JPH1175890A - Evaluation of disease-treating medicine with t-cell clone - Google Patents

Abstract

PROBLEM TO BE SOLVED: To evaluate a medicine for treating a disease such as allergy, atopic dermatosis, autoimmune disease or T-cell lymphoma in good reliability by using as a culture cell for the evaluation a T-cell clone obtained by a production method using a B-cell as a main feeder cell. SOLUTION: This method for evaluating a medicine for treating a disease comprises using a T-cell clone as a culture cell for the evaluation. The T-cell clone is obtained by a production method 9-77416 (Japanese patent application number) using a B-cell (preferably an EB virus-transformed cell treated with mitomycin) as a main feeder cell, is low the danger of infection, and can reduce the change of the nature of the cell itself.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for evaluating a therapeutic agent for a disease such as a drug using a T cell clone as a cultured cell for evaluation. More specifically, the present invention relates to a method for evaluating a therapeutic agent for a disease such as allergy, atopic dermatitis, an autoimmune disease, or a T cell lymphoma using a T cell clone as a culture cell for evaluation.

[0002]

2. Description of the Related Art T cells are the main cells that regulate the immune system. In recent years, in a system in which T cells are separated from a patient's diseased tissue or blood, and the T cells are cultured in vitro, diseases such as allergies, autoimmune diseases, and the like that may be caused by abnormalities in the immune system are considered. Attempts have been made to apply them to the evaluation of disease therapeutic agents such as T-cell lymphomas and to the analysis of their disease states. T cells are recognized antigens and lectins having a T cell activating action (concanavalin A (hereinafter referred to as conA),
It is known that phytohemagglutinin (hereinafter referred to as PHA) and interleukin 2 (hereinafter referred to as IL-2) enhance the proliferative power. It is also possible to obtain usable amounts of cells. Furthermore, if an experiment using cloned T cells can be performed, more detailed information on the evaluation of a therapeutic agent for a disease and analysis of a disease state can be obtained. (Hereinafter, the cultured cells used for the evaluation method by culturing the disease therapeutic agent are abbreviated as “cultured cells for evaluation”)

[0003] For example, in atopic dermatitis, which is one of allergic diseases, mononuclear cells are separated from peripheral blood of a patient sensitized to house dustmite antigen (hereinafter referred to as HDM), and HDM and IL are isolated. There is a report that cloned T cells that were cultured and proliferated under stimulation with -2 (EA
Wierenga et al. Journal of Immunology, 144, (12), 465
1-4656, 1990). According to this, HDM-specific reactive T cell clones derived from patients with atopic dermatitis are helper T cells, and are thought to induce humoral immunity of the antibody-producing system.
It was reported to belong to the type subtype. Allergic reactions and helper T cell type 2 subclass (hereinafter Th2 cells)
The relationship between is being clarified, and in the field of allergy, evaluation using cloned Th2 cells has been actively conducted.

[0004] As a method for cloning T cells, usually, T cells are diluted to a very low density in a medium, and seeded so that one cell per well is placed in a microculture plate to obtain clone cells. A limiting dilution method is used. When performing this limiting dilution method, it is necessary to add feeder cells to support cell growth. In addition, it is necessary to add feeder cells in a timely manner during the expansion culture of T cells after cloning. The feeder cell is a cell in which the proliferation activity is inactivated while maintaining the metabolic activity, and a cell capable of inducing the proliferation of T cells is used.

As feeder cells, peripheral blood mononuclear cells (E. Maggi et al. Journal of Immunology. 148, (7), 214)
2-2147, 1992), a mixture of peripheral blood mononuclear cells and a B cell line transformed with 10% of the EB virus (EA Wierenga et al. Journal of Immunology, 144, (1)
2), 4651-4656, 1990; H. Yssel et al. Journal of Immunol
ogy, 148, (3), 738-745, 1992). In addition, as a method for inactivating the proliferation activity of feeder cells, it is known to use irradiation or treatment with mitomycin C (hereinafter referred to as MMC).

[0006] If a T cell clone that reflects the pathology of a disease can be obtained as a cultured cell for evaluation, it is useful for screening for a therapeutic agent for a disease, analyzing the mechanism of action, and evaluating pathological analysis of a disease. However, the current T cell cloning method cannot easily produce and establish T cell clones. That is, in cloning, a large number of feeder cells are required, but it is difficult to collect a large amount of peripheral blood mononuclear cells with a large burden on the donor. There is a problem that performing is not convenient. Further, the feeder cells can inactivate the proliferation activity by irradiation, but have a problem that a special device is required. If there is no equipment,
By using the MMC treatment method, the growth activity of feeder cells can be inactivated. However, this MMC processing also requires MM
Culture is performed for 30 minutes to 1 hour in the presence of C, but since peripheral blood mononuclear cells adhere to the culture plate during this culture, it is difficult to collect peripheral blood mononuclear cells after MMC treatment. There is also a problem.

Heretofore, generally, as a screening method and an evaluation method for a therapeutic agent for a disease such as a drug, an experimental animal is generally used.
Evaluation method in an n vivo system and using animal cells such as animal tissue cells and blood cells as culture cells for evaluation
An evaluation method using an in vitro system is known and widely used for research and development of pharmaceuticals and the like. However, in the evaluation method using different animal species, there is a problem that animal experiment data between different species cannot always be directly applied to the animal to be treated and interpreted from the viewpoint of species difference from the human or animal to be treated. In addition, in recent years, from the viewpoint of animal welfare and the growing criticism of animal experiments, an in vitro evaluation method using human-derived cultured cells is desired.

[0008] Using human-derived cultured cells for evaluation
As an evaluation method in vitro, for example, an evaluation method using peripheral blood mononuclear cells (Monouclear cells: MNC) of atopic dermatitis patients has been reported in connection with a therapeutic agent for allergic disease (Japan). Dermatological Association Magazine, No. 10
Vol. 6, No. 4, pp. 395-402, Japanese Dermatological Association 19
1996). In addition, as a method for evaluating the direct action of a drug on T cells, an evaluation method using cedar-specific T cells of a cedar pollinosis patient as culture cells for evaluation (Japanese Journal
al of Pharmacology, 61, 31-39, 1993), an evaluation method using T cell clones as culture cells for evaluation (“Cytokine and allergy”, Medical Review Co., Ltd., 19)
1996).

[0009] However, in the evaluation method using peripheral blood mononuclear cells as culture cells for evaluation, it is necessary to use the material for evaluation as soon as possible after collecting the material. There is a problem that the burden on the side) is large. On the other hand, a T cell clone is a useful culture cell for evaluation that can improve the problem of the evaluation method using the above peripheral blood mononuclear cells as a culture cell for evaluation. However, when a conventional feeder cell is used, there is a problem that the production and establishment of a T cell clone cannot be easily performed as described above.

[0010] Furthermore, when these T cells and T cell clones are used in an evaluation system as culture cells for evaluation, dangerous T cell infectious viruses such as HIV (human immunodeficiency virus) and HTLV (human T cell leukemia virus) are used. However, there is a safety problem that the risk of these dangerous virus infections from the cultured cells for evaluation always accompanies the evaluation experimenter. In any conventional method for evaluating a therapeutic agent for a disease using cultured cells, in the culture or cloning of T cells, peripheral blood mononuclear cells containing about 70% of T cells are used as feeder cells. There is a risk of viral infection of T cells.

[0011] In addition, as a result of infection of T cells with these viruses, changes in properties such as sensitivity of the T cells themselves to a therapeutic agent for a disease also occur. Because of the influence on the evaluation system due to the change in the properties of the cultured cells for evaluation, there is a problem related to reliability that it is difficult to always reproduce a stable and reliable evaluation result. Even in the case of evaluation of a therapeutic agent for a virus-infected T cell, it is preferable to use a T cell clone infected with a virus other than the target virus as a culture cell for evaluation because of the above-mentioned safety and reliability. is not.

[0012]

The invention described in claims 1, 2, 3 and 4 is intended for screening of therapeutic agents for diseases using cultured cells for evaluation, analysis of the mechanism of action, etc., for the evaluation of T cell infectious viruses and the like. Can reduce the risk of infection to the experimenter due to the above, improve the safety of the production of cultured cells for evaluation, and the handling involved in the evaluation test. In addition, the sensitivity of T cells themselves due to dangerous T cell infectious virus infection, etc. An object of the present invention is to provide a highly reliable evaluation method capable of reducing property changes and constantly reproducing a stable evaluation result.

[0013]

[Means for Solving the Problems]

(1) The present invention relates to a method for evaluating a therapeutic agent for a disease, which uses a T cell clone obtained as a culture cell for evaluation using a B cell as a main feeder cell. (2) The present invention relates to a method for evaluating a therapeutic agent for a disease, wherein the T cell clone according to (1) is used as a cultured cell for evaluation, wherein the main feeder cells are EB virus-transformed cells. (3) The present invention relates to a method for evaluating a therapeutic agent for a disease, wherein the T cell clone according to the above (2) is used as a culture cell for evaluation, wherein the EB virus transformed cell is an EB virus transformed cell treated with mitomycin. (4) The method for evaluating a therapeutic agent for a disease using the T cell clone as a culture cell for evaluation according to the above (1), (2) and (3), wherein the therapeutic agent for a disease acting on T cells is evaluated. .

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The present inventors have found that by using B cells as main feeder cells, conventional T cell cloning can be effectively performed, and since B cells have no adhesiveness, they can be used for MMC treatment. It has been found to be suitable.
In addition, it has been found that a T cell clone derived from a disease patient obtained by a T cell cloning method using B cells as a main feeder cell well reflects the pathology of the original disease (Japanese Patent Application No. 9-77416). ).

Further, in screening of therapeutic agents for diseases, analysis of mechanism of action, etc., conventional T cell clones are evaluated by using T cell clones obtained by using B cells as main feeder cells as culture cells for evaluation. The risk of infection by T-cell infectious virus, etc., can be reduced as compared to the use as cultured cells for use, and the change in properties of the cultured cells for evaluation can be reduced, and stable and reliable evaluation results can be reproduced. To complete the present invention.

[0016] The culture cell for evaluation of the present invention is used.
A method for producing a T cell clone obtained by using B cells as main feeder cells (Japanese Patent Application No. 9-77416).
) Will be described below. In producing T cell clones, B cells are used as the main feeder cells. Here, “main” preferably means 30% of the total number of feeder cells.
(V / V)% or more, more preferably 50 (V / V)% or more, still more preferably 70 (V / V)% or more, and most preferably peripheral blood mononuclear cells and the like. , And using only B cells as feeder cells.

For the cloning of the T cell clone, a limiting dilution method, a soft agar method and the like can be used, and the method is not particularly limited, but the limiting dilution method is preferred because it is simple. B cells used as feeder cells include, for example, B cells collected from blood, transformed cells (B cell lymphoma patient-derived B cells).
Cell lines, cells transformed with B cell infectious virus) and the like.

It is preferable to use EB virus-transformed cells as B cells, since a large amount of cells can be easily obtained. The EB virus-transformed cells were obtained by separating a mononuclear cell layer containing B cells from blood by a specific gravity centrifugation method, and culturing the mononuclear cell layer in the presence of a culture supernatant of EB virus-infected cells.
It can be obtained by infecting B cells with the EB virus. The EB virus-infected cells are not particularly limited, and known and available cells such as B958 can be used.

Irradiation or treatment with mitomycin C (MMC) is preferably performed to inactivate the growth potential of the EB virus-transformed cells. 3000 for irradiation
It is preferable to irradiate a gamma ray of up to 6000 rad. MMC
In the case of treatment, for example, 30 to 70 μg / ml, more preferably 40 to 60 μg / ml, particularly preferably 50 μg / ml of M
In the presence of MC, the cells are cultured at 30 to 40 ° C., particularly preferably at 37 ° C. for 30 minutes to 1 hour.
It is preferable to use a method of washing off MC. MM is useful because it does not require radiation that is harmful to the human body and is simple.
C treatment is more preferred.

T cells to be cloned obtained by a production method characterized in that B cells are the main feeder cells
The cells are not particularly limited, but T cells collected from blood are preferred. Furthermore, as described later, T cells derived from a diseased part such as an invasive T cell of a diseased part or a diseased lesion for the purpose of evaluating a therapeutic agent for a disease are particularly preferable. T cells are activated under growth factor stimulation, and are preferably diluted to a density of 5 to 20 cells / ml in a culture solution.

The culture solution is not limited as long as it contains nutrients necessary for the proliferation of T cells. Amino acids, vitamins, nucleic acid bases, etc. are added to a culture solution derived from an organism such as serum, or a balanced salt solution. If necessary, a synthetic medium containing fetal bovine serum or bovine serum albumin can be used.
Preferred are I 1640 medium, AIM-V medium and the like. Commercially available products can be used as these media.

In culturing T cells, it is preferable to first add a T cell growth factor to the T cells diluted with the culture solution. T-cell diluted with culture solution is 0.05-0.2ml per well
It is preferable to sow one by one. As T cell growth factors,
It is preferable to use interleukin 2 (IL-2), phytohemagglutinin (PHA), or the like. The concentration of IL-2 is preferably 5 to 200 U / ml as the final concentration during culture, and PHA is 0.1 to 10 μL as the final concentration during culture.
g / ml is preferred. Culture plates of various sizes can be used for the culture vessel.

Next, the above-mentioned B cells (feeder cells) are added, preferably suspended in a culture solution. The number of feeder cells is preferably 1 × 10 ⁴ to 5 × 10 ⁵ per well, more preferably 7 × 10 ⁴ to 2 × 10 ⁵ per well. The feeder cells are preferably diluted with a culture solution to be used in a volume of 0.05 to 0.2 ml per well. Thereafter, the culture is continued for 3 days to 3 weeks, and preferably 30 to 80 (V / V)% of the medium, more preferably 40 to 60 (V / V)% of the medium, Preferably, half the volume is replaced with fresh medium containing growth factors.
The frequency of the subsequent medium exchange becomes shorter as the cells grow, but is preferably once every two to ten days. The temperature of the culture is preferably from 30 to 40 ° C, particularly preferably 37 ° C.

When sufficient proliferation of T cells is observed, it is preferable to transfer the cells to a culture vessel having a larger hole. At this time, the concentration of the feeder cells was 5 × 1 / ml of the medium.
It is preferred to use those from 0 ⁵ containing 5 × 10 ⁶ cells,
It is preferable to add the solution so that the liquid volume per hole is 1 to 1.5 ml. Thereafter, until the required amount of T cell clone is obtained, preferably in the same manner as described above,
0 to 80 (V / V)%, more preferably 40 to 60 (V / V)
V)%, particularly preferably half, is replaced once every 2 to 10 days with fresh medium containing growth factors at the same concentration as above. It is preferable to add 1 × 10 ⁴ to 5 × 10 ⁵ feeder cells per well once every 10 to 20 days. The temperature of the culture is preferably from 30 to 40 ° C, particularly preferably 37 ° C. At this stage, generally 2
It is preferable to use a 4-well culture plate.

The T cell clone obtained as described above well reflects the properties of the initial T cells (mother cells) and the pathology of the disease. Screening of the therapeutic agent of the present invention,
In the evaluation of the mechanism of action analysis, etc., the T cell clone produced or established as described above is used as an evaluation culture cell for evaluation.

Screening of a therapeutic agent for a disease and analysis of the mechanism of action using a T cell clone derived from a lesion such as an infiltrating T cell at a diseased lesion for the purpose of evaluating a therapeutic agent for a disease as a culture cell for evaluation It is preferable to use it for the evaluation of the condition and the like and the analysis of the disease state. For example, in the case of T-cell lymphoma, tumorigenic T cells have high proliferative activity and proliferate predominantly in the above method, and many of the obtained T cell clones are tumorigenic T cells. The infiltrating T cells in the lesion are generally activated, and are derived from T cells collected from the lesion (eg, skin of T cell lymphoma, joint of rheumatism, lung of asthma, skin of atopic dermatitis, etc.). Cell clones reflect pathology. Furthermore, in the case of an allergic disease, an autoimmune disease, or the like, an antigen-specific T cell clone can be obtained by using the disease-causing antigen as a T cell growth factor in the above method. It is particularly preferable to use T cell clones reflecting the pathology of these primary diseases as cultured cells for evaluation.

Examples of diseases involving T cells include:
The above allergic diseases, autoimmune diseases, T-cell lymphoma, graft-versus-host disease (GVHD), and the like,
It is not limited to these diseases. As the therapeutic agent for diseases, therapeutic agents acting on T cells (for example, immunosuppressants,
Antitumor agents, etc.) and therapeutic agents expected to have therapeutic effects on diseases involving T cells. However, it is not limited by the therapeutic agent.

In order to evaluate a therapeutic agent for a disease, first, cultured cells for evaluation are cultured together with the therapeutic agent for a disease to be evaluated, and after a certain period of time or over time, one or both of the cells and / or the culture solution is removed. to recover. Next, the recovered cells are quantitatively or qualitatively measured or detected, or components derived from the cells themselves or from the culture solution are quantitatively or qualitatively measured or detected. Using the change in the measured value or the detected value as an index, the action of the disease therapeutic agent on the cultured T cells for evaluation is evaluated, and this is used as the evaluation of the disease therapeutic agent.

The measurement or detection of cells includes counting of cells, measurement or detection of cell proliferation activity, and the like. The cells can be counted by a hemocytometer or flow cytometry. Cell proliferation activity can be measured using commercially available cell proliferation detection kits and cell proliferation quantification kits (BrdU labeling &
Detection Kit 3, Boehringer Mannheim)
Can be used to measure or detect the presence or absence. In addition, as a cell-derived component from the cell itself, for example,
Cell surface substances such as cell surface antigens (eg, HLA-DR, CD25, etc., which are markers for T cell activation);
These measurements or detections can be performed by flow cytometry, immunostaining and the like.

The components derived from the cells from the culture solution include T
Cell-derived physiologically active substances (eg, cytokines such as interleukin-4 and interferon-γ in the case of atopic dermatitis and allergic diseases). These physiologically active substances are obtained by a qualitative method or a quantitative method by an immunological assay such as a radioimmunoassay (RIA), an enzyme immunoassay (EIA, ELISA), and a chemiluminescence immunoassay (CLIA). The presence or absence or its concentration can be measured.

Although specific examples of the measuring object and measuring means have been described above, the present invention is limited to the measuring object and measuring means as long as a change in the evaluation cell itself or a component derived therefrom can be detected and measured by the action of the therapeutic agent for disease. Not something.

In the evaluation of the therapeutic agent for the disease, not only the culture group to which the evaluated therapeutic agent is added but also a positive control group (a therapeutic agent with a known effect), a negative control group (including no added therapeutic agent), etc. Can be added to the evaluation, and it is preferable to compare and evaluate.

In the culture of the cultured T cells for evaluation,
An appropriate T cell stimulant using lectin or a specific antigen can also be added. At the time of specific antigen stimulation, it is preferable to add antigen-presenting cells such as monocytes, macrophages, and B cells in an amount corresponding to 10% to 10 times of T cells. In addition, the number of cells and the volume of the medium are appropriately determined according to the examination items.

[0034]

In the above-described method for evaluating a therapeutic agent for a disease, the production of a T cell clone used as a culture cell for evaluation of the present invention can be carried out by using a B cell as a main feeder cell. Can be easily manufactured and established. Furthermore, by producing T cell clones using B-cells as main feeder cells,
The risk of infection of the experimenter with dangerous T-cell infectious virus such as HIV (human immunodeficiency virus) and HTLV (human T-cell leukemia virus) can be reduced. Production of cultured cells for evaluation and handling accompanying evaluation tests Safety can be improved.
In addition, since a change in properties such as sensitivity of the T cells themselves due to dangerous T cell infectious virus infection can be reduced, a stable evaluation result can always be reproduced. Thereby, not only the reproducibility of the evaluation result in the facility but also the reproducibility between the facilities can be improved, and the standardization of the evaluation method becomes possible.

[0035]

The present invention will be described below with reference to examples. (1) Cloning Peripheral blood from an atopic dermatitis patient sensitized to HDM was applied to a vacuum blood collection tube (VECUTAINER, B) coated with heparin.
ecton Dickinson). T from this peripheral blood
Mononuclear cells, including cells, can be separated from commercially available separation agents (Lymphoprep, Nyco
med) according to the protocol. The obtained peripheral blood mononuclear cells (about 70 (V /
V) is suspended in RPMI1640 medium (manufactured by GIBCO) supplemented with fetal bovine serum at a concentration of 10 ⁶ cells / ml so that the concentration of HDM is 10 μg / ml.
/ ml, and inoculated in a U-bottom 96-well culture plate (manufactured by COSTER) at 0.1 ml per well. 5% CO ₂ 37
The cells were cultured for 5 days under the condition of ° C., and on the 5th day, 0.1 ml of AIM-V medium (manufactured by GIBCO) containing 100 U / ml of IL-2 (manufactured by BTI) was added per well. Five more days later, half of the medium (0.1 ml) was replaced with AIM-V medium containing 100 U / ml of fresh IL-2. Five days later, the grown cells were cloned by the limiting dilution method.

That is, cells were cultured at 10 cells / ml in AIM-V medium.
, And seeded in a U-bottom 96-well culture plate at 0.1 ml per well. Furthermore, MMC treatment (MMC concentration 50
added to the medium so as to [mu] g / ml, after 30 minutes incubation, 3 times with phosphate buffer wash) B cells lines 10 ⁶ cells / ml to 1
Add 0.1 ml per hole, IL-2 50U / ml, PHA
(PHA L4, manufactured by Wako Pure Chemical Industries, Ltd.) 1 μg / ml
Was added. One week later, half of the medium was replaced and the culture was continued. Thereafter, half of the medium was replaced once every three days. After sufficient growth was observed, 10 ⁶ MMC treated B cell lines, 5
AIM-V containing 0 U / ml IL-2, 1 μg / ml PHA
The cells were transferred to 1 ml of the medium and expanded in a 24-well culture plate.

The B cell line used in the above method was prepared by obtaining human peripheral blood mononuclear cells by the above method, culturing the cells in the culture supernatant of B958 cells, which are EB virus-infected cells, and infecting them with the EB virus. Was obtained by transformation. Specifically, B958 cells were cultured in RPMI1 supplemented with 10% fetal calf serum.
640 medium, and the culture supernatant was centrifuged (40
0 × g, 5 minutes) and a pore size of 0.45 μm
(MILLEXHV, manufactured by MILLIPORE). Human peripheral blood mononuclear cells at a concentration of 10 ⁶ cells / ml B95
The cells were suspended in 8-cell culture supernatant and cultured for 2 hours. Two hours later, the medium was replaced with RPMI1640 medium supplemented with 20% fetal bovine serum and 0.2 μg / ml cyclosporine, and the culture was continued. One week later, half of the medium was replaced with fresh 20% fetal calf serum, 0.2 μl.
The medium was replaced with RPMI1640 medium supplemented with g / ml cyclosporin. Then, after 3 days and 6 days, half of the medium was exchanged.
After 10 days, replace the medium with 10% fetal bovine serum RPM
The culture was continued while changing the medium to I1640 medium and increasing the culture volume in accordance with the cell growth.

(2) Analysis of the Properties of the Obtained T Cell Clones The properties of the obtained T cell clones were analyzed by detecting cell surface antigens by flow cytometry. Specifically, 2 (V) was set to be 1 × 10 ⁷ T cell clones / ml.
/ V)% human serum-containing phosphate buffered saline solution, and 0.1 ml of this solution was added with a fluorescently labeled antibody (Pharmingen). As the fluorescently labeled antibody added, FI
TC (fluorescein isothiocyanate) labeled anti-human CD4 antibody and PE (phycoerythrin) labeled anti-human CD
8 antibody combination and FITC-labeled anti-human CD4
Using a combination of the 5RA antibody and the PE-labeled anti-human CD45RO antibody, 20 μl each was used. 1 at room temperature with shading
After 5 minutes of reaction, 1 ml of ice-cold 2% human serum phosphate buffered saline was added, and an automatic cell screener (Cyto ACE-
150, manufactured by JASCO Corporation). As a result, all T cell clones derived from atopic dermatitis patients obtained by pre-stimulation of house dustmite antigen were CD4
Positive, CD8-negative helper T cells and CD45
The cells were RO-positive and CD45RA-negative memory type cells.

(3) Examination of clonality Next, the clonality of the cells was determined by using the T cell receptor (hereinafter referred to as T cell receptor).
M) of the beta chain variable region (hereinafter referred to as Vβ)
RNA expression was examined by the RT-PCR method. 10 ⁶
Kit for separating mRNA from individual cells (MICRO-FAS
(T TRACK, manufactured by Invitrogen) according to the protocol to separate mRNA. cDNA synthesis kit (cDNA CYCLE, manufactured by Invitorogen)
A cDNA is synthesized using Proc. Natl. Acad. Sci.
USA, Vol. 86, pp8941-8945 (November 1989), in the presence of a TCR β chain fixed region (hereinafter Cβ) primer and 22 types of TCR Vβ primers as described in Table 1
Using aq DNA polymerase (TAKARA Taq, manufactured by Takara Shuzo Co., Ltd.), heat denaturation 94 ° C., 45 seconds, annealing 60 ° C., 45 seconds, extension reaction 72 ° C., 1 minute 30
The PCR reaction was performed under the conditions of 35 cycles for seconds. PCR
The sample after the reaction was subjected to electrophoresis on a 2% agarose gel, stained with ethidium bromide, and then detected by irradiation with ultraviolet light. All the obtained T cell clones expressed only one kind of TCR Vβ mRNA, and it was considered that there was no problem in clonality.

(4) Analysis of subclass of helper T cells The subclass of helper T cells was analyzed by the cytokine production pattern under lectin stimulation. T
Cell clones were suspended in AIM-V medium at a concentration of 10 ⁶ cells / ml, and 0.2 ml was inoculated on a 96-well culture plate. 1μ
After culturing for 24 hours under the stimulation of g / ml PHA, the culture supernatant was collected and interleukin 4 (IL-4), which is a type 2 subtype cytokine, and interferon γ, which is a type 1 subtype cytokine ( The amount of IFN-γ) produced was measured using an ELISA kit (Quantikine, manufactured by R & D). The results are shown in Tables 1, 2 and 3. Many of the T cell clones derived from atopic dermatitis patients obtained by pre-stimulation with house dust antigen were type 2 subtypes with high interleukin 4 production and low interferon gamma production. This result indicates that the T cell clone obtained by this method reflects the pathology of the atopic dermatitis of the primary disease. For this reason, this clone was used as a cultured cell for evaluating the following therapeutic agent for diseases.

[0041]

[Table 1]

[0042]

[Table 2]

[0043]

[Table 3]

(5) Evaluation method of therapeutic agent for disease The representative synthetic corticosteroid drug dexamethasone (Dex) used in the treatment of atopic dermatitis was evaluated by the cytokine production evaluation system of the above T cell clone (4). To evaluate the therapeutic agent for this disease. In the same manner as in the above operation (4), one T cell clone was placed in AIM-V medium.
0 were suspended in a concentration of ⁶ / ml, 0 to 96-well culture plate.
2 ml was seeded. 1 μg / ml PHA and Dex 10-
The cells were added at a concentration of 9 to 10-7 M, cultured for 24 hours, and the culture supernatant was recovered. The amount of IL-4, a cytokine considered to be strongly involved in the pathology of atopic dermatitis, was determined by ELISA. The measurement was performed using a kit (Quantikine, manufactured by R & D). Table 4 shows the results. Dex dose-dependently suppressed IL-4 production of T cell clones from atopic dermatitis patients. This showed an immunosuppressive effect, which is the mechanism of action of Dex as a therapeutic agent for atopic dermatitis.

[0045]

[Table 4]

[0046]

The method for evaluating a therapeutic agent for a disease according to any one of claims 1 to 4, is a method for screening a therapeutic agent for a disease using a cultured cell for evaluation and evaluating the mechanism of action, etc. The risk of infection to cells can be reduced, and the safety of the production of cultured cells for evaluation and handling accompanying evaluation tests can be improved. In addition, since a change in properties such as sensitivity of the T cells themselves due to dangerous T cell infectious virus infection can be reduced, a stable evaluation result can always be reproduced. This allows
It is possible to improve not only the reproducibility of the evaluation result within the facility but also the reproducibility between the facilities, and standardization of the evaluation method becomes possible. Further, it can be used for research use of various drugs.

Claims (4)

[Claims] 1. A method for evaluating a therapeutic agent for a disease, wherein a T cell clone obtained by a production method comprising B cells as main feeder cells is used as a cultured cell for evaluation. 2. The method according to claim 1, wherein the main feeder cells are EB virus-transformed cells, using the T cell clone as a culture cell for evaluation. 3. The EB virus-transformed cell is a EB virus-transformed cell treated with mitomycin.
A method for evaluating a therapeutic agent for a disease, wherein the T cell clone described above is used as a cultured cell for evaluation. 4. The method for evaluating a therapeutic agent for a disease using a T cell clone as a cultured cell for evaluation according to claim 1, wherein the therapeutic agent for a disease acting on T cells is evaluated.