WO2004039968A1

WO2004039968A1 - Immortalized dendritic cell line originating in bone marrow

Info

Publication number: WO2004039968A1
Application number: PCT/JP2003/013951
Authority: WO
Inventors: Toshiyuki Takai; Masuo Oginata; Yumi Ito; Kozue Ito; Shin Ebihara
Original assignee: Japan Science And Technology Agency
Priority date: 2002-10-30
Filing date: 2003-10-30
Publication date: 2004-05-13

Abstract

It is intended to provide an immortalized dendritic cell line sustaining the functions and properties inherent to dendritic cells, a method of establishing the same, a method of screening a useful substance by using the above immortalized dendritic cell line, and a cell vaccine comprising the above immortalized dendritic cell line as the main component. Namely, an immortalized dendritic cell line having an ability to take up an antigen, an ability to present an antigen and an ability to introduce CTL activity is established by hemolyzing bone marrow cells of a transgenic mouse carrying a large T antigen gene of a temperature-sensitive SV40 mutant strain tsA58 transferred thereinto, removing lymphocytes and Ia-positive cells therefrom, culturing the thus obtained cells in the presence of GM-CSF to induce dendritic cells, repeatedly subculturing the cells 10 times or more, and thus expressing myeloid molecules and leukocyte molecules on the cell surface.

Description

Immortalized dendritic cell line derived from bone marrow

The present invention relates to an immortalized dendritic cell line derived from bone marrow. More specifically, the present invention relates to a transgenic mouse transfected with the large T antigen gene of a temperature-sensitive mutant tsA58 of SV40 (tMs s SV40). Dendritic cells (DCs) derived from the bone marrow of LTT g mice)

The present invention relates to an immortalized dendritic cell line that can be established, a method for producing the cell line, and use thereof. The immortalized dendritic cell line of the present invention can be applied to in vitro analysis of dendritic cells, development of vaccine therapy using dendritic cells, and study of modification and enhancement of immune response. Background art

In the past, animals were mainly used for testing and researching the safety and efficacy of pharmaceuticals.However, instead of using animals from the viewpoint of animal welfare, the efficacy and safety of pharmaceuticals were studied in vitro using cultured cells. Research is being conducted at the practical level of technology for testing and researching the technology. For example, animal tests have been carried out after preliminarily testing using primary cultured cells collected from biological tissues or immortalized cells (established cells) that grow indefinitely. However, primary cells proliferate well in the early stage, but gradually stop growing with subculture and eventually die (this phenomenon is called cell senescence). In addition, it has been pointed out that the characteristics of primary cells change with passage, in addition to the fear that their characteristics will differ each time they are collected from living tissue. In particular, if the growth rate is very slow or derived from micro-organs, primary cells that are sufficient for testing may be obtained. And is said to be very difficult.

On the other hand, immortalized cells that have acquired the ability to proliferate indefinitely while avoiding cell senescence during repeated passage of primary culture will have stable and uniform characteristics. Many cells lose some or all of the morphology and functions that cells originally have in living organisms. Therefore, it has been considered difficult to accurately reflect the original characteristics of the tissue from which the cell line is derived in a test using such an immortalized cell line. Thus, oncogenes such as the ras gene and the c-myc gene, the adenovirus ElA gene, the SV40 virus large T antigen gene, and the human papillomavirus HPV16 gene were introduced into primary cells. Attempts have been made to establish immortalized cells by transforming the cells, maintaining the vigorous proliferative ability of the primary cells continuously, and further subculturing the cells without losing the inherent characteristics of the cells. However, even in such immortalized cells, some functions have already been lost when primary cells are prepared and these oncogenes and large T antigen genes are introduced, depending on the target organ. However, it was difficult to obtain immortalized cells in the strict sense of maintaining the original function. In particular, it has been extremely difficult to prepare and establish primary cells when the growth rate is very slow or derived from micro-organs.

On the other hand, instead of introducing oncogenes and large T antigen genes into individual cells using gene transfer technology for animal individuals that has been established in recent years, gene transfer that stably integrates these genes into chromosomes An animal is created, primary cells are prepared from an organ of an animal that already has an oncogene or a large T antigen gene in the cell at the time of generation of the individual, and immortalized cells are obtained by subculture. Methods for establishing have been reported. In particular, immortalized cells obtained from ts SV40 LTT g mouse organs can be manipulated by changing the temperature to control their growth and development of differentiation traits. (Transgenic Research 4, 215-225, 1995, Genes to Cells, 2, 235-244, 1997, Exp.Cell Res., 197, 50-56, 1991, Exp.Cell Res., 209, 382-387, 1993, Exp.Cell Res., 218, 424-429, 1995, Blood, 86, 2590-2597, 1995, J. Cell.Physiol., 164, 55-64, 1995, Exp.Hematol , 27, 1087-1096, 1999).

Dendritic cells (DCs), on the other hand, are dendritic cell populations derived from hematopoietic stem cells and are widely distributed in vivo. Immature dendritic cells recognize and take in foreign substances, such as viruses and bacteria, that have invaded their tissues, and generate peptides by digestion and degradation in the process of migrating to lymphoid organ T cells. By binding to MHC molecules and presenting them on the cell surface, they act as antigen-presenting cells that activate antigen-specific T cells and induce an immune response (Ann. Rev. Immunol. 9, 271-296, 1991, J. Exp.Med., 185, 2133-2141, 1997) ₀ Thus, DCs are very important for the initiation of T cell responses that can elicit T cell-dependent early immune responses. (Nature, 392, 245-252, 1998; Annu. Rev. Immunol., 18, 767-811, 2000). DCs born in the bone marrow are immature and are phagocytosically distributed to various tissues in the body. The immature DCs take up the antigen, mature and migrate to secondary lymphoid organs. It accumulates in the T cell area and selectively activates antigen-specific T cells circulating in the body to drive an immune response. However, these detailed mechanisms in DC in vivo are still unknown and need to be analyzed in vitro. The use of granulocyte-macophage phage colony-stimulating factor (GM-CSF) in the mouse in vitro mouth system allows the induction of functional DCs in the initial culture, but has a longevity of at least 2 months. (J. Exp. Med., 175, 1157-1167, 1992). Recently, long-term culture (more than 12 months) of DC from the spleen, a secondary lymphoid organ of mice, has been performed in a GM-CSF dependent manner. (D1 cells: J. Exp. Med., 185, 317-328, 1997), but DC cell lines have been established from primary lymphoid tissues such as bone marrow that are immature and do not take up antigen. It has not been.

Dendritic cells (D C) are important cells that drive the immune response. However, the analysis of the dynamics of DC in vivo and its application to cancer immunostimulation have just begun, and many unclear points remain. DCs can be cultured by preparing them from living organisms, but their lifespan is limited.Even if they are cultured in the presence of cytokines such as GM-CSF, they can survive for as long as about one month and then die. I do. Until now, it has been extremely difficult to produce dendritic cells that continue to proliferate stably, and there is no simple method for establishing dendritic cells. The prospect was not standing. That is, an object of the present invention is to provide an immortalized dendritic cell line that retains the functions and characteristics inherent to dendritic cells, a method for establishing the same, a method for screening a useful substance using the immortalized dendritic cell line, and a method for screening the same. An object of the present invention is to provide a cell vaccine containing an immortalized dendritic cell line as a main component.

The present inventors have conducted intensive studies to solve the above problems, and after hemolyzing bone marrow cells of ts SV40 LTT g mice, removing lymphocytes and Ia-positive cells, and obtaining the obtained cells. Dendritic cells are induced by culturing in the presence of GM-CSF, and subculture is repeated 10 times or more.The established immortalized dendritic cell line expresses myeloid and leucosite molecules on the cell surface. The present inventors have confirmed that DCs have inherent properties such as an ability to take up an antigen, an ability to present an antigen, and an ability to induce CTL activity, thereby completing the present invention. Disclosure of the invention

That is, the present invention provides an immortalized dendritic cell characterized by being derived from bone marrow. The strain (Claim 1), which expresses a myeloid molecule and a leucosite molecule on the cell surface, and has an antigen uptake ability, an antigen presentation ability, and an ability to induce CTL activity. The immortalized dendritic cell line (Claim 2) or can be grown at 33 ° C, but the growth is suppressed at 37 ° C, The immortalized cell line according to claim 1 or 2, A dendritic cell line (Claim 3), an immortalized dendritic cell line according to any one of claims 1 to 3, which has an ability to respond to LPS stimulation (Claim 4), and a rodent The immortalized dendritic cell line according to any one of claims 1 to 4, which is characterized in that it is an origin (claim 5), and the immortalized cell line according to claim 5, wherein the rodent is a mouse. The present invention relates to a dendritic cell line (Claim 6) and an immortalized dendritic cell line TDC (FE RM BP-08527) (Claim 7).

The present invention also provides a method for removing lymphocytes and Ia-positive cells after hemolyzing bone marrow cells of a transgenic mouse into which the large T antigen gene of the temperature-sensitive mutant tsA58 of SV40 has been introduced. The obtained cells are cultured in the presence of GM-CSF to induce dendritic cells.Subculture is repeated at least 10 times, expressing myeloid molecules and leucosite molecules on the cell surface, and taking up antigen. A method for producing an immortalized dendritic cell line, which comprises establishing a cell line having the ability to present antigen, the ability to present antigen, and the ability to induce CTL activity (claim 8), and growth at 33 ° C. The method for producing an immortalized dendritic cell line according to claim 8, characterized in that the growth is suppressed at 37 ° C, and a cell line capable of responding to LPS stimulation is established. 9) The immortality according to any one of claims 1 to 7, in the presence of a test substance. A method for screening a substance that promotes or suppresses maturation of dendritic cells, comprising culturing a denatured dendritic cell line, and measuring and evaluating the expression level of the maturation marker protein in the cell line (claim 10). ) and the marker one protein, Mieroi de molecules, Roikosai preparative molecular, I one a ^b, that CD 8 6 is及Pi Z or CD 4 0 A method for screening a substance that promotes or suppresses maturation of dendritic cells according to claim 10 (claim 11), and the immortalized tree according to any one of claims 1 to 7, in the presence of a test substance. A method for screening a substance that promotes or suppresses cell growth in dendritic cells (Claim 12), which comprises culturing a dendritic cell line, and measuring and evaluating the degree of proliferation of the cell. An LPS stimulation of the immortalized dendritic cell line according to any one of claims 1 to 7, and measurement and evaluation of the amount of IL-112 produced by the cell, whereby the activation of dendritic cells is promoted. Alternatively, the present invention relates to a method for screening an inhibitor (Claim 13).

The present invention further provides a substance for promoting maturation of dendritic cells obtained by the screening method according to claim 10 or 11 (claim 14), and the screening method according to claim 12. A cell growth promoting substance in dendritic cells (Claim 15), a dendritic cell activation promoting substance obtained by the screening method according to Claim 13 (Claim 16), and Claims 1 to 7 The immortalized dendritic cell line according to any one of the above, wherein the cell vaccine (claim 17) or the immortalized dendritic cell line can grow at 33 ° C. The immortalized dendritic cell line according to claim 17, wherein the cell vaccine is an immortalized dendritic cell line in which proliferation is suppressed in 37, and the immortalized dendritic cell line is an antigen. Or an immortalized dendritic cell line into which an antigen-IgG immune complex has been incorporated. That claims 1-7 or 1-8 wherein the cell vaccines (claims 1-9) and relates 請 Motomeko 1 9, wherein the cell vaccine antigen is characterized in that the tumor antigen (0 Claim 2). BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a photograph showing the results of Wit-Giemsa staining of dendritic cells of the immortalized dendritic cell line of the present invention and dendritic cells of B6 mouse.

FIG. 2 shows the MTT assay for the immortalized dendritic cell line of the present invention. FIG. 4 is a view showing the results of measuring the proliferation ability while changing the temperature conditions according to the present invention.

FIG. 3 is a view showing the results of measuring the requirement of GM-CSF by MTT assay in the growth of the immortalized dendritic cell line of the present invention. FIG. 4 is a diagram showing the results of analysis of the expression levels of representative myeloid molecules on the immortalized dendritic cell line of the present invention and dendritic cells of B6 mice by FACS.

FIG. 5 is a diagram showing the results of analysis of the expression levels of representative leucocyte molecules on the dendritic cell surface of the immortalized dendritic cell line of the present invention and the B6 mouse by FACS.

FIG. 6 shows the results of FACS analysis of the antigen uptake ability of the immortalized dendritic cell line of the present invention and the dendritic cells of B6 mice.

FIG. 7 is a graph showing the results of FACS analysis of dendritic cell maturity in response to LPS stimulation of immortalized dendritic cell lines of the present invention and dendritic cells of B6 mice.

FIG. 8 shows the results of measuring the amount of IL-12p70 produced by dendritic cells of the immortalized dendritic cell line of the present invention and the dendritic cells of B6 mice in response to LPS stimulation by ELISA. FIG.

FIG. 9 is a diagram showing the results of FVAS analysis of the maturity 2 days after OVA was incorporated into the immortalized dendritic cell line of the present invention and the dendritic cells of B6 mice.

FIG. 10 shows the results of the ability of the immortalized dendritic cell line of the present invention and dendritic cells of B6 mice to present antigen to OVA-T cells.

FIG. 11 is a diagram showing the time-course anti-OVA antibody titer of OVA transferred to the immortalized dendritic cell line of the present invention and the dendritic cells of B6 mice.

FIG. 12 shows the results of immunohistochemical staining (anti-GL-7 antibody) of the spleen germinal center approximately three weeks after the transfer of the dendritic cells of the dendritic cell line of the present invention. It is a photograph.

FIG. 13 is a diagram showing the results of comparative examination of the in vivo CTL activity by transferring the dendritic cells of the dendritic cell line of the present invention.

FIG. 14 is a diagram showing the results of comparing the expression levels of MHC class I / OVA peptide complexes in the immortalized dendritic cell line of the present invention and dendritic cells of B6 mice.

FIG. 15 is a view showing the results of enhanced antitumor activity in vivo by transferring the immortalized dendritic cell line of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

The immortalized dendritic cell line of the present invention may be any immortalized dendritic cell line derived from bone marrow, and can be grown at 33 ° C, and can grow at 37 ° C. A cell line to be suppressed is preferable, and a cell line having characteristics inherent to DC is more preferable except for this temperature sensitivity. For example, cell lines that express myeloid and leucosite molecules on the cell surface and have antigen uptake, antigen presentation, and CTL activity induction, or dendritic cells mature by stimulation with LPS Suitable examples include a cell line that is activated and has the ability to respond to LPS stimulation, such as producing IL-112, and a cell line having these properties in combination. Further, a specific example of such an immortalized dendritic cell line is the immortalized dendritic cell line TDC. This TDC line is deposited at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary under the depository number. FE RM BP—085-27 (transferred from FE RM P—19044 deposited on September 26, 2002) has been deposited under the Budapest Treaty. The origin of the immortalized dendritic cell line of the present invention is not particularly limited, but the immortalized dendritic cell line obtained from an animal such as a rodent such as a mouse has an abundant disease model in a mouse, Pharmacological review It is preferred because it is widely used for its value. Hereinafter, a method for producing the immortalized dendritic cell line of the present invention will be described by taking a mouse method as an example.

The immortalized dendritic cell line of the present invention derived from a mouse can be obtained, for example, by subjecting bone marrow cells of a ts SV40 LTT g mouse to hemolysis using ammonium chloride, for example, an anti-CD4 antibody, an anti-CD8 antibody, an anti-I one a ^b antibody, anti-rat I g using antibody and Usagi complement, complete R PM I medium cells depleted lymphocytes and I a-positive cells containing the mouse recombinant GM- CSF of 2 0 ng / mL (5% FCS) to induce dendritic cells (DC), repeat subculture 10 times or more, express myeloid molecules and leucosite molecules on the cell surface, It can be obtained by establishing a cell line having the ability to present antigen and the ability to induce CTL activity.

The ts SV 40 LTT g mouse can be produced as follows. Plasmid p SV in which ts A58 ori (I) deleted the SV40 replication origin (ori) and opened into pBR322 by opening all 12 DNAs with the restriction oxygen BamHI ts A58 (-) — 2 (O noT. et al., Cytotechnology 7, 165-172, 1991) was amplified in large amounts in E. coli according to a conventional method, and the amplified plasmid was digested with the restriction enzyme BamHI. To remove a portion of the vector to prepare a DNA fragment having the large T 钪 protogene of tsA58. The DNA fragment containing the promoter of the large T antigen gene was transfected into mouse totipotent cells according to a conventional method, whereby the large T antigen gene of the temperature-sensitive mutant tsA58 of SV40 was transferred to all cells. A transgenic mouse, ie, a transgenic mouse, can be produced. In such a transgenic mouse, the tsA58 large T antigen gene is expressed in all its somatic cells. Specific examples of the totipotent cells include fertilized eggs and early embryos, as well as ES cells having pluripotency. '' And all As a method for introducing DNA into competent cells, known gene transfer methods such as a microinjection method, an electric pulse method, a ribosome method, and a calcium phosphate method can be used.

The nuclei of the totipotent cells (cultured cells) of the above mice are transplanted into non-fertilized enucleated eggs and initialized (nuclear transfer). An antigen gene can be introduced. In addition, the eggs obtained by microinjecting the large T antigen gene of tsA58, a temperature-sensitive mutant of SV40, into the male pronucleus of fertilized eggs at the pronuclear stage were transplanted into the oviduct of the foster mother. After obtaining the offspring, the offspring having the injected gene are selected, and an individual having the gene integrated stably is obtained. Thus, transgenic mice, ie, transgenic mice, that have been integrated into the chromosomes of these cells can be efficiently produced.

The immortalized dendritic cell line of the present invention retains a permanent growth ability at 33 ° C, suppresses growth at 37 ° C, and stops growth at 39 ° C. It has the characteristic of being able to control the expression of the differentiation traits of the plant. In addition, this immortalized dendritic cell line shows good proliferation at 33 ° C. even after being subcultured for 7 months or more, and retains its function as a dendritic cell. The immortalized dendritic cell line of the present invention can continue to proliferate stably, and can activate T cells due to the antigen uptake ability and antigen presentation ability of the dendritic cells. In addition to being useful as a vaccine, it can be used to study the induction and modification of immunity and therapy using dendritic cells. Further, as shown below, it can be used for screening useful substances for dendritic cells.

As a screening method in the present invention, the above-described immortalized dendritic cell line of the present invention is cultured in the presence of a test substance;

0 Child, Roikosai preparative molecular, I one A ^b, and a screening method of the mature promoting or inhibiting substances in dendritic cells to measure and assess the degree of expression of CD 8 6, CD 4 mature markers Isseki protein such as 0, the Measuring the degree of cell line proliferation-screening method for cell growth promoting or inhibiting substances in dendritic cells to be evaluated, LPS stimulation of the cell line, measuring and evaluating the amount of IL-12 produced by the cell And a method for screening a substance that promotes or suppresses the activation of dendritic cells. The present invention also includes a substance for promoting maturation in dendritic cells, a substance for promoting cell growth in dendritic cells, and a substance for promoting activation of dendritic cells, which are obtained by the above-described screening method.

The screening of substances for promoting or inhibiting maturation in dendritic cells is performed by culturing immortalized dendritic cell lines in the presence of various concentrations of a test substance, and measuring the amount of marker protein expressed after culturing for a certain period of time. It is performed by detecting and measuring, and comparing and evaluating with a control cultured in the absence of the test substance. For example, myeloid molecules and leucosite molecules, which are maturity marker proteins expressed on the surface of dendritic cells, can be measured by immunochemical detection using a specific antibody in a conventional manner. In addition, it can also be measured by detecting the expression level of mRNA corresponding to these by a conventional method. The screening for substances that promote or suppress cell growth in dendritic cells is performed by culturing immortalized dendritic cell lines in the presence of various concentrations of test substances, and measuring the number of cells and cell morphology after culturing for a certain period of time. · Analyzed and compared with that of a control cultured in the absence of the test substance. Screening for dendritic cell activation-promoting or -suppressing substances is performed by culturing immortalized dendritic cell lines in the presence of various concentrations of test substances, and culturing for a certain period of time. Is measured and compared with that of a control cultured in the absence of the test substance. The cell vaccine of the present invention is not particularly limited as long as it comprises the above-mentioned immortalized dendritic cell line of the present invention as a main component. A dendritic cell line is preferable, and such a human-derived immortalized dendritic cell line is obtained by isolating a dendritic cell line from human peripheral blood or bone marrow, and obtaining a large T of a temperature-sensitive mutant tsA58 of SV40. The large T antigen gene of the temperature-sensitive mutant tsA58 of SV40 was introduced into the human embryonic stem cells (ES cells) by introducing the antigen gene and repeating subculture, or The cells can be differentiated into dendritic cell strains and established by repeating subculture. In addition, immortalized dendritic cell lines can grow at 33 ° C, but growth is suppressed at 37 ° C, or immortalized dendritic cell lines Or an antigen-IgG immune complex is preferred. The cellular vaccine of the present invention can be used as an antigen-presenting cell that stimulates T cells, which takes up an antigen in vivo or in vitro and presents an antigenic peptide on the cell surface after modification. For example, a cell vaccine of the present invention comprising a suspension of the immortalized dendritic cell line of the present invention is to be inoculated as a therapeutic vaccine into the human body, but growth is suppressed in 37. Therefore, safety is high. Usually, heat treatment, radiation treatment, mitomycin C treatment, etc. are required to enhance the safety as a cell vaccine, but the immortalized dendritic cell line of the present invention requires such cell inactivation treatment. It is unnecessary and can be grown at 33 ° C, but it can be said that it is an extremely safe vaccine because growth is suppressed at 37.

The cell vaccine of the present invention, in particular, a cell vaccine mainly comprising a human-derived immortalized dendritic cell line, is a cell vaccine that can be transferred to humans, and is used for various tumors such as leukemia, liver cancer, lung cancer, gastric cancer, and colon cancer, and It can be advantageously used for infectious diseases caused by various viruses and bacteria. Cell of the present invention The dose of Kuching varies depending on the patient's age, weight, gender, type of cancer, degree of progression of the cancer, symptoms, etc., and cannot be determined unconditionally. A small amount can be administered to the patient. The cell vaccine of the present invention can be used for patients themselves, but can be administered to a large number of MHC-compatible patients of the same type due to the development of bone marrow banks and cord blood banks. Hereinafter, the present invention will be described more specifically with reference to examples, but the technical scope of the present invention is not limited to these examples. Example 1 (Production of transgenic mouse)

Transgenic mice transfected with the DNA of the temperature sensitive mutant tsA58 of SV40 were created by the following procedure.

(Preparation of transgene)

For microinjection, a genomic DNA of SV40 temperature-sensitive mutant tsA58 modified by genetic engineering was used. The genomic DNA of tsA58 was opened with the restriction enzyme BamHI, introduced into the BamHI site of pBR322, and the SίiI sequence was converted to SacII to convert SV40 to A DNA clone with an origin of replication (ori) deleted, ori (-) p SV ts A58 ori (-)-2 (Ohno T. et al., Cytotechnolgy, 165-172, 1991) The DNA for introduction was prepared according to the procedure described above. That is, after digesting pSVtsA580ri (1) -2 of plasmid DNA obtained by large-scale amplification in Escherichia coli with a restriction enzyme BamHI (Takara Shuzo), After separation by agarose electrophoresis (1% gel; Boehringer), the gel was dissolved, and then subjected to phenol / chloroform treatment and ethanol precipitation treatment to recover DNA. The recovered purified DNA is It was dissolved in a buffer (10 mM Tris_HCl containing I mM EDTA; pH 7.6) to obtain a solution containing 170 g / m 1 of purified DNA. This DNA solution was diluted with a buffer for injection (10 mM Tris-HCl containing 0.1 mM EDTA; pH 7.6) to a concentration of 5 g / m 1 and diluted with DNA for injection. A solution was prepared. The prepared DNA solution was stored at -20 until injection.

(Creation of transgenic mouse)

Microinjection of the above prepared DNA solution for injection into mouse pronucleus fertilized eggs was performed as follows. Sex-matured 8-week-old Wistar mice are bred under a light-dark cycle of 12 hours (4: 00 to 16: 00 light hours), temperature of 23 ± 2 C, humidity of 55 ± 5%, and vaginal smear. The female estrous cycle was observed by, and the date of hormone treatment was selected. First, 150 IU Zkg of pregnant serum serum gonadotropin (PMS gonadotropin (PMS G), manufactured by Nippon Zenjak) was intraperitoneally administered to female mice for 48 hours. Later, 75 IU / kg of human chorionic gonadotropin (manufactured by Sankyo Organ Co., Ltd .; human chorionic gonadotropin: hCG) was administered, followed by superovulation and cohabitation with males. The mating was performed. Pronuclear stage fertilized eggs were collected by tubal perfusion 32 hours after hCG administration. For fallopian tube perfusion and egg culture, mK RB solution (Toyoda Y. and Chang M., J. Reprod. Fertil., 36, 9-22, 1974) was used. The collected fertilized eggs were enzymatically treated at 37 ° C for 5 minutes in mKRB solution containing 0.1% hyaluronidase (Sigma; Hyaluronidase Typel-S) to remove cumulus cells. and washed 3 times with a liquid to remove the enzyme, to DNA injection operation C 0 ₂ - and stored in - (95% of a ir, 3 7 ° C, saturated humidity 5% C_〇 ₂₎ incubator. The DNA solution was injected into the male pronucleus of the mouse fertilized eggs prepared in this manner. Transplanted 2 2 8 eggs into 9 foster parents And gave birth to obtain 80 offspring. The injected DNA was introduced into mice by PCR using DNA prepared from tails obtained by cutting the tails immediately after weaning. [Primers used: tsA58_lA, 5'-TCCTAATGTGC AGT CAGGT G—3, (corresponding to 1365 to 1384 sites: SEQ ID NO: 1), tsA58-1B, 5′-ATGAC GAGCTTTGGC ACT TG—3 ′ (1571 to 1559 Equivalent to 0 site: SEQ ID NO: 2)]. As a result, out of the 20 offspring (6 males, 8 females, and 6 sexes unknown) in which the gene was transfected, survived until the age of 12 weeks after the sexual maturity period. Transgenic mice (male lines: # 07-2, # 07-7-5, # 09-19-6, # 12-23, # 19-19, female lines: # 09-9-7, # 11) 1, # 1 2—5, # 1 2—7, # 1 8—5, # 1 9—8). These G. Generation transgenic mice and Wistar mice were bred, and two lines (# 07-7-2, # 07-7-5) of male huaunda and three lines of female founder (# 09-9-7, # 11-16) , # 1918), confirmed the gene transfer to the next generation and beyond. Example 2 (Separation and preparation of DC from mouse bone marrow)

Two strains of mice were used, C57BL / 6 (B6 mouse) and temperature-sensitive SV40T antigen transgenic mouse (ts SV40 LTT g mouse; B6 background). All the mice were 6-8 week old females. Mouse bone marrow cells Te to 0. 1 44 M chloride Anmoniumu to erythrocyte lysis treatment, anti-CD 4 antibodies, anti-CD 8 antibodies, anti-I one A ^b antibody, anti-rat I g antibody (respectively TIB 2 0 7, 2 Lymphocytes and Ia-positive cells were removed using 11, 15 4 and 2 16: Amerikan Type Culture Collection) and Egret complement (Cedarlane). Mouse recombinant GM- CSF in the cells ^{IX 1 0 6 / well 2 0} ng / mL

Five Using a complete RPMI medium (5% FCS) containing (Peprotech), the cells were cultured in a 24-well plate, and dendritic cells (DC) were induced 6 days later. DC is 3 3 ° from ts SV 4 0 LTT g mouse (:, after induction with 5% C_〇 ₂ conditions, more than 1 0 times passaged ^{5 X 1 0 5 / mL /} well repeated (4-5 Culture medium was changed every day, subcultured every 3 weeks), and cultured for more than 7 months.DCs from B6 mice were isolated by isolating bone marrow cells by the above method and inducing DCs. , 3 7 ° C, 5% C_〇 cultured in ₂ conditions, used for the analysis at that point. example 3 (embodiment according Lai Togimuza staining observed)

The DC obtained in Example 2 was stained with Wright-Giemsa. DCs originating from two strains, B6 mouse and ts SV40 LTT g mouse, were adhered to slide glass by site spin, and the light Giemsa method (light stain and Giemsa stain, both manufactured by Merck) ) And visualized. The results are shown in Figure 1. As a result, the cell size of tsSV40LTTg DC (SV40TB6) was larger than that of B6 mouse DC (primary culture B6). Example 4 (Proliferation ability at different temperatures)

MTT (3- (4,5-dimethyltiazol-2-yl) -2,5-diphenyltetrazo-1ium bromide) is cleaved by the dehydrogenase of the inner mitochondrial membrane and has a purple-red MT. Generate T-iormazan. Based on the fact that this color reaction is proportional to the proliferative capacity of the cells, the MTT assay at ts SV40 LTT g at different temperatures (33 ° C, 37 ° C, 39 ° C) Was measured for its proliferation ability. MTT Atsushi was prepared by adding 10 L of a 5 mg / mL MTT (Sigma) solution to the above-mentioned 2 ng / mL mouse recombinant GM—CSF (Peprotech

6 It was added to the cell suspension 1 0 0 2 L containing Company Ltd.), 9 6 well plates 7. plated cells at ^{5 X 1 0 3/1 0} 0 L / ell, 1 MTT solution during measurement 0 / Wells were added and measurements were made over time. The result is shown in figure 2. As a result, the cells had the highest proliferative ability at 33 ° C at which the SV40 T antigen was expressed. Example 5 (GM—CSF requirement)

For examining the requirement of granulocyte macrophage colony stimulating factor (GM-CSF) for DC of ts SV 40 LTT g, MTT assay was performed under the same conditions as in Example 4. That is, 10 L of a 5 mg / mL MTT (Sigma) solution was added to a cell suspension containing mouse recombinant GM-CSF (Peprotech) at concentrations of 20, 10, 2, and OngZmL, