JPH08289699A - Model animal for autoimmune disease and its preparation - Google Patents

Abstract

PURPOSE: To obtain an autoimmune disease model animal useful for a morbid state investigation and the development of a treating agents by culturing a pathological lymphocyte derived from an autoimmune disease patient together with a T-lymphocyte activating material to obtain an activated T-lymphocyte and transferring the activated lymphocyte into an immunodeficiency model animal. CONSTITUTION: This autoimmune disease model animal such as an autoimmune disease model mouse having the symptom of a disease such as synovial membrane proliferation, fibrosis or phagedena in bone and cartrilage peculiar to serious autoimmune diseases is obtained by transferring an activated T- lymphocyte obtained by culturing a pathological T-lymphocyte such as a synovial T-lymphocyte (SFT) derived from an autoimmune disease patient such as RA patient together with a T-lymphocyte activating material as an autoantigen such as collagen type II (C II) capable of introducing autoimmunization which causes autoimmune diseases into an immunodeficiency model animal.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an autoimmune disease model animal and a method for producing the same. More specifically, the present invention provides a pathogenic T that is directly involved in the development of human autoimmune disease.
Activated T lymphocytes obtained by culturing lymphocytes with a T lymphocyte activating reagent, and autoimmune disease models that develop autoimmune diseases obtained by transferring the activated T lymphocytes into immunodeficiency model animals The present invention relates to an animal and a method for producing the animal.

[0002]

2. Description of the Related Art The model animal for autoimmune disease according to the present invention means that when a pathogenic T lymphocyte is transferred to an immunodeficiency model animal, the model animal is immunodeficient and the T lymphocytes are not rejected. Accepted is an animal that has developed a human autoimmune disease as a result of its immune system being reconstituted by human T lymphocytes.

Conventionally, severe combined immunodeficiency (SCID)
Various attempts have been made to transfer human hematopoietic stem cells and lymphocytes to mice and reconstitute them with human blood cells.
-Hu has been reported variously [JR Frey et a
l., J. Exp. Med., 175, p1067 (1992); GM Fulop et
al., J. Immunol., 136, p4438 (1986)]. In addition, instead of normal cells, attempts have been made to reproduce human diseases by transfecting lymphocytes and cancer cells involved in human autoimmune diseases and using them as disease model mice [TD Gep
pert et al., Arthritis Rheum., 33, p599 (1990)].
However, reproduction of human autoimmune diseases by lymphocyte transfer is not always successful.

For example, systemic lupus erythematosus (SL)
E) In a report in which patient lymphocytes were transferred to SCID mice, production of various autoantibodies was observed, but the renal and vascular lesions characteristic of typical human SLE were not successfully reproduced [ MA Duchosal et al., J. Exp. Med., 17
2, p985 (1990)].

On the other hand, it has been reported that CSF of human multiple sclerosis (MS) was introduced into the ventricles of SCID mice to induce MS-mimicking limb paralysis. Y. Saeki et al., Proc. Natl. Acad. Sci.
USA, 89, p6157 (1992)]. However, SC of T lymphocytes in the synovial fluid of human rheumatoid arthritis (RA) patients
Even when injected locally into the knee joint of ID mice, only a very slight synovitis was observed [Saeki et al., Annual Meeting of the Immunological Society of Japan, Vol. 22, p400 (1992)].

As described above, it is considered difficult to induce the severe proliferative synovitis, which often leads to the thickening of synovium, the formation of pannus, and the destruction of bone and cartilage, which are often observed in RA. It has been considered that it is far from the establishment of the RA model animal.

[0007]

As described above, the conventional model animal for autoimmune disease has an autoimmune disease, especially RA.
Was insufficient as a disease model animal. The present invention
The object of the present invention is to overcome this drawback and to establish a disease model animal closer to human pathological conditions and a method for producing the disease model animal.

[0008]

Under these circumstances, the present inventors have conducted diligent research, and as a result, after culturing pathogenic T lymphocytes derived from patients with autoimmune disease with T lymphocyte activating substances, By transferring the obtained activated T lymphocytes to an immunodeficiency model animal, a model animal that develops a pathological condition of a severe autoimmune disease was successfully prepared, and the present invention was completed.

[0009] Furthermore, intraperitoneal administration is effective as a cell transfer route, but if necessary, direct administration to the knee joint is used in combination to more efficiently produce the target autoimmune disease model animal. be able to.

[0010]

In order to prepare an autoimmune disease model animal of the present invention, first, a pathogenic T derived from an autoimmune disease patient is used.
Lymphocytes are used. For example, rheumatoid arthritis (R
When preparing an onset model animal of A), it is preferable to use synovial fluid T lymphocytes (SFT) of RA patients.
The T lymphocytes are obtained by purifying joint synovial fluid collected from patients with autoimmune disease according to a conventional method, for example, the sheep erythrocyte rosette method.

However, even if the purified pathogenic T lymphocytes thus obtained are directly transferred to an immunodeficiency model animal, it is not possible to obtain a model animal exhibiting a desired disease condition. Therefore, activated T lymphocytes are prepared by culturing the T lymphocytes together with various T lymphocyte activators before transfer to the immunodeficiency model animal. Examples of T lymphocyte activators used include mitogens such as T cell lectins or anti-T cell antibodies, and self-antigens such as type II collagen (CII).

Mitogen (mitogen) is a substance having the property of activating lymphocytes and inducing and promoting cell division. T cell lectin, which is a preferred example of the mitogen used in the present invention, has the property of binding to sugar chains existing on the surface of T lymphocytes. When T cell lectin is mixed and cultured with T lymphocytes, the T cell lectin becomes T
T lymphocytes are activated by binding to sugar chains existing on the surface of lymphocytes and inducing division and proliferation of T lymphocytes. An example of a T cell lectin is phytohemagglutinin (PHA).

Another preferred example of the mitogen used in the present invention is an anti-T cell antibody. More preferably, an anti-CD3 antibody is used. here,
CD3 is a group of molecules that form a T cell receptor complex by non-covalently binding to a T cell receptor capable of recognizing an antigen on the surface layer of T lymphocytes, and play a role in initiation of signal transduction related to antigen recognition. . When an anti-CD3 antibody, which is an antibody specific to CD3, is reacted with T lymphocytes, CD3 existing on the surface layer of T lymphocytes is stimulated and T
Lymphocytes are activated by inducing mitotic proliferation.

Other suitable T lymphocyte activators include autoantigens. An autoantigen is a component of the body and is an antigen capable of inducing the production of autoantibodies that cause autoimmune diseases. As a preferred example of the autoantigen used in the present invention, for example, when the autoimmune disease is RA, it is presumed to be one of the autoantigens of RA.
Type II collagen (CII) can be used. CII
When cultured with RA synovial fluid T cells (SFT) derived from RA patients, CII specifically reacts with certain T lymphocytes, activating proliferation and proliferation of the T lymphocytes, thereby activating SFT. Be made

Activated T lymphocytes can be obtained by culturing the above various T lymphocyte activators with pathogenic T lymphocytes derived from patients with autoimmune diseases. Then, by transferring the activated T lymphocytes to an immunodeficiency model animal, it becomes possible to prepare a model animal that develops a desired autoimmune disease condition.

An example of the immunodeficiency model animal used in the present invention is a severe combined immunodeficiency (SCID) mouse.

According to the results of the present inventor, even if SFT of RA patients is used, strong arthritis does not occur when SFT that has not been previously activated by PHA or the like is transferred. Therefore, activation of SFT before transfer is considered to be an essential requirement for the onset of arthritis. Also, normal people and R
Arthritis does not occur when peripheral blood T lymphocytes (PBT) of patient A are stimulated and transferred, and even after synovial lymphocytes (SF) of non-rheumatic osteoarthritis (OA) patients are transferred after stimulation. Arthritis does not develop. From these results, it is important to culture-stimulate and transfer the SFT of RA patients, and T lymphocytes reactive with the joint autoantigen are accumulated in the joints of RA patients and activate them. Is assumed to be a necessary condition. This is considered to be a very effective method applicable not only to RA but also to the production of model animals for many other autoimmune diseases.

On the other hand, even when SFT derived from RA patients is transferred after stimulation with a T lymphocyte activator, strong arthritis may not develop due to individual differences in RA patients and the number of transferred cells. When the number of transferred cells is large (~ 1 x 10
^{In 7} ), some patients develop strong arthritis only by intraperitoneal administration of activated SFT. However, when the number of SFT cells to be transferred is as small as 2 to 5 × 10 ⁶ cells, weak arthritis may develop only by intraperitoneal administration. Therefore, in order to effectively and strongly develop arthritis, it is preferable to use intraperitoneal administration as well as intra-articular direct administration in combination.

As described above, according to the present invention, an autoimmune disease model animal having a pathological condition peculiar to a serious autoimmune disease could be produced. Rheumatoid arthritis (R
A) It was possible to prepare a model mouse exhibiting a unique disease state, that is, synovial proliferation, fibrosis, or erosion of bone and cartilage.

According to the results of the present inventors, human T lymphocyte-specific monoclonal antibody MT- was localized in the local arthritis induced in SCID mice by transferring activated SFT.
Only T lymphocytes reactive with 1 (Bioscience Products) were proliferating and infiltrating. Further, not only the right knee joint directly injected with SFT, but also the unrelated left knee joint and ankle joint were found to have strong arthritis. From these results, it was revealed that the recognized arthritis was caused by human SFT. Furthermore, it was shown that the arthritis was not due to physical damage at the infusion site of cells, but was a polyarthritis similar to that of human RA. In addition, the finding of GVHD, which is often a problem in SCID mice, was investigated in various organs (liver, kidney, parotid gland,
Small intestine).

Hereinafter, the present invention will be described in more detail with reference to examples, but these examples do not limit the scope of the present invention.

[0022]

[Examples] 1) Separation of synovial fluid T lymphocytes (SFT) from RA patients Collect 15 to 40 ml of synovial fluid from RA patients. T lymphocytes contained in the synovial fluid are purified by sheep red blood cell (SRBC) rosette method. That is, about 0.2 to 0.3 g of hyaluronidase powder was added to the synovial fluid stored overnight in ice and well mixed, and the mixture was heated in warm water at 37 ° C for 30 minutes to 1 hour.
After filtering the cells with a mesh, RPMI culture medium is added and washed twice by centrifugation (1600 rpm, 10 minutes) to remove the supernatant. After mixing the resulting precipitate (containing T lymphocytes) and loosening it, AET (2-amino ethyl isothiouronium bromide h)
1 ml of sheep red blood cells (AET-SRBC) suspended at 10% in RPMI culture medium supplemented with 10% fetal calf serum (FCS). After mixing well, centrifuge at 1600 to 1800 rpm for 1 to 2 minutes, and leave on ice for 1 to 1.5 hours. Then, after mixing and filtering with a mesh, overlay 2 to 3 ml of Ficoll-Conray liquid, which is dispensed into 3 to 4 test tubes, 3.0 ml each, at room temperature, 1600 to 1800 rpm, 20 to 30 minutes. Centrifuge. After removing the supernatant, the precipitate is collected and centrifuged once in an RPMI solution. Tris-hydrochloric acid solution (hemolyzed) was added to this sediment to form SRBC.
Hemolyze and count the number of cells. The yield of SFT is 5 × 10 ⁶ to 2 × 10 ⁷ cells per patient.

2) Activation of SFT SFT was prepared at 5 × 10 ⁶ cells / ml in RPMI culture medium supplemented with 10% FCS, and 1 each was prepared in a 24-well culture dish.
Incubate ~ 2 ml. At this time, as the T lymphocyte activator, PHA prepared in RPMI1640 culture medium supplemented with 10% FCS
(Gibco) 10 μg / ml, anti-CD3 antibody (OKT3; ATC
C, Rocville, ML) 5 μg / ml, or human type II collagen (CII) 40 μg / ml, and added at 37 ° C., 5% CO ₂ 3
Incubate for days. After culturing, the cells are centrifuged and washed twice with Hanks solution, and then used for the transfer experiment.

3) Transfer of cells The activated SFT obtained in 2) is transferred to an immunodeficiency model animal. Severe combined immunodeficiency (SCID) mice were used as immunodeficiency model animals. Six-week-old SCID mice are transfected with 1 to 5 × 10 ⁶ cells per mouse into the knee joint and the abdominal cavity at a ratio of 1: 4. After the activated SFT was prepared at 2 × 10 ⁷ cells / ml, 50 μl thereof was directly injected into the right knee joint using a tuberculin syringe. Then 200 μl is injected intraperitoneally. Moreover, PHA-activated peripheral blood mononuclear cells (PBMC; 2 × 10 ⁶ to 1 × 10 ⁷ cells) derived from RA patients and PHA-activated joint synovial lymphocytes (SF; 0.3 to 0.8 × 10 ⁶ cells), PHA-activated normal individual-derived peripheral blood T lymphocytes (PBT; the ^{5 × 10 6 ~1 × 10 7} ) were inoculated in the same manner as described above SCID mice.

4) Histological search 6 weeks after cell transfer, the mice were sacrificed, the joints of the limbs were fixed with formalin, decalcified with 0.5N EDTA (pH7.4), and stained with hematoxylin / eosin. Go and observe the tissue. PHA (phytohemagglutinin), OK
SFT activated with various T lymphocyte activators such as T3 (anti-CD3 antibody) and CII (human type II collagen)
As a result, the histological findings of arthritis induced in SCID mice were searched for three indicators of synovial proliferation, fibrosis, and erosion of bone and cartilage, and the severity thereof is shown based on the scores in Table 1. Shown in 1.

[0026]

[Table 1]

As is clear from FIG. 1, even in SFT derived from RA patients, when activation is not performed before transfer, almost no symptoms specific to arthritis occur. On the other hand, PHA, OK
It was confirmed that strong arthritis was induced when SFT activated by T lymphocyte activating substances such as T3 and CII was transferred. On the other hand, peripheral blood lymphocytes of RA patients (PBM
C), synovial lymphocytes (SF) in non-rheumatic osteoarthritis (OA) patients and peripheral blood T lymphocytes (PB) in normal subjects
When T) was stimulated and transferred with a T lymphocyte activating substance such as PHA, almost no symptom peculiar to arthritis was induced.

[0028]

INDUSTRIAL APPLICABILITY As described above, T lymphocytes derived from patients with autoimmune diseases are treated with T cells such as mitogens or autoantigens.
After being activated by culturing with a lymphocyte activating substance, the resulting activated T lymphocytes are transferred into an immunodeficiency model animal to obtain an autoimmune disease model animal having a serious autoimmune disease pathology. It has become possible to manufacture efficiently. The use of the model animal can be an extremely effective means for studying pathological conditions of autoimmune diseases and screening and development of therapeutic agents.

[Brief description of drawings]

FIG. 1 shows the onset of arthritis in SCID mice due to transfer of T lymphocytes derived from patients with autoimmune disease.

Front page continuation (72) Inventor Hanping 1-1-1 Honjo, Kumamoto City, Kumamoto Prefecture Kumamoto University (72) Inventor Takafumi Omura 2-2-1 Honjo, Kumamoto City, Kumamoto Prefecture

Claims (16)

[Claims] 1. An autoimmune disease model mouse having a pathological condition peculiar to a serious autoimmune disease. 2. The autoimmune disease model mouse according to claim 1, wherein the autoimmune disease is rheumatoid arthritis (RA). 3. The autoimmune disease model mouse according to claim 1 or 2, wherein the pathological condition is synovial proliferation, fibrosis, or bone and cartilage erosion. 4. A method of activating T lymphocytes derived from a patient with autoimmune disease, which comprises culturing pathogenic T lymphocytes together with a T lymphocyte activator, which is capable of inducing pathologies specific to autoimmune diseases. Manufacturing method. 5. The method for producing activated T lymphocytes according to claim 4, wherein the autoimmune disease patient has RA. 6. The method for producing activated T lymphocytes according to claim 4, wherein the pathogenic T lymphocytes are synovial fluid T lymphocytes (SFT) derived from RA patients. 7. The method for producing activated T lymphocytes according to claim 4, wherein the T lymphocyte activator is mitogen. 8. The method for producing activated T lymphocytes according to claim 7, wherein the mitogen is an anti-T cell antibody. 9. The mitogen is a T cell lectin such as phytohemagglutinin (PHA).
A method for producing the activated T lymphocytes described. 10. The method for producing activated T lymphocytes according to claim 4, wherein the T lymphocyte activator is an autoantigen capable of inducing the production of autoantibodies that cause autoimmune diseases. 11. The type II collagen (C
II) The method for producing activated T lymphocytes according to claim 10. 12. A method for producing an autoimmune disease model animal, which comprises transferring the activated T lymphocytes obtained by the method according to any one of claims 4 to 11 to an immunodeficiency model animal. 13. The method for producing an autoimmune disease model animal according to claim 12, wherein the transfer route of the activated T lymphocytes to the immunodeficiency model animal is intraperitoneal administration. 14. The autoimmune disease according to claim 12, wherein the route of transfer of the activated T lymphocytes to the immunodeficiency model animal is a combination of intraperitoneal administration and direct administration to the knee joint. Method for producing model animal. 15. The method for producing an autoimmune disease model animal according to claim 12, wherein the immunodeficiency model animal is a severe combined immunodeficiency (SCID) mouse. 16. An autoimmune disease model animal obtained by the method according to claim 12.