, both fractions that are not adsorbed to concanavalin A Sepharose are obtained. Furthermore, the active fraction may be subjected to preparative isoelectric focusing using Ultrodesox or FPLC-Mon
o It can also be obtained by performing Q anion exchange chromatography. For storage, freeze-drying is preferred.

The cellular DNA synthesis inhibitor of the present invention thus obtained is a proteinaceous substance having the following properties.

+11 When the culture supernatant is salted out with ammonium sulfate, activity is observed in the 0-50% saturated ammonium sulfate fraction, and almost no activity is observed in the 50-90% saturated fraction.

(2) Molecular weight: The molecular weight is 45.000 to 70.000 as determined by gel filtration using a column of Sephacryl S-200 (manufactured by Pharmacia) (Figure 1).

(3) It is non-adsorbable to immobilized concanavalin A-Sepharose (Figure 2).

(4) Isoelectric point: The isoelectric point is 4.5 to 5.5 by performing preparative isoelectric focusing (pH 3 to 10) using Ultrodesox (LKB Co., Ltd.). (3rd
figure).

(5) Anion exchange chromatography
Anion exchange chromatography using L CMono Q column (manufactured by Pharmacia) was performed at pH 8 and 0 to 1.
By using a linear concentration gradient of sodium chloride of M, elution is achieved at a salt concentration of 0.5 to 0.7 M (Figure 4).

(6) Physicochemical properties: Insensitive to deoxyliponuclease, ribonuclease A treatment, and periodate oxidation, but sensitive to trypsin, α-chymotrypsin, and proase treatment, and stable in the pH range of 2 to 7. However, it is deactivated by heat treatment at 90°C for 30 minutes (first
table).

Table 1: Physicochemical properties of cellular DNA synthesis inhibitor In addition, other biochemical properties include: (7) Addition of 2-mercaptoethanol and Rehamisole does not inhibit the activity; (8) N-acetyl- The activity is not inhibited by the addition of D-glucosamine, N-acetyl-D-galactosamine, and α-methyl-D-mannoside. (9) The activity is not inhibited by the addition of L-arginine and L-ornithine.

00) Biological Activity 2 The cellular DNA synthesis inhibitor of the present invention has the following biological activities.

b) Cell D using 'H-thymidine uptake of rat bone marrow cells, mouse bone marrow cells, and rat thymidocytes as an indicator
Suppresses NA synthesis.

) Inhibits the proliferation of human, Laso1~ and mouse T cells stimulated with T cell mitogens such as Concanavalin A (Con A) and Fi1-hemagglutinin (P)IA).

C) Proliferation of human, rat, and mouse B cells stimulated with B cell mitogens such as Hawkelyde mitogen (PWM) and Staphylococcus aureus colawan I (SAC) and immunoglobulin G (
IgG) or IgM production.

2) Cultured tumor cells derived from humans, mice, or rats (
CCRF-5B, RPMI B226, H3B-
2, K 562, N5-1, SI'-2, L 1210
, P 388 , U 937 , RPMI 1788, etc.) has antitumor activity such as suppressing DNA synthesis and proliferation using 3H-thymidine incorporation as an indicator.

e) Suppresses IL2-dependent proliferation of human and mouse-derived T cells.

f) No cytotoxicity is shown within 24 hours against actinomycin D-treated mouse 929 cells.

[Action and effect of the invention]

The cellular DNA synthesis inhibitor of the present invention has the properties shown above and is novel, different from conventional human TIII-derived inhibitory lymphokines. Furthermore, this factor suppresses DNA synthesis in cells across a wide range of animal species, including humans, rats, and mice.

Therefore, this factor is useful for suppressing transplant rejection in patients who have received xenogeneic or allogeneic transplants such as bone marrow, kidney, and heart transplants.
Alternatively, it can be applied to the treatment of systemic lupus erythematosus disease (SLD), autoimmune diseases such as rheumatoid arthritis, allergies, lymphocyte proliferation abnormalities such as leukemia, and cancer. Furthermore, this factor can be widely used as a reagent in the medical and pharmaceutical fields.

When the cellular DNA synthesis inhibitor of the present invention is used as a medicine, it can be formulated according to ordinary formulation techniques, for example, by mixing the drug substance with carriers, excipients, diluents, etc. It can be provided in the form of capsules, injections, etc.

〔Example〕

EXAMPLES The present invention will be specifically described below with reference to Examples and Experimental Examples, but the present invention is not limited by these Examples and Experimental Examples.

Example 1 (a) Production and purification of cellular DNA synthesis inhibitor using human peripheral blood lymphocytes Human peripheral blood lymphocytes obtained by density gradient centrifugation using Ficoll Puncture (manufactured by Pharmacia) were mixed with 2% beef tallow. Child serum (heat inactivated), kanamycin 60μg/ml, N2
-Hydroxyethylpiperazine-N''-2-ethanesulfonate ()IEPES'') 10rr+R containing M
Culture at 37°C for 48 hours in air containing 5% carbon dioxide in the presence of 10 μg/ml concanavalin A (Sigma) and 5 x 10 M of 2-mercaptoethanol in P old 1640 medium. . Thereafter, the culture supernatant (h-Con) obtained by removing cells by centrifugation
Solid ammonium sulfate was added to A Sup ) at 4°C, and the precipitates were fractionated into a 50% saturated ammonium sulfate fraction and then a 90% saturated ammonium sulfate fraction. After collection by refrigerated high-speed centrifugation, phosphate buffer (pH 7)
, 4) and reduce the volume to about 1/100th. Phosphate buffer (p
Dialyze overnight against H7,4), sterilize by filtration, and store at 4°C. The thus obtained concentrated h-Con A Sup
was used for purification after measuring its activity using rat bone marrow cells. The activity is concentrated in the O~50% saturated ammonium sulfate fraction.

Using a column (IX90cai) of Sephacryl S-200 (manufactured by Pharmacia), a phosphate buffer solution (pH 7,4
), a molecular weight calibration curve was prepared using standard proteins (Pharmacia!!: ribonuclease A, chymotrypsinogen A, ovalbumin, bovine serum albumin), and each percentage obtained by gel filtration was When the molecular weight was measured by activity evaluation using rat bone marrow cells, the activity peak was eluted between ovalbumin and bovine serum albumin, as shown in Figure 1.
The molecular weight was 45,000-70,000.

Fractionation was performed using a Sephacryl S-200 column, the obtained active fraction was concentrated by ultrafiltration, and a column (lX
Affinity chromatography is performed using a 7.5 cm). Elution begins with phosphate buffer (1) H7,4)
This is carried out using 75 ml to obtain a concanavalin A non-adsorbable fraction. Next, phosphate buffer'/P1. (pH 7,4) 5
Elute with 0 ml L' to obtain a concanavalin A adsorbent fraction. After dialysis with phosphate buffer overnight for each portion, the activity was evaluated using rat bone marrow cells.
As shown in FIG. 2, activity was observed in both concanavalin A non-adsorbable fractions.

Concanavalin A non-adsorbable fraction was concentrated by ultrafiltration and dialyzed overnight against deionized water, followed by Ultrodex (
(manufactured by LKB) 4g, Ampholine pH 3-10 (LKB
Mix 5+wl and deionized water, approximately 100m
1 is prepared, and isoelectric focusing is performed at 4° C. for 6 to 8 hours with a constant power of 18 W. Immediately after the electrophoresis is completed, the gel is fractionated into 30 fractions, the pH of each fraction is measured, and then the protein is eluted from the gel with deionized water. After further dialysis with phosphate buffer (p+-47,4) for one night, the activity was evaluated using rat bone marrow cells.
It was recognized in the range of 5.5.

In addition, both concanavalin A non-adsorbable fractions were concentrated by 1 µl extrafiltration, dialyzed overnight against deionized water, and 500 µl
/ is fractionated by FT'LC-Mono Q anion exchange chromatography (manufactured by Pharmacia).
Trishydroxyaminemethane 30mM, pH 8.0)
After adding the sample, at a flow rate of 1 ml/min,
Elute with 10 ml of Tris-HCl buffer followed by a linear concentration gradient (10 ml) of 0-1.0 M NaCl. 1m each
Both portions of the sample were collected and dialyzed against phosphate buffer (pH 7, 4) for one night, and the activity was evaluated using rat bone marrow cells. As shown in FIG. 4, the activity was 0.1. 5-0.7
It was observed in both portions of M that were eluted with sodium chloride.

(b) Physicochemical and biochemical properties The partially purified cellular DNA synthesis inhibitor was examined for deoxyribonuclease, ribonuclease A, trypsin, α-chymotrypsin, proase treatment, periodate oxidation, temperature, and thermostability. . Deoxyribonuclease (manufactured by Sigma) 50 μg/ml, ribonuclease A (manufactured by Sigma >50 μg/ml, trypsin (manufactured by Sigma) 50 μg/ml, α-chymotrypsin (manufactured by Sigma) 50 μg/ml, proase (manufactured by Sigma) 50 μg/ml of each cell DNA synthesis inhibitor was added and treated at 37°C for 3 hours, and for the trypsin treatment group, soybean trypsin inhibitor 150
After adding μg/ml at the end of treatment, activity evaluation using rat bone marrow cells was performed. Also, periodate oxidation
Add 5mM of sodium periodate (manufactured by Wako Pure Chemical Industries, Ltd.),
Leave it in a cool, dark place for 3 hours, and at the end of the treatment, use ShōIJ! 5
%w/v was added, left to stand for 30 minutes, and then dialyzed overnight against phosphate buffer (pH 7, 4), and activity was evaluated using rat bone marrow cells.

As a result, as shown in Table 1 above, the cellular DNA synthesis inhibitor is stable against deoxyribonuclease, ribonuclease treatment and periodate oxidation, but trypsin,
It was sensitive to α-chymotrypsin and proase treatment. Furthermore, although it is stable at pH 2 to 8, it is deactivated by heat treatment at 90° C. for 30 minutes.

Furthermore, the influence of each substance on the activity of the cellular DNA synthesis inhibitor of the present invention was investigated.

During the reaction, 10-'M 2-mercaptoethanol, 5
, crg/ml Levamisole, 50m, MN-acetyl-D-glucosamine, 50mM N-acetyl-
D-galactosamine, 50mM α-methyl-D-mannoside, 10mM L-arginine and 10mM
When L-ornithine was added and the activity was evaluated using rat bone marrow cells, the activity of the cellular DNA synthesis inhibitor of the present invention was not inhibited by any of the substances.

The activity of this factor is L-arginine (0.5-20mM)
It is different from arginase because it is not inhibited even by the addition of I,-ornithine (0.5-20mM). It is also different from various proteases because it is not inhibited by the addition of protease inhibitors such as soy bean trypsin inhibitor. Furthermore, this factor is also known as lymphotoxin derived from human peripheral blood lymphocytes, tumor necrosis factor (TNF), or interferon-T.
It is a novel cellular DNA synthesis inhibitor that differs in molecular weight, non-adsorption to concanavalin A Sepharose, and various physicochemical and biochemical properties.

Example 2: Production using human T cell hybridomas Human peripheral blood lymphocytes obtained by specific gravity centrifugation were adjusted to 6 5XIO cells/ml in RPMI 1640 medium containing 10% tallow serum, and Con A was added at 10 pg/ml. ,
After culturing for 3 days at 37° C. and 5% carbon dioxide, the cells were washed three times with RP old 1640 medium containing no serum and Con A to obtain concanavalin A-stimulated human peripheral blood lymphocytes. These cells are resistant to 8-azaguanine and the T-T leukemia cell line CCRF-CEM or the same MOLT-4 is blood-free? After mixing at a ratio of 1:1 in ffRPMr 1640 medium and centrifuging at 400Xg for 10 minutes to form a cell mass, 1 ml of 45% polyethylene glycol 4000 was added to each cell over about 1 minute to induce a fusion reaction. After washing once with serum-free RPMI 1640 medium, the concentration was adjusted to 1×10 6 cells/ml with RPMI 1640 medium containing 10% tallow serum, and 10 opl each was added to a 96-well flat bottom multi-well plate. After 24 hours, Hyboxanthin lXl0-'M1 aminopterin 4 x 1
RPM+ 1640 medium (HAT medium) containing 10% tallow serum containing 0-7M and thymidine 1.6XIO-'M
was added in an amount of 100 μβ, and thereafter, approximately half of the medium in each well was replaced with IIAT medium every 2 to 3 days. After 2 to 4 weeks, for wells with hybridoma growth, hyboxanthin 1 x 10-'M, thymidine 1.6
RPM containing lO% tallow serum containing XlO-'M [16
40 medium (HT medium), and thereafter the same medium was replaced every 2 to 3 days. After culturing in HT medium for about 2 weeks, the medium was replaced with RP old 1640 medium containing 10% tallow serum, and an assay using 3H-thymidine incorporation into rat bone marrow cells as an indicator was performed. Cells in wells in which suppressive activity was observed were immediately cloned by limiting dilution, and the suppressive activity contained in the culture supernatant of the obtained clones was measured by activity evaluation using rat bone marrow cells.

By the above method, 8-azaguanine-resistant CCRF-C
Hybridoma clone HC-6 using IEM as a parent strain and biridoma clone IIM-53 using 8-azaguanine-resistant MOLT-4 as a parent strain were established. When we investigated the optimal cell concentration and culture time for the production of cellular DNA synthesis inhibitors, we found that serum-free RPMI
It was revealed that the maximum amount of cellular DNA synthesis inhibitory factor was produced in the supernatant after culturing at 2×10' cells/ml in 1640 medium for 72 hours. Cell D of the present invention produced in the culture supernatant of this hybridoma clone
The NA synthesis inhibitor can be purified according to the method of Example 1 and the like.

Example 3: T8 antigen-positive suppressor T cells - produced from the cytotoxic T cell subpopulation Human peripheral blood lymphocytes obtained by density gradient centrifugation using Ficoll-paque and a snake rosette formation reaction can be developed. 1:1 with sheep red blood cells treated with 0.14 M of the known 2-aminoethylisothiouronium bromide:
After mixing at a ratio of 100:1, centrifuge and leave at 4°C for 2 hours.
Form an E rosette. Thereafter, using Ficollvac, the cells were separated into E rosette-negative cells and E rosette-positive cells, and the sheep red blood cells were hemolyzed by hypotonic treatment.
E rosette positive T cell fraction was obtained. On the other hand, E rosette-negative cells were adhered to a plastic Petri dish to obtain a non-adherent B cell fraction and an adherent table tennis fraction, respectively.

Furthermore, the E rosette-positive T cell fraction was analyzed using the bunning method CR using 0KT8 and 0KT4 monoclonal antibodies.
e1nherz et al., Cl1n, Immunol.

1m+5unopath1.21$ 257 pages 19
(1981), T8 antigen-positive and T4 antigen-positive T
Separated into cell subgroups. First, E. roseso) II T cells were reacted with mouse anti-0KT8 or 0KT4 monoclonal antibody at 4°C for 1 hour, and then placed in a plastic dish to which goat anti-mouse immunoglobulin antibody had been attached in advance. Deposition was achieved by incubation at 37°C for 1 hour. After collecting non-adherent cells, they were gently washed twice with phosphate buffered saline, and adherent cells (T8-positive or T4-positive cells) were collected from the dish by vigorous pipetting.

Various cell fractions obtained by the above method, namely E rosette positive T cell fraction, E rosette negative plastic dish non-adherent B cell fraction, E rosette negative plastic dish adherent monocyte fraction. fraction, and T8 antigen or T4 antigen positive T cell fraction, respectively, in 5×1
06 cells/ml? Adjust to the seismic intensity, Con A l
The cells were stimulated with Opg/ml for 48 hours at 37°C, and the culture supernatant was obtained.

The activity of the cellular DNA synthesis inhibitor in the Con A-stimulated cell culture supernatant obtained using these various cell fractions was measured using rat bone marrow cells.
Activity to suppress cellular DNA synthesis by 80% or more was observed in the cell fraction. On the other hand, E rosette-negative nonadherent cells (B
No activity of this factor was observed in E rosette-negative adherent cells (!11 cell fraction).

Furthermore, a strong cellular DNA synthesis inhibitory activity was observed in the culture supernatant obtained by stimulating the T8 antigen-positive (T4 antigen-negative) '■' cell fraction with Con A; No activity of this factor was observed in the la culture obtained by this method. From the above results, this cell DNA
The synthesis suppressor is a T8 antigen-positive suppressor T cell.
It has become clear that this is produced by a subpopulation of cytotoxic T cells.

Experimental Example Regarding the biological activity of the cellular DNA synthesis inhibitor of the present invention,
The experiment was conducted as follows. The activity of this factor was evaluated using rat bone marrow cells as follows.

Experimental Example 1: Activity measurement method using rat bone marrow cells A 96-well microtest plate (manufactured by Corning) was used as a culture vessel, containing 10% heat-inactivated tallow serum and kanamycin 6oμg/ml'/r; RP old 16
Adjust rat bone marrow cell suspension (5X
10b pieces/ml) O, 1ml and diluted sample 0.1ml
were mixed and cultured at 37°C for 12 hours in air containing 5% carbon dioxide. Then, 3H-thymidine (specific activity 5 Ci/m
mol (manufactured by Amajam) at a concentration of 0.5 μci/well, and after further culturing for 4 hours, a cell harvester was added.
Cells were collected using a glass fiber filter. The residual radioactivity in the cells was measured using a toluene chiller and a liquid scintillation counter.
The activity of this factor is determined from comparison with the control. Furthermore, the percentage inhibition of 'H-thymidine uptake can be calculated from the following 2 and used as an index of the activity of this factor.

Furthermore, if necessary, the titer can be determined by plotting the dilution of the sample and the inhibition percentage on normal probability paper, etc., determining the dilution that gives a 50% inhibition percentage of H-thymidine uptake, and determining the titer in units of (
It can be displayed as [1].

In the following biological activity evaluation, when 'H-thymidine incorporation is used, the remaining intracellular radioactivity of 'H-thymidine and the inhibition percentage of uptake are calculated in the same manner as the above method, and the present cell DNA synthesis trial is calculated. This was used as an indicator of the activity of the regulatory factor.

Experimental Example 2: Inhibitory effect on DNA synthesis and antibody production of human peripheral blood lymphocytes caused by mitogens Human peripheral blood lymphocytes obtained by specific gravity centrifugation were
5X1 in RPMI 1640 medium containing % tallow serum.
After adjusting to 0' pieces/ml, various my-1-dienes [Staphylococcus aureus colawan I (SA
C) 0.003% Pork Ride Mitogen (P
WM) 1/100 dilution, phytohemagglutinin (
PIIA) 5 pg/ml concanavalin A
(Con A) 5 μg, 7 ml] and 100 μβ were added to each well of a 96-well flat-bottom multi-well plate in which a sample (100 μm) containing a cellular DNA synthesis inhibitor had been previously placed. After culturing at 37° C. and 5% carbon dioxide for 48 to 72 hours, 311-thymidine was added at 0.5 μC/well, and the cells were further cultured under the same conditions for 4 hours. Cells were collected using a cell harvester, and the radioactivity incorporated into the cells was measured using a liquid scintillation counter. The results are shown in Table 2. In addition, in the table below, the numbers in parentheses indicate the inhibition percentage (%), and the SE
M indicates standard deviation.

Table 2: Inhibition of DNA synthesis in human peripheral blood lymphocytes induced by various mitogens As is clear from the table, samples containing cellular DNA synthesis inhibitors have a higher level of 3H- Thymidine uptake was inhibited by 50-70%.

Regarding Staphylococcus aureus colawan■ and Hawkelyde mitogen, after culturing for 6 days under the above conditions, the culture supernatant was collected, and the immunoglobulin M (IgM) I contained in the supernatant was collected. of,
In addition, immunoglobulin G (
When IgG) I was quantified by enzyme-linked immunosorbent assay (ELISA), the third
As shown in the table, the amount of 1 gM produced and the amount of IgG produced were 60% each depending on the sample containing the cellular DNA synthesis inhibitor.
~80%, and 20-40% inhibition.

Table 3: Suppressive effect on IgM antibody production and natural IgG antibody production of human peripheral blood lymphocytes induced by P or SAC Experimental example 3: Suppressive effect on DNA synthesis and cell proliferation of cultured tumor cells Various cultured tumors Cells (CCRF-3B, RPMI 8
226.11SB-2, K562, N5-1.5P
-2) was adjusted to 3 lXIO/ml in RPMI 1640 medium containing 10% tallow serum, and cell D
A sample (100 μm) containing the NA synthesis inhibitor was placed in each well of a 96-well flat-bottom multi-well plate.
0μβ was added at a time. 24 at 37°C and 5% carbon dioxide
After culturing for an hour, 0.5 μCi/well of 3H-thymidine was added, and the cells were further cultured for 4 hours under the same conditions. Cells were collected using a cell harvester, and the radioactivity incorporated into the cells was measured using a liquid scintillation counter. Table 4 shows the results.

As is clear from Table 4, Table 2, Suppressive Effect on 'H-Thymidine Uptake of Various Cultured Cells, the sample containing the cellular DNA synthesis inhibitor of the present invention has a higher 'H-thymidine uptake than the control that does not contain the same factor.
Thymidine uptake was inhibited by 40-90%.

In addition, when CCRF-SB, RPMI 8226, and RP old 1788 were cultured for 72 hours under the above conditions, the number of viable cells was measured by Trivan blue staining, and as shown in Table 5, the cells of the present invention Samples containing DNA synthesis inhibitors were found to inhibit proliferation of these cells by 50-70% compared to controls.

Table 5: Suppressive effect on proliferation (increase in cell number) of various cultured cells 1 Below 1 Margin 1 Experimental example 4: I+-2 dependent mouse cell line CTLL-2 induced by interleukin-2 (IL2) Inhibitory effect on DNA synthesis of L-2-dependent mouse cell line CTLL-2 at 10%
IX1 in RP old 1640 medium containing % tallow serum.
After adjusting to 0' cells/ml, recombinant human interleukin-'l (r-IL-2) was added (200 U/ml).
lO01! to each well of a 96-well flat-bottomed multi-well Brake 1, into which a sample (100 μe) containing a cellular DNA synthesis inhibitor had been placed in advance. Added e each time.

After culturing for 20 hours at 37°C and 15% carbon dioxide,
3H-thymidine was added at 0.5 μCi/well, and the cells were further cultured for 4 hours under the same conditions. Cells were collected using a cell harvester, and the radioactivity incorporated into the cells was measured using a liquid scintillation counter. The results are shown in Table 6.

Table 6: Inhibitory effect on IL2 response of interleukin-2 (NL2) dependent mouse cell line CTLL-2 As is clear from the table, the response of CTLL-2 to IL2 is It was suppressed by 50-60%.

[Brief explanation of drawings]

Figure 1 shows the elution pattern by gel filtration using Sephacryl S-200, and Figure 2 shows the elution pattern of immobilized concanavalin A.
Figure 3 shows the migration pattern by affinity chromatography using Sepharose, Figure 3 shows the migration pattern by preparative isoelectric focusing, and Figure 4 shows the migration pattern by FPLC.
- Linear analysis of common salt (0-1,0M) using Mono O column? The elution patterns by rotational gradient are shown respectively. In each figure, ←−→ indicates the percentage inhibition of tritium thymidine incorporation, which indicates the activity of a cellular DNA synthesis inhibitor (
%), □ is absorbance at 280 nm (0, D, zao)
, --.- represents the pH, and --.- represents the linear concentration gradient due to common salt, respectively. FIG. 5 shows the production of the cell 1) NA synthesis inhibitor of the present invention when human peripheral blood lymphocytes were fractionated into various cell populations.

Claims (1)

[Claims] A cellular DNA synthesis inhibitor derived from human lymphocytes and characterized by the following properties. (a) has a molecular weight of 45,000 to 70,000 (gel filtration chromatography); (b) is non-adsorbable to immobilized concanavalin A-Sepharose; (c) has an isoelectric point of 4.5 to 5. 5, (d) eluted at a salt concentration of 0.5 to 0.7 M in FPLC-MonoQ anion exchange chromatography, (
e) insensitive to deoxyribonuclease, ribonuclease, periodate treatment, sensitive to trypsin, α-chymotrypsin, proase, (f) pH 2-7
(g) Inactivated by treatment at 90°C for 30 minutes.