WO2008126940A1

WO2008126940A1 - Novel t cell

Info

Publication number: WO2008126940A1
Application number: PCT/JP2008/057460
Authority: WO
Inventors: Keishi Fujio; Tomohisa Okamura; Kazuhiko Yamamoto
Original assignee: The University Of Tokyo
Priority date: 2007-04-10
Filing date: 2008-04-10
Publication date: 2008-10-23
Also published as: JPWO2008126940A1

Abstract

A CD4-positive, CD25-positive regulatory T cell, which is a known regulatory T cell, cannot control an autoimmune disease by itself satisfactorily. Thus, the object is to search a regulatory T cell other than a CD4-positive, CD25-positive regulatory T cell, which can control an autoimmune disease satisfactorily. A study was made on the relationship between a regulatory T cell and Egr-2 gene which has been reported to be a transcription factor involved in T cell anergy. As a result, a novel T cell can be isolated, and the activities of the T cell can be elucidated.

Description

Specification

New T cells

Technical field

The present invention relates to providing a novel τ cell population derived from a mammal. More specifically, the present invention relates to providing a novel regulatory rod cell population derived from mammals. Background art

The living body precisely distinguishes between self and non-self by the immune system. Organism's immune system is inherently tolerant to its constituent proteins, but under certain conditions an immune response to the self-protein may occur. As a result of the immune response to, autoimmune diseases occur. Currently, collagen diseases, rheumatoid arthritis, etc. are known as autoimmune diseases, but it is becoming clear that autoimmune responses are also involved in arteriosclerosis, cancer, and neurodegenerative diseases. Autoimmune response is thought to be involved in the pathology of various diseases.

For the treatment of such autoimmune diseases, non-antigen-specific treatment methods such as administration of corticosteroids and administration of immunosuppressants are used. However, these currently applied therapies also cause severe problems such as opportunistic infections because they cause systemic high immune suppression. Such a situation also occurs when an immunosuppressant is administered after organ transplantation. Therefore, in the field of treatment of autoimmune diseases and organ transplantation, there is a strong demand for immunosuppressive treatments specific to autoantigens that cause autoimmune diseases and immunosuppressive treatments specific to transplanted antigens. Yes.

Recently, it has been found that regulatory atory cells (regulatory T cells) are important in the formation of autoimmune tolerance specific to self-antigens.

Mammalian T cells are a type of lymphocyte that refers to the progenitor cells produced in the bone marrow that have been differentiated and matured through selection in the thymus. It is known that 70-80% of lymphocytes in peripheral blood are T cells. This T cell is characterized by expressing CD4 or CD8 as a cell surface marker molecule. T4 cells that express CD4 function as helper T cells that induce the functional expression of other T cells, induce the differentiation and maturation of B cells, and induce antibody production by producing lymphoin, etc. . On the other hand, CD8 positive T The cell functions as a CTL (killer T cell) that destroys virus-infected cells. In addition to such T cells, it has been shown that CD4 positive regulatory T cells have a function of suppressing the activity of other T cells. Regulatory T cells known so far include regulatory T cells (Foxp3 Treg cells) that express CD4 and CD25 molecules on the cell surface and express the transcription factor Foxp3 (Non-patent Document 1). It was known to be the most common. On the other hand, in addition to Foxp3Treg cells, regulatory T cells that produce IL-10 (T _R 1 cells) (Non-patent document 2), regulatory T cells that produce TGF3 (T _H 3 cells) (Non-patent document 3) However, the expression profile of the cell surface antigen of these cells is unknown, and its substance has not been clarified.

These regulatory T cells had the function of suppressing the activity of other T cells, and as a result were expected to be responsible for autoimmune tolerance in vivo. However, even in mice lacking a functional Foxp3 gene or in mice lacking the Aire gene, which is responsible for central immune tolerance in the thymus, there are obvious disorders in the central nervous system, joints, small intestine, endocrine glands, etc. Not allowed (Non-Patent Document 4). This suggests that CD4 + CD25 + regulatory T cells alone may not be able to adequately control autoimmune diseases.

The Egr-2 (Early Growth Response Gene-2) gene is 2862 bp (CDS), also called Krox20, NGF1-B, Zfp-25, Zfp-6, NGFl-) 3 (nerve growth factor inducible gene 1- / 3) Is a molecule that is cloned from mouse and human as a Zinc-finger type transcription factor (Non-patent document 5; Non-patent document 6). This gene has been reported to be a transcription factor associated with T cell anagenesis (refractory) (Non-patent Document 7). However, none of the known regulatory T cells mentioned above was associated with this.

Non-Patent Document 1 Sakaguchi S., et al., J Immunol 1995; 155: 1151-1164 Non-Patent Document 2 Groux H. A, et al., Nature 1997; 389: 737-742

Non-Patent Document 3 Chen Y., et al., Science 1994; 265: 1237-1240

Non-Patent Document 4: Chen Z. et al., Pro atl. Acad. Sci. USA, 2005, 102, 14735-14740

Non-Patent Document 5: Chavrier P, et al., Mol. Cell. Biol. 8 1988, pp. 1319-1326 Non-Patent Document 6: Joseph LJ, et al., Proc. Nat l. Acad. Sci. USA 85 1988, 7164-716

Non-Patent Document 7: Saf ford M. et al., Nat. Immunol., 2005, 6, 472-480 Disclosure of the Invention

Problems to be solved by the invention

In the present invention, CD4 positive CD25 positive regulatory T cells, which are conventionally known regulatory T cells, cannot sufficiently control autoimmune diseases. Therefore, CD4 can sufficiently control autoimmune diseases. The objective is to search for regulatory T cells other than positive CD25 positive regulatory T cells.

Means for solving the problem

The inventors of the present invention examined the relationship between the Egr-2 gene reported to be a transcription factor related to T cell anergy (refractory) and regulatory T cells, and thus developed novel T cells. It was shown that the above problems can be solved by isolating and clarifying the action.

Specifically, in the present invention, CD4 positive (CD4 +), CD25 negative (CD25-), CD45RB negative (low positive) (CD45RB- (low)), and LAG-3 positive (LAG-3 +) or Isolate new T cells from mammals with CD4 positive (CD4 +), CD25 negative (CD25-), CD45R0 positive (CD45R0 +), and LAG-3 positive (LAG-3 +) cell surface antigen properties. By providing this, the above problems can be solved.

CD4 positive (CD4 +), CD25 negative (CD25-), CD45RB negative (low positive) (CD45RB- (low)), and LAG-3 positive (LAG3 +) or CD4 positive (CD4 +), CD25 isolated in the present invention Novel T cells with negative (CD25-), CD45R0 positive (CD45R0 +), and LAG-3 positive (LAG-3 +) properties are shown to have a function as regulatory T cells. Specifically, the function as a regulatory T cell is to suppress the activation reaction of other T cells, and its characteristics include anaphylaxis (refractory).

These cells are derived from any mammal (eg, human, primate, Inu, cat, etc.), and may be isolated from any mammal.

The present invention also provides from a mammalian lymphocyte population or T cell population that: (i) CD4 is positive; Collecting a certain cell (CD4 +), (ii) collecting a CD25 negative cell (CD25-), (iii) a CD45RB negative (low positive) cell (CD45RB- (low)) or CD45R0 By collecting the cells (CD45R0 +) positive for, and (iv) collecting the cells (LAG3 +) positive for LAG-3, by performing steps (i) to (iv) in any order. , CD4 positive (CD4 +), CD25 negative (CD25-), CD45RB negative (low positive) (CD45RB-(low)), and LAG-3 positive (LAG-3 +) or CD4 positive (CD4 +), CD25 negative (CD25 -), A method for isolating mammalian T cells having the cell surface antigen characteristics of CD45R0 positive (CD45R0 +) and LAG-3 positive (LAG-3 +) can be provided. ,

In this method, the mammal may be any mammal (eg, human, primate, Inu, cat, etc.).

In the present invention, a mammalian lymphocyte population or T cell population may be collected from peripheral blood and then the steps (i) to (iv) may be performed as they are, or a mammalian lymphocyte population or T cell population. May be collected from peripheral blood and the lymphocyte population or T cell population may be expanded before the steps (i) to (iv) are performed.

In the present invention, the T cell population isolated by performing the steps (i) to (iv) may be used as it is, or the steps (i) to (iv) are performed. A more isolated T cell population of the present invention may be used after being expanded.

In the present invention, CD4 positive (CD4 +), CD25 negative (CD25-), CD45RB negative (low positive) (CD45RB- (low)), and LAG-3 positive (LAG3 +) or CD4 positive (CD4 +), CD25 To provide a pharmaceutical composition comprising mammalian T cells having negative (CD25-), CD45R0 positive (CD45R0 +), and LAG-3 positive (LAG-3 +) cell surface antigen characteristics. it can. By administering this pharmaceutical composition, it is possible to induce functional suppression of pathogenic T cells that cause inflammation. Therefore, the pharmaceutical composition of the present invention is capable of eliminating an antigen by an antigen-antibody reaction. It can be used to treat various diseases caused. In the present invention, various diseases caused by an immune reaction against an autoantigen or due to an antigen being eliminated by an antigen-antibody reaction include, for example, autoimmune diseases (eg, rheumatoid arthritis, collagen disease, inflammatory bowel disease) , Multiple sclerosis, type 1 diabetes, etc.) or transplant organ rejection. In the present invention, the aforementioned CD4 positive (CD4 +), CD25 negative (CD25-), CD45RB negative (low positive) (CD45RB- (low)), and LAG-3 positive (LAG3 +) or CD4 positive (CD4 +), CD25 Screen for compounds to increase the number of mammalian T cells with negative (CD25-), CD45R0 positive (CD45R0 +), and LAG-3 positive (LAG-3 +) cell surface antigen properties A method is also provided. The method for screening the target compound having the above-mentioned action is:

(a) CD4 positive (CD4 +), CD25 negative (CD25-), CD45RB negative (low positive) (CD45RB- (low)), and LAG-3 positive (LAG3 +) or CD4 positive (CD4 +), CD25 negative (CD25- ) Culturing T cells derived from mammals having CD45R0 positive (CD45R0 +) and LAG-3 positive (LAG-3 +) cell surface antigen characteristics in the presence of a test compound;

(b) measuring the change in the expression of the Egr-2 gene or the change in the expression of the LAG-3 gene in the cultured T cells;

(c) Compounds that lead to increased Egr-2 gene expression or LAG-3 gene expression are CD4 positive (CD4 +), CD25 negative (CD25-), CD45RB negative. (low positive) (CD45RB- (low )), And LAG-3 positive (LAG-3 +) or CD4 positive (CD4 +), CD25 negative (CD25-), CD45R0 positive (CD45R0 +), and LAG-3 positive (LAG-3 +) cell surfaces Selecting as a compound that increases T cells from mammals having antigenic properties;

It is characterized by comprising.

The invention's effect

Since the regulatory T cells of the present invention can induce local immune tolerance, the side effects caused by systemic suppression of immune function such as administration of immunosuppressants have been plagued so far. Various diseases such as autoimmune diseases and rejection of transplanted organs can be treated without causing the above-mentioned side effects in the patient body. Brief Description of Drawings

FIG. 1 shows a schematic diagram of a retroviral vector pMX-Egr2-IRES-GFP and a control vector mock pMX-IRES-GFP vector used in the present invention.

[Fig. 2] Fig. 2 shows mouse CD4-positive splenocytes infected with Mock (pMX-IRES-GFP) or Egr2 (p X-Egr2-IRES-GFP) using a flow cytometer and GFP expression as an indicator. The process of separating and collecting in 4 groups (negative, low, mid, high) is shown.

[Fig. 3] Fig. 3 shows the results of real-time PCR analysis of cDNA synthesis from each of the 4 groups of cells collected and collected in Fig. 2, and the expression at the Egr2 mRNA and LAG-3 mRNA levels. Show.

[Fig. 4] Fig. 4 shows that co-transfer of OVA-specific T cell receptor (D01 1.10) significantly enhances its suppressive effect and enables antigen-specific immunosuppression. It is.

[Fig. 5] Fig. 5 shows the spleen mononuclear cells of mice co-transfected with Egr2 and D01 1.10 by flow cytometry. After lymphocyte and CD4 positive cell gate, D01 1.10 T cell receptor This is the process of developing each cell population separately using the KJ-1 antibody and GFP.

[Figure 6] Figure 6 shows that Egr2 (pMX-Egr2-IRES-GFP) and DO 1 1.10 (pMX-DOTAE, pMX-DOTBE) co-transfected cells were treated with pMX-Egr2-IRES-GFP. Overexpression,

It is a figure which shows that the expression of LAG-3 is further enhanced.

[Fig. 7] Fig. 7 shows that among mouse spleen mononuclear cells, LAG-3 surface antigen is CD4 positive among CD25 negative cells (Fig. 7a) and CD45RB negative to CD45RB weak positive cells (Fig. 7b). It is a figure which shows that expression is enhanced.

FIG. 8 is a graph showing that expression of Egr2 mRNA, IL-10 mRNA, and LAG-3 mRNA is enhanced in mouse LAG-3 expression cells that are CD45RB negative to CD45RB weak positive.

[FIG. 9] FIG. 9 shows cells gated in CD4 positive CD45RB ex low cell group, LAG-3,

Expanded on CD25, CD4 + CD25- LAG3 + CD45RB ex l ow (Egr2- Treg), CD4 + CD25- LAG3-

FIG. 4 shows the results of separating and collecting three groups of cell populations, CD45RB ex low and CD4 + CD25 + CD45RB ex low.

[FIG. 10] FIG. 10 shows that Egr2 expression was strongest in CD4 + CD25-LAG3 + CD45RB ex low (Egr2-Treg), and the same expression tendency was not observed in Foxp3 expression.

[Fig. 1 1] Fig. 1 1 shows cells gated in CD4 positive CD25 negative cells group developed with LAG-3 and CD45RB, and each cell group was separated and collected by flow cytometry as shown in the figure. Show process It is a figure.

FIG. 12 shows that Egr2 expression is strongest in CD4 + CD25-LAG3 + CD45RB ex low, and Egr2-Treg different from the known Foxp3-Treg exists.

[Fig. 13] Fig. 13 shows that CD4 + CD25-LAG3 + CD45RBex low (Egr2-Treg) is still present after type II collagen immunization, and further enhancement of I-10 expression occurs in “Egr2-Treg” after immunization FIG.

[FIG. 14] FIG. 14 shows a process in which CD4 + CD25- CD45RB ex low cell population (Thyl.2 +) is divided into high, low and nega '3 groups by LAG-3 expression as suppressor cells. FIG.

[FIG. 15] FIG. 15 shows that Egr-2Treg cells suppress cell division of a cytotoxic T cell subset (CD45RBhi cells), and the suppressive ability depends on LAG-3 expression.

[Fig.16] Fig.16 shows the suppression of enteritis caused by CD4 + CD25- CD45RBM cells administration in CD4 + CD25-LAG3 + CD45RB ex low (Egr2-Treg) cells in RAG 卜 /-mice. It is a figure which shows what can be done.

[Fig. 17] Fig. 17 shows CD4 + CD25-LAG3 + CD45RB ex low (Egr2-Treg) in the histology of the large intestine. ) It is a figure showing that the improvement was observed by co-transfer of cells.

[Figure 18] Figure 18 shows that CD4 + CD25- LAG3 + CD45RB ex low (Egr2-Treg) cells are also present in the splenocytes of Scurfy mice lacking the functional Foxp3 gene as analyzed by flow cytometry. FIG.

FIG. 19 is a view showing that Egr2-Treg cells of Scurfy mice have an in vitro suppressive ability, and that the suppressive ability is attenuated by LAG-3 neutralizing antibody.

[Figure 20] Figure 20 shows the results of intraperitoneal administration of C57BL / 6 mouse spleen CD4 + CD25- CD45RBhi cells 1X10 ⁵ cells to Rag 卜 /-mice. It is a figure which shows suppressing the enteritis in.

FIG. 21 is a schematic diagram showing a technique for producing a shRNA-Egr2 bone marrow chimeric mouse in which Egr2 gene expression is suppressed. [FIG. 22] FIG. 22 is a diagram showing that shRNA-Egr2 bone marrow chimeric mice develop marked thickening of the intestinal tract, which is characteristic of the onset of enteritis.

[Fig. 2 3] Fig. 23 shows that CD4 + CD25- LAG3 + CD45RB ex l ow (Egr2-Treg) cells were hardly observed in At MT mice lacking B cells, and Egr2- Treg cells were introduced by adoptive introduction of B cells. It is a figure which shows that this cell population is induced | guided | derived by B cell dependence by growing.

[Figure 24] Figure 24 shows flow cytometry of LAG-3 expression and CD45RB expression in thymus and spleen of OT-11 TCR transgenic mice with or without RIP-mOVA transgene. FIG.

[Figure 25] Figure 25 shows a normal TEa offspring (left) gated with CD4 + CD25 "T cells,

FIG. 4 shows flow cytometry of LAG-3 expression and CD45RB expression in spleen and Peyer's patch (PP) of 0T-I I (middle) and TEa TCR transgenic mice (right).

[Fig. 26] Fig. 26 shows the number of Egr-2 positive cells in the follicle. FIG. 4 shows the results of immunohistochemical examination of cells expressing Egr-2 molecules in spleen tissue sections derived from TEa mice, 0T-I I mice and 7BL / 6 mice.

[FIG. 27] FIG. 27 shows that spontaneous development of colitis is inhibited in TEa TCR transgenic mice by adoptive transfer of CD4 ⁺ CD25 "LAG3 + T cells.

[Fig. 28] Fig. 28 shows that LAG-3 surface antigen is enhanced in CD25 negative cells (Fig. 28a) and CD45R0 positive cells (Fig. 28b) among human tonsil cells. FIG.

[FIG. 29] FIG. 29 shows the expression of Egr2 mRNA, CD45R0 positive human LAG-3 expressing cells,

It is a figure which shows that the expression of IL-10 mRNA and LAG-3 mRNA is enhanced.

BEST MODE FOR CARRYING OUT THE INVENTION

The inventors of the present invention first examined whether the Egr-2 gene, a gene reported to be a transcription factor associated with T cell anergy (refractory), is associated with the activity of regulatory T cells. Went. Specifically, the Egr-2 gene was introduced into CD4 + T cells, and the traits were introduced When T cells were transferred to mice, it was revealed that delayed hypersensitivity reaction to foreign antigens was remarkably suppressed. This indicates that the Egr-2 gene is associated with the activity of regulatory T cells. It was also shown that co-introduction of antigen-specific T cell receptor with Egr-2 gene enhances its suppressive ability and induces antigen-specific suppression.

Based on this finding, we examined the gene expression profile of T cells into which Egr-2 gene was introduced in detail. It was found that the expression of the protein on the cell surface was enhanced. Using these characteristics as a clue, we searched for a cell population that highly expresses Egr-2 in cells present in the mouse spleen. As a result, we found that the expression level of Egr-2 mRNA in CD4 positive CD25 negative CD45RB negative LAG-3 positive T cells. Was found to be extremely high.

Since T cells having such cell surface antigen characteristics have never been known, the present inventors have developed a new T cell population having the characteristics of CD positive CD25 negative CD45RB negative LAG-3 positive. A detailed analysis was performed, considering that it is a regulatory T cell that plays an important role in the immune tolerance induction mechanism of T cells and can sufficiently control autoimmune diseases.

As a characteristic of this cell, IL-10, which is known as a cytokine with strong anti-inflammatory action, is highly expressed, and as a characteristic of the cell itself, in vitro, CD4 positive CD45RB highly expressed naive T cell division is observed. In vivo, in vivo enteritis model in which naive T cells with high expression of CD4 + CD45RB are transferred to RAG1-mouse mice, the onset of enteritis is induced by transfer of CD4 + CD25 negative CD45RB negative LAG-3 + T cells. It became clear to suppress. From these cell characteristics, T cells with cell surface antigen characteristics of CD positive (CD4 +), CD25 negative (CD25-), CD45RB negative (low positive) (CD45RB- (low)), and LAG3 positive (LAG3 +) It was suggested that it is a regulatory Γ cell.

In addition, based on the knowledge of the mouse described above, a search was made for a cell population that highly expresses Egr-2 for cells present in the human tonsils. As a result, CD4 positive CD25 negative CD45R0 positive LAG-3 positive T cells The mRNA expression level of 2 was found to be extremely high. The CD45RB negative (low positive) memory T cell population in mice is the CD45R0 fluorescence intensity in humans. However, the fact that the above-mentioned T cells were obtained from rabbits was consistent with this conventional finding. The CD4 positive CD25 negative CD45R0 positive LAG-3 positive T cells, like mouse CD4 positive CD25 negative CD45RB negative LAG-3 positive T cells, also show high IL-10 expression. It was done.

The inventors of the present invention named these cells “Egr-2 Treg cells” because these cells have the feature that the expression level of Egr-2 is extremely high.

The “Egr-2 Treg cells” obtained in the present invention were compared with the regulatory T cells “Foxp3 Treg cells” (CD4-positive CD25-positive Foxp3-positive T cells) known so far. As a result, high expression of the Foxp3 gene was not observed in the “Egr-2 Treg cell” of the present invention, whereas the high Egr-2 gene was found in the conventionally known regulatory T cell “Foxp3 Treg cell”. It became clear that it showed a contrasting characteristic that no expression was seen. From these results, it was considered that the “Egr-2 Treg cells” of the present invention and the “Foxp3 Treg cells” of the prior art are independent T cell populations different from each other.

A mouse called scur fy, which lacks the functional Foxp3 gene, is known to cause autoimmune-like disease with inflammatory cell infiltration into various organs and die within 4 weeks of birth. When T cells in the spleen of this scur fy mouse were examined, the same T4 subcells with the characteristics of CD4 positive CD25 negative CD45RB negative LAG-3 positive as the "Egr-2 Treg cells" of the present invention proliferated remarkably. It was shown that T cell subsets with this cell surface antigen profile were collected and examined for function in vitro. As a result, these cells suppressed the cell division of naive T cells with high expression of CD4 + CD45RB in vitro, and RAGl transfected with naive T cells with high expression of CD4 + CD45RB causing intestinal inflammation in vivo. -When transferred to mice, it was found to have an activity to suppress the onset of enteritis. Judging from the commonality of these cell surface antigen profiles and the common function of cells, T cell subsets with CD4-positive CD25-negative CD45RB-negative LAG-3 positive characteristics in scur fy mice are `` Egr-2 "Treg cells". Judging from the fact that “Egr-2 Treg cells” are prominently present in scur fy mice even though scur fy mice lack the Foxp3 gene, “Egr-2 Treg cells” are It was confirmed that it has independent characteristics. On the other hand, Egr-2 gene knockout mice are known to have the characteristics of embryonic lethality that develops symptoms such as impaired formation of the hindbrain and myelination of peripheral nerves and die during fetal period ( Genes Dev. 7 1993, pp. 207-2084). Therefore, the present inventors produced a bone marrow chimeric mouse in which a sh-RNA sequence that suppresses the expression of the Egr-2 gene was expressed in bone marrow cells using a retrovirus vector. The mouse died approximately 4 weeks after bone marrow transplantation, and histological examination revealed that it developed enteritis. Thus, the Egr-2 gene deficiency reduces the number of CD4-positive, CD25-negative, CD45RB-negative, LAG-3-positive cells that should be present in the body, resulting in the mechanism of regulatory T cells such as enteritis. It was shown to develop clinical symptoms associated with reduced performance.

Taken together these findings, CD4 positive CD25 negative CD45RB negative LAG-3 positive T cells or CD positive CD25 negative CD45R0 positive LAG-3 positive T cells are active in vitro or in vivo While the Tgr cell body and its transcription factor Egr-2 gene plays an important role in its differentiation, this regulatory T cell is shown to be independent of the functional control by the Foxp3 gene. It was done.

Based on the above findings, in one embodiment of the present invention, the present invention provides a novel regulatory T cell as a CD positive CD25 negative CD45RB negative LAG-3 positive T cell or CD4 positive CD25 negative CD45R0 positive LAG-3 positive T cell, "Egr-2 Treg cells" are provided. As described above, these cells are a cell population belonging to a T cell subset different from “Foxp3 Treg cells” which are regulatory T cells known so far.

In the present invention, the term “Egr-2” refers to the protein having the amino acid sequence represented by GenBank Accession No. P 1 1 161 and the nucleotide sequence encoding it for human “Egr-2”. For Egr-2, the protein having the amino acid sequence indicated by GenBank accession number NP_034248 and the nucleotide sequence encoding the protein, and for rat “Egr-2”, the amino acid indicated by GenBank accession number NP—446085. A protein having a sequence and a nucleotide sequence encoding the protein are respectively referred to.

In the present invention, when detecting the presence or absence of expression of the Egr-2 gene, mRNA expressed from the Egr-2 gene or cDNA prepared therefrom is used as a saddle, and in the case of human gcaccagc tg tctgacaaca tctac (SEQ ID NO: 1) as a forward primer and agcaaagctg ctgggatatgg (SEQ ID NO: 2) as a reverse primer, for mice agccgt t tec ctgtcctctg (SEQ ID NO: 3) as a forward primer and gtccctcacc acctccactt (SEQ ID NO: 4) can be detected as a reverse primer by amplification according to a general PCR protocol.

On the other hand, when referring to “LAG-3” in the present invention, for human “LAG-3”, the protein having the amino acid sequence represented by GenBank accession number P_002277 and the nucleotide sequence encoding it are as follows: For mouse “LAG-3”, refer to the protein having the amino acid sequence indicated by GenBank accession number P_032505 and the nucleotide sequence encoding it. For rat “LAG-3”, GenBank accession number NP. — Refers to the protein having the amino acid sequence represented by 997678 and the nucleotide sequence encoding it, respectively.

In the present invention, when detecting the presence or absence of expression of the LAG-3 gene, mRNA expressed from the LAG-3 gene or cDNA prepared therefrom is used as a saddle type, and in humans atctgcagga acagcagctcaa (SEQ ID NO: 5) is forwarded. Reverse primer as a primer and agggatccag gtgacccaaag (SEQ ID NO: 6) as reverse primer, for mice t tgct tctgg gactgctttg (SEQ ID NO: 7) as forward primer and gccactgtct ggttgatgtt g (SEQ ID NO: 8) As a primer, it can be detected by amplification according to a general PCR protocol.

On the other hand, the term “Foxp3” in the present invention refers to the protein having the amino acid sequence represented by GenBank accession number NP-054728 and the nucleotide sequence encoding the same for human “Foxp3”, and for mouse “Foxp3” It refers to the protein having the amino acid sequence represented by GenBank accession number NP-473380 and the nucleotide sequence encoding it.

In the present invention, when detecting the presence or absence of the expression of the Foxp3 gene, mRNA expressed from the Foxp3 gene or cDNA prepared therefrom is used as a saddle type, and in the case of a mouse, cagctgccta cagtgcccctag (SEQ ID NO: 9) is used as a forward primer and catttgccag cagtgggtag (SEQ ID NO: 10) as a reverse primer and a general PCR protocol Can be detected by amplification according to

The above-mentioned “Egr-2 Treg cells” are CD4, CD25, CD45RB or CD45R0, and LAG-3 cell surface antigens, CD4 positive, CD25 negative, CD45RB negative ( (Low positive) or CD45R0 positive, and LAG-3 positive as an indicator, isolated from a leukocyte population or T cell population collected from a living body as a single cell population it can. Specifically, the “Egr-2 Treg cell” of the present invention can be isolated by sorting with CD5, CD25, CD45RB or CD45R0 and LAG-3 in a cell saw. Sorting based on the expression of CD4, CD25, CD45RB or CD45R0, and LAG-3 may be done in any order.

In humans, the lymphocyte population or T cell population obtained from the peripheral blood of the living body can be used as the T cell population that is the source of ^ 81 "-268 cells". Specifically, peripheral blood mononuclear cells are separated by extracorporeal circulation (pheresis) using the blood component separation device of peripheral blood, and four types of CD4, CD25, CD45RB or CD45R0, and LAG-3 The Egr-2 Treg cells can be isolated and collected using Celso overnight. In addition, “Egr-2 Treg cells” can be separated and collected using a magnetic cell separation device such as Isolex or CliniMACS instead of Cell Soryu.

The above-mentioned lymphocyte population or T cell population may be subjected to cell sorter treatment for the above-mentioned CD4, CD25, CD45RB or CD45RO, and LAG-3 as it is to isolate “Egr-2 Treg cells”. If the number of cells is insufficient, the above-mentioned lymphocyte population or T-cell population is expanded to a desired level, and then subjected to cell sorter treatment for the above-mentioned CD4, CD25, CD45RB or CD45R0, and LAG-3. “Egr-2 Treg cells” may be isolated. In such cases, lymphocyte populations or T cell populations can be grown by culturing in the presence of CD3.CD28 costimulation, TGF-beta and IL-2.

In addition, the above-mentioned CD81, CD25, CD45RB or CD45RO, and LAG-3 can be used as they are. If the number is insufficient, the isolated _§ 1-2 ₆ _§ cells can be further _expanded by culturing in the presence of CD3 'CD28 co- _stimulation , TGF- _beta and IL-2. . In addition, for treatments that require strong organ migration, they were isolated using the above method.

By introducing a T cell receptor gene against an organ-specific antigen into “Egr-2 Treg cells” using a retrovirus vector, the antigen-specific therapeutic effect can be enhanced.

In the present invention, cells that are positive for the cell surface antigen CD4 are sorted based on the fact that the cells are clearly one group in which the CD4 fluorescence intensity is enhanced more and more by Celso using an antibody against CD4. Can be isolated. As antibodies to CD4, Al 1 ophycocyan in (APC) labeled anti-CD antibody (available from BDbioscience, Beckman Coulter, AbD Serotec, etc.) for humans, FITC labeled anti-CD4 antibody (BD bioscience) for mice Available from Beckman Coulter, BioLegend, etc.).

In the present invention, cells that are negative for the cell surface antigen CD25 are sorted based on the fact that the cells are a population obtained by removing one population in which the CD25 fluorescence intensity is clearly enhanced by a cell sorter using an antibody against CD25. Can be isolated. As for antibodies against CD25, allophycocyanin-Cyanin-7 (APC-Cy7) labeled anti-CD25 antibody (available from BD bioscience, BioLegend, etc.) for humans, and APC-labeled anti-CD25 antibody (BD bioscience) for mice. Available from BioLegend, etc.).

According to the present invention, cells that are negative (low positive) for the cell surface antigen CD45RB are 70% of cells that are relatively strongly expressing CD45RB among CD4 positive cells according to CELSO overnight using an antibody against CD45RB. The antibody against CD45RB can be isolated by using Phycoerythrin (PE) as an antibody against CD45RB (hereinafter, synonymous with “CD45RB ex low” in the main text). ) Labeled anti-CD45RB antibody (available from BD bioscience, AbD Serotec, BioLegend, etc.) For mice Fluorescein isothiocyanate (FITC) labeled anti-CD45RB antibody (available from BD bioscience, BioLegend, Dako, etc.) In the case of rabbits, the CD45RB negative (low positive) cell population can also be sorted as a single population with enhanced CD45R0 fluorescence intensity. versus Clearly by cell sorting evening one to use that antibody Isolation can be performed by sorting based on the fact that the CD45R0 fluorescence intensity is enhanced. As an antibody against CD45R0, a PE-labeled anti-CD45R0 antibody (available from BD bioscience, etc.) can be used for humans.

The cells positive for the cell surface antigen LAG-3 in the present invention are based on the fact that the cells are a group in which the LAG-3 fluorescence intensity is clearly enhanced by a cell saw using an antibody against LAG-3. It can be isolated by separating it. As an antibody against LAG-3, ATT0 488-labeled anti-LAG-3 antibody (available from Alex is Biochem ls) is available for humans, and PE-labeled anti-LAG-3 antibody (BD pharmingen) for mice. Available from companies).

As described above, since the “Egr-2 Treg cell” of the present invention has a function of causing local immune tolerance as a regulatory T cell, the present invention provides an isolated “Egr-2 Treg cell”. ”As a constituent component, for treating various diseases caused by an immune reaction against a self-antigen or due to an antigen being eliminated by an antigen-antibody reaction, such as various autoimmune diseases, transplanted organ rejection, etc. A pharmaceutical composition can be provided.

When the isolated “Egr-2 Treg cell” of the present invention is used in a pharmaceutical composition for treating various autoimmune diseases, transplant organ rejection, etc., 2 × 10 ⁶ cells / kg to 4 This can be done by intravenous administration of 100 to 500 ml of a cell suspension of X 10 ⁶ cels / kg. The isolated “Egr-2 Treg cells” of the present invention administered in this manner are carried throughout the body in the bloodstream and can induce local immune tolerance.

In the present invention, “various diseases caused by antigens being eliminated by antigen-antibody reaction” mainly refers to diseases such as rejection of transplanted organs, but is not limited thereto. The term “various diseases caused by immune responses to self antigens” refers to autoimmune diseases such as rheumatoid arthritis, collagen disease, inflammatory bowel disease, multiple sclerosis, type 1 diabetes.

The present invention also provides a method of screening for a compound capable of inducing the proliferation of the isolated ^ _§1 "-26 £ cell" of the present invention. Specifically, (a) §1 of the present invention "-2 1 ^ a _§ cell", step culturing in the presence of a test compound; (b) cultured "Egr-2

Changes in Egr-2 gene expression or LAG3 gene expression in Treg cells (C) selecting a compound that leads to increased expression of Egr-2 gene or increased expression of LAG3 gene as a compound that increases “Egr-2 Treg cells” of the present invention; A method for screening a compound that increases “Egr-2 Treg cells” is provided.

Such compounds can also be used to grow "Egr-2 Treg cells" isolated in vitro or immunize by growing "Egr-2 Treg cells" in vivo. It can also be used to induce tolerance.

Example

Example 1:

The purpose of this example is to clarify the mechanism by which the transcription factor Egr-2, which is expected to be related to T cell analogy, is related to the T cell suppressive ability. The Egr-2 gene was introduced into T cells, and the effects of Egr-2 transformed T cells were examined.

First, the full-length Egr2 cDNA obtained from the cDNA library was introduced into the retroviral vector pMX (Onishi M, et al., 1996 Exp. Hematol. 24: 324-329) and pMX-Egr2-IRES-GFP A retroviral vector was prepared (Figure 1). Using the same method, we created a retrovirus vector that introduced Foxp3, D0TAE, and D0TBE (D0TAE and D0TBE correspond to the specific T cell receptor α-chain for chicken ovalbumin (OVA), respectively] cDNA). Since Egr2 and Foxp3 are used to evaluate the expression of transferred genes by GFP (green fluorescent protein) expression,

(internal ribosomal entry site) / GFP was co-introduced. The mock pMX-IRES-GFP vector was used as a control (Fig. 1). Each plasmid was transfected into a packaging cell line, PLAT-E cells, to obtain genetically modified retrovirus-producing cells. The retrovirus obtained from the culture supernatant was then infected into splenocytes using RetroNectin (Takara, Japan).

Mock (pMX-IRES-GFP) or Egr2 (pMX-Egr2-IRES-GFP) retrovirus vector obtained in this way was infected with mouse spleen cells to express Egr2, and the resulting mouse CD4-positive spleen Using flow cytometry, cells were divided into 4 groups each using GFP expression as an indicator.

Separated and collected (negative, low, mid, high) (Fig. 2). Then, cDNA synthesis was performed from each cell of each of the 4 groups separated and collected in Fig. 2, and Egr2 mRNA and LAG-3 mRNA were synthesized. Expression at the level was examined by real-time PCR (Figure 3). The mRNA expression is a relative expression level normalized with / 3 -actin. The real-time PCR method was analyzed using the same method. As a result, LAG-3 expression was enhanced along with Egr2 expression (Fig. 3).

Furthermore, a delayed hypersensitivity reaction to chicken ovalbumin (OVA) was examined to evaluate the ability of Egr2 to suppress in vivo. Specifically, a mixture of OVA 200 g and complete Freund's adj uvant (CFA, Chondrex or BD Di fco) was intradermally applied to the tail of Balb / c mice (6-8 weeks old). Injected. Six days after the first immunization, isolated splenic mononuclear cells (Balb / c mice (6-8 weeks old)) were treated with pMX-Egr2- IRES-GFP, pMX-Foxp3-IRES-GFP, pMX-DOTAE, 72 hours after each infection with pMX-DOTBE retrovirus, CD8, -CDl lb, -CD19 positive cells were biotinylated using a MACS column (Milttenyi Biotec), and CD19 antibody and Strepton were biotinylated. After negative selection with avidin-coupled magnetic beads and CD4 positive cells were concentrated, 1 × 10 ⁷ cells / mouse was transferred by intravenous injection from the tail of a mouse immunized with 0VA. Each experimental group consists of Mock (pMX-IRES-GFP), D011.10 (pMX-D0TAE, pMX-DOTBE), Foxp3 (pMX-Foxp3-IRES-GFP), Egr2 (pMX-Egr2-IRES-GFP) Retroviruses were infected as shown (n = 12 animals in each group). 48 hours after transfer of each retrovirus-infected cell, booster immunization with OVA20 / xg / PBS100 l on the right foot and PBS 100 / ^ at the same time on the left foot, Δ footpad (right footpad thickness- Left foot pad thickness) 匪 was measured and used as an index of delayed hypersensitivity reaction (Δ foot pad (24 hours after reimmunization-before reimmunization) mm).

As a result, Egr2 suppressed delayed hypersensitivity reaction like Foxp3. In addition, co-transfer of 0VA-specific T cell receptor significantly enhanced the suppressive effect, indicating that antigen-specific immunosuppression was possible (Fig. 4).

In addition, spleen mononuclear 24 hours after booster immunization of mice co-transfected with Egr2 (p X-Egr2-IRES-GFP) and DO 11.10 (pMX-DOTAE, pMX-DOTBE) under the same experimental conditions as in Fig. 2. The cells were separated and analyzed by flow cytometry. Spleen mononuclear cells are lymphocytes and flow cytometry

After CD positive cell gate, it was developed with D011. 10T cell receptor specific antibody 26-26 antibody and GFP, and each cell population was separated and collected as shown in the figure. In addition, the cells isolated and collected in Fig. 3 CDNA synthesis was performed, and expression at the Egr2 mRNA, LAG-3 mRNA, and Foxp3 mRNA levels was examined by real-time PCR. As a result, the transferred cells overexpress Egr2 with PMX-Egr2-IRES-GFP, enhance the expression of LAG-3, and co-express the OVA-specific T cell receptor. Expression was further enhanced, indicating that an antigen-specific T cell receptor signal is important for the ability to suppress Egr-2 (Figures 5 and 6).

These results indicate that Egr2 induces regulatory T cell-like activity in vivo. In addition, Egr2-transformed sputum cells express LAG-3 significantly, and the expression level of Egr2 and LAG-3 are positively correlated. Furthermore, LAG-3 expression is consistent with antigen-specific T cell reception. It became clear that it was strengthened more.

Example 2:

In this example, cell sorting was performed for the purpose of collecting T cells expressing the Egr-2 gene.

Mouse spleen mononuclear cells were analyzed with a flow cytometer using CD4-Cy7AP CD25-AP CD45RB-FIT LAG-3-PE. As a result, LAG-3 surface antigen showed enhanced expression in CD25 negative cells (Fig. 7a) CD45RB negative to CD45RB weak positive cells (Fig. 7b) among CD4 positive cells (Egr-2 Treg cells). Foxp3-Treg is defined as a CD4-positive and CD25-positive T cell group, and is a cell population different from Egr2-Treg.

In addition, CD4 positive T cells were isolated and collected using flow cytometry according to the presence or absence of LAG-3 expression. After cDNA synthesis, LAG-3 mRNA, Egr2 mRNA, Foxp3 mRNA, and IL were synthesized by real-time PCR. -10 mRNA expression was examined, and enhanced expression of Egr 2 mRNA, IL-10 mRNA, and LAG-3 mRNA in LAG-3 expressing cells was observed (Fig. 8). Foxp3 expression did not show significant correlation with LAG expression.

Next, the cells gated in the CD4 positive CD45RB ex low cell group were expanded with LAG-3 and CD25, and then CD4 + CD25- LAG3 + CD45RB ex low (Egr2- Treg), CD4 + CD25- LAG3- CD45RB ex l ow,

Three cell populations, CD4 + CD25 + CD45RB ex low, were isolated and collected (FIG. 9).

CDNA was synthesized from the cells isolated and collected in Fig. 9, and the expression of Egr2 mRNA and Foxp3 mRNA was examined by real-time PCR. As a result, CD4 + CD25- LAG3 + CD45RB ex l ow (Egr2-Treg) showed the strongest expression of Egr2, and Foxp3 expression did not show the same expression tendency (Fig. 10).

In addition, cells gated in CD4 positive CD25 negative cell groups are expanded with LAG-3 and CD45RB, and each cell group is separated and collected by flow cytometry as shown in the figure, synthesized cDNA, and real-time PCR The expression of Egr2 mRNA and Foxp3 mRNA was examined by the method. As a result, it was shown that Egr2 expression was strongest in CD4 + CD25- LAG-3 + CD45RB ex low, and there was Egr2-Treg different from the known Foxp3-Treg (Fig. 11, Fig. 12).

Example 3:

In this example, Egr2-Treg was examined using a type II collagen-induced arthritis onset model. ,

DBA-1J mice (6-8 weeks old) were injected intradermally with a mixture of sushi-derived collagen type II and complete Freund's adjuvant (complete Freund's adjuvant, CFA, Chondrex or BD Di fco) (dayO) ), And then boosted with type II collagen (day 21) to induce type II collagen-induced arthritis. After induction of arthritis, each cell group was separated and collected by the same method as in Fig. 11 and analyzed by real-time PCR. As a result, CD4 + CD25-LAG3 + CD45RB ex low (Egr2-Treg) still existed after immunization with type II collagen, and further enhancement of IL-10 expression in “Egr2-Treg” was observed after immunization (FIG. 13). still,

Egr-2 remained highly expressed even after the onset of arthritis.

Example 4: '

In this example, CD4 + in vitro using spleen cells from C57BL / 6 mice.

The inhibitory ability of CD25-LAG-3 + CD45RB ex low (Egr2-Treg) was examined.

As a naive T cell subset, CFSE-labeled CD4 + CD25-CD45RB high cell population (Thyl. H) 5X10 ⁴ cells and spleen cells (Thy 1.2+) lX10 ⁵ cells irradiated with 1500 rad as antigen-presenting cells were used. As suppressor cells, the CD4 + CD25- CD45RB ex low cell population (Thyl.2 +) was separated and collected into 3 groups of high, low and nega by LAG-3 expression (each group 5X10 ⁴ cells) (Fig. 14) . Each cell group was co-cultured for 72 hours in the presence of soluble CD3 antibody as shown in the figure, and the attenuation of CFSE expression in cells gated with Thyl.1 + CD4 + was evaluated as an indicator of CD4 + CD25- CD45RB high cell population proliferation. went. As a result, Egr-2 Treg cells suppressed cell division of the naive T cell subset (CD45RBhi cells), and the suppression ability was dependent on LAG-3 expression (Fig. 15). Example 5:

In this example, C57BL / 6 mouse spleen CD4 + CD25- CD45RBhi cells 1X10 ⁵ cells were intraperitoneally administered to Rag 卜 / − mice to produce an inflammatory enteritis model.

Enteritis was assessed by weight loss and histology. Body weight was 100% on the day of cell transfer (dayO). Each group consists of 3 groups of CD4 + CD25- CD45RBhi cells 1X10 ⁵ cells only, CD4 + CD25- CD45RBhi cells 1X10 ⁵ cells + CD4 + CD25-LAG3 + CD45RB ex low (Egr2-Treg) cells IX 10 ⁵ cells and PBS only (control) Evaluated. In RAG 卜 /-mice, enteritis caused by C57BL / 6 mouse spleen CD4 + CD25- CD45RBhi cell administration could be suppressed by co-transfer of CD4 + CD25- LAG3 + CD45RB ex low (Egr2-Treg) cells (Fig. 16) . In addition, in the histology of the large intestine, CD4 + CD25- LAG3 + CD45RB ex low (Egr2-Treg) cells co-transferred with inflammatory cell infiltration and intestinal wall thickening, etc., observed with single administration of CD4 + CD25- CD45RBhi cells. (Fig. 17).

Example 6:

Foxp3 is currently thought to be a mass gene for suppressor T cells. Therefore, in this example, Scurfy mice lacking the Foxp3 functional gene were used.

A study on Egr2-Treg was conducted.

First, spleen cells of Scurfy mice lacking the functional gene of Foxp3 were analyzed by flow cytometry using CD4-Cy7AP CD25-APC and CD45RB-FIT LAG-3-PE. As a result, CD4 + was also detected in spleen cells of Scurfy mice.

CD25- LAG3 + CD45RB ex low (Egr2- Treg) cells were shown to be present (Figure 18). In addition, as a naive T cell subset, CFSE-labeled C57BL / 6 mouse CD4 + CD25-

The CD45RB high cell population (Thyl.) 5X10 ⁴ cells was used as antigen-presenting cells, and whole splenocyte (Thyl.2 +) 1X10 ⁵ cells of 1500 rad irradiated C57BL / 6 mice were used. Suppressor cells include Scurfy mouse CD4 + CD25- CD45RB ex low cell population (Thyl.2 +)

2.5 × 10 ⁴ cells were used. Each cell group was co-cultured for 72 hours in the presence of soluble CD3 antibody as shown in the figure, and the CFSE expression attenuation of cells gated with Thyl. II CD4 + was CD4 + CD25- CD45RB high The cell population was evaluated as an indicator of cell division and proliferation. A system in which LAG-3 neutralizing antibody was added to the culture supernatant under the same conditions was also examined. As a result, it was shown that Egr2-Treg cells of Scurfy mice lacking the functional Foxp3 gene have suppressive ability in vitro, and the suppressive ability is attenuated by LAG-3 neutralizing antibody (Fig. 19). .

Furthermore, C57BL / 6 mouse spleen CD4 + CD25- CD45RBh i cells 1X10 ⁵ cells were intraperitoneally administered to Ragl − / − mice to prepare a model for the development of inflammatory bowel disease. Enteritis was assessed by weight loss and histology. Body weight was defined as 100% on the day of cell transfer (dayO). Each group consists of C57BL / 6 mouse spleen CD4 + CD25- CD45RBhi cells 1X10 ⁵ cells only, C57BL / 6 mouse spleen CD4 + CD25- CD45RBhi cells 1 X 10 ⁵ cells + Scurfy mouse CD4 + CD25- LAG-3 + CD45RB ex low (Egr2 -Treg) Cells were evaluated in 3 groups: 1 x 10 ⁵ eel Is and PBS only (control). As a result, it was shown that enteritis in the inflammatory enteritis onset model of Ragl-/-mice was also suppressed in vivo (Fig. 20).

From the above, it was shown that Egr2- Treg cells are a novel inhibitory T cell population that exerts suppressive ability independent of Foxp3 function, and that the suppressive ability is mediated by LAG-3.

Example 7:

In this example, in order to analyze the function of the Egr2 gene, a shRNA-Egr2 retrovirus was prepared, the Egr2 gene was silencing, and mouse phenotype analysis was performed. Specifically, bone marrow cells of C57BL / 6 mice treated with 5FU were collected, infected with shRNA-Egr2 retrovirus, and then transferred to irradiated C57BL / 6 mice, so that shRNA-Egr2 bone marrow chimeric mice were Fabricated (Fig. 21). As a result, LAG-3 expression was suppressed by silencing Egr2 gene expression, and shRNA-Egr2 bone marrow chimeric mice showed marked thickening of the intestinal tract. . After bone marrow transplantation, he developed severe enteritis and died in about 4 weeks.

Example 8:

In this example, in order to clarify the mechanism by which Egr2-Treg cells of the present invention are induced in vivo, an induction experiment of Egr2-Treg cells was performed.

Finely cut the spleen and Peyer's patch of C57BL / 6 mice or B cell-deficient mice / χΜΤ mice (Jackson Laboratory, Bar Harbor, ME) into small pieces of tissue. CD4 + T cells were collected from genase (Sigma-Aldrich, St. Louis, MO) and PE-anti-LAG-3 monoclonal antibody (LAG-3-PE) was used as described in Example 2 for these cells. The analysis was performed on a flow cytometer using the FITC-anti-CD45RB monoclonal antibody (CD45RB-FITC). As a result, in C57BL / 6 mice, both spleen-derived cells and Peyer's patch-derived cells have T cells with the same phenotype as the Egr2-Treg cells of the present invention (ie, LAG-3 + CD45RB ex low). However, it was revealed that no T cells with LAG-3 + CD45RB ex low were present in jMT mice (Fig. 23).

From this result, it was shown that B cells may have some function in inducing differentiation of Egr2-Treg cells in vivo. In this example, it is further a B cell-deficient mouse; On the other hand, B cells collected from the spleen of C57BL / 6 mice were transplanted to examine whether the Egr2- Treg cells of the present invention were induced in the living mouse.

C57BL / 6 mouse sputum cells can be obtained from MACS using a single cell suspension of spleen from C57BL / 6 mice using the S cell isolation kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer's protocol. Purification was done by negative selection. The purity of B cells sorted by MACS was> 98%. 2X10 ⁷ B cells purified in this way were injected intravenously into / MT mice. Control mice were irradiated with PBS. Nine weeks after cell transplantation, mice were sacrificed and spleen cells were analyzed by flow cytometry. As a result, in MT mice transplanted with B cells from C57BL / 6 mice, T cells having the same phenotype (LAG-3 + CD45RB ex low) as the Egr2-Treg cells of the present invention were significantly compared to ζΜΤ mice. (Fig. 23). Therefore, it has been clarified that the presence of B cells is indispensable for inducing differentiation of the Egr2-Treg cells of the present invention in vivo.

Example 9:

Next, it was known that Foxp3 ⁺ Treg cells are required to have higher affinity agonist interactions with self-peptide / MHC expressed by thymic stromal cells to induce differentiation. In this example, it was examined whether CD4 + CD25— LAG3 + T cells were induced to differentiate through the same thymic selection process as Foxp3 ⁺ Treg cells.

TCR Transgenic 0T-II mice (Taconic) and RIP-mOVA mice (Jackson Laborat ory) Transgenic TCRs that express membrane-bound OVA under the control of the rat insulin gene promoter in the islets and thymus while simultaneously recognizing OVA peptides in the context of IA ^b RIP-mOVA / OT-I I double-transgenic mice were also generated that also express V a 2 and V) 3 5.1 / 5. In this RIP-mOVA / OT-I I double-transgenic mouse, the frequency of CD4 + CD25 "LAG3 + T cells, as opposed to the increased frequency of CD4 + CD25 + Treg cells in these organs And the spleen did not increase (Figure 24), where the top and bottom panel plots are CD4 + V j3 5.1 / 5.2 + and CD4 ⁺ CD25 "V / 3 5.1 / 1 respectively. 5. Gated for 2 ⁺ T cells.

On the other hand, 0T-II transgenic mice contained as many CD4 + CD25- LAG3 + T cells as normal mice in the spleen and Peyer's patches, but Ι-Ε α-chain-specific IA ^b -restricted transgenig TEa transgenic mice expressing TCR had few CD4 ⁺ CD25- LAG3 ⁺ T cells, especially in the spleen (Figure 25).

Furthermore, TEa mice also contained very few ½r-2-expressing cells in splenic follicles in histological examination (FIG. 26). In this figure, each bar indicates the number of measurements from one follicle. This data was collected from 3 mice in each group. All error bars indicate standard deviation; an asterisk indicates <0. 01.

Taken together these results indicate that Egr2-Treg cells have a high affinity interaction with the autoantigen peptide / MHC complex in the thymus for differentiation. It became clear that it was a cell. Interestingly, TEa transgenic mice developed spontaneous enterocolitis at 10 weeks of age in the breeding environment of the inventor, and this animal could be used as a model for colitis. (Figure 27). In this figure, representative macroscopic photographs (left) and hematoxylin of the colon of TEa mice, the colon of TEa mice adopting CD4 + CD25- LAG3 + T cells, and the colon (control) of normal Tea littermates -Shows eosin tissue section (right). In addition, when CD4 + CD25— LAG3 + T cells were transplanted from normal C57BL / 6 mice into TEa transgenic mice, the spontaneous development of colitis in TEa transgenic mice was markedly suppressed. (Figure 27). These findings suggest that CD4 + CD25 "LAG3 + T cells have therapeutic potential to modulate colitis-induced T cell responses.

Example 10:

In this example, based on the mouse knowledge obtained in Example 2, cell sorting was performed for the purpose of collecting T cells expressing the Egr-2 gene from human tonsil-derived cells. .

Human tonsil-derived cells were analyzed by flow cytometry using CD4-Cy7AP CD25-APC, CD45R0-FITC, and LAG-3-PE. Since it was known that the CD45RB negative (low positive) cell population could be sorted as a population with enhanced CD45R0 fluorescence intensity in rabbits, the CD45RB- Instead of FITC, CD45R0-FITC was used in this example.

As a result, enhanced expression of LAG-3 surface antigen was observed in CD25 negative cells (Fig. 28a) and CD45R0 positive cells (Fig. 28b) among CD4 positive cells (Egr-2 Treg cells).

Furthermore, CD4 positive T cells were isolated and collected using flow cytometry according to the presence or absence of LAG-3 expression. After cDNA synthesis, LAG-3 mRNA, Egr2 mRNA, Foxp3 mRNA, and IL were synthesized by real-time PCR. -10 When mRNA expression was examined, enhanced expression of Egr 2 mRNA, IL-10 mRNA, and LAG-3 mRNA in LAG-3 expressing cells was observed (Fig. 29). Foxp3 expression was not significantly correlated with LAG expression.

Industrial applicability

Currently, to treat various diseases caused by the elimination of antigens due to unwanted antigen-antibody reactions, such as autoimmune diseases and transplanted organ rejection, administration of immunosuppressive agents, etc. However, side effects including severe infections associated therewith have been a major problem. However, administration of the regulatory T cells of the present invention can induce local immune tolerance. Therefore, autoimmune diseases and transplanted organs caused by immune responses to self-antigens or elimination of antigens by unwanted antigen-antibody reactions without causing side effects as described above in the patient's body. Various diseases such as rejection can be treated.

Claims

The scope of the claims

1. CD4 positive (CD4 +), CD25 negative (CD25-), CD45RB negative '14. · (low positive) (CD45RB- (low)), and LAG-3 positive (LAG3 +) or CD4 positive (CD4 +), CD25 negative An isolated T cell from a mammal with cell surface antigenic properties of (CD25-), CD45RO positive (CD45R0 +), and LAG-3 positive (LAG3 +).

2. The isolated T cell according to claim 1, which has a function as a regulatory T cell.

3. The isolated T cell according to claim 1 or 2, wherein the function as a regulatory T cell is a function of inducing T cell tolerance (tolerance).

4. The isolated T cell according to any one of claims 1 to 3, wherein the mammal is a human.

5. From a mammalian lymphocyte population or T cell population,

(i) collecting CD4 positive cells (CD4 +);

(i i) collecting cells that are negative for CD25 (CD25-);

(i i i) collecting CD45RB negative (low positive) cells (CD45RB- (low)) or CD45R0 positive cells (CD45R0 +); and

(iv) collecting a cell (LAG3 +) positive for LAG-3;

By performing the steps (i) to (iV) in any order, CD4 positive (CD4 +), CD25 negative (CD25-), CD45RB negative (low positive) (CD45RB- (low)), and LAG-3 Mammalian origin with positive (LAG-3 +) or CD4 positive (CD4 +), CD25 negative (CD25-), CD45R0 positive (CD45R0 +), and LAG-3 positive (LAG-3 +) cell surface antigen properties Of isolating T cells.

6. The method for isolating a mammal-derived T cell according to claim 5, wherein the steps (i) to (iv) are performed after collecting a mammalian lymphocyte population or T cell population from peripheral blood. Law.

7. The mammalian lymphocyte population or T cell population is collected from peripheral blood, and the lymphocyte population or T cell population is expanded, and then the steps (i) to (iv) are performed. Of isolating mammalian T cells.

8. The mammal-derived T according to any one of claims 5 to 7, wherein the T cell population of the present invention isolated by performing the steps (i) to (iv) is used as it is. Isolate cells how to.

9. The mammalian origin according to any one of claims 5 to 7, wherein the T cell population of the present invention isolated by performing the steps (i) to (iv) is used after being expanded. A method of isolating the sputum cells.

10. The method for isolating a mammal-derived sputum cell according to any one of claims 5 to 9, wherein the mammal is baboon.

1 1. CD4 positive (CD4 +), CD25 negative (CD25-), CD45RB negative (low positive) (CD45RB- (low)), and LAG-3 positive (LAG-3+) or CD4 positive (CD4 +), CD25 negative A pharmaceutical composition comprising an isolated T cell from a mammal having (CD25-), CD45R0 positive (CD45R0 +), and LAG-3 positive (LAG-3 +) cell surface antigen properties.

12. The pharmaceutical composition according to claim 11, for treating various diseases caused by an immune reaction against a self-antigen or caused by elimination of an antigen by an antigen-antibody reaction.

1 3. The pharmaceutical composition according to claim 12, for treating autoimmune disease or transplanted organ rejection.

14. The pharmaceutical composition according to claim 13, wherein the autoimmune disease is selected from rheumatoid arthritis, collagen disease, inflammatory bowel disease, multiple sclerosis, type 1 diabetes and the like.

1 5. (A) CD4 positive (CD4 +), CD25 negative (CD25-), CD45RB negative (low positive) (CD45RB- (low)), and LAG-3 positive (LAG-3+) or CD4 positive (CD4 +) Culturing T cells derived from mammals with cell surface antigen characteristics of CD25 negative (CD25), CD45R0 positive (CD45R0 +), and LAG-3 positive (LAG-3 +) in the presence of the test compound Process;

(c) Compounds that increase Egr-2 gene expression or increase LAG-3 gene expression are CD4 positive (CD4 +), CD25 negative (CD25-), CD45RB negative (low positive) (CD45RB- (low) ), And LAG-3 positive (LAG3 +) or CD4 positive (CD4 +), CD25 negative (CD25-), CD45R0 positive (CD45R0 +), and LAG-3 positive (LAG3 +) cell surface antigens Selecting as a compound that increases the number of T cells; CD positive (CD4 +), CD25 negative (CD25-), CD45RB negative (low positive) (CD45RB- (low)), and LAG-3 positive (LAG-3+) or CD4 positive (CD4 +), CD25 negative A screening method for compounds that increase T cells derived from mammals having (CD25-), CD45R0 positive (CD45R0 +), and LAG-3 positive (LAG-3 +) cell surface antigen properties.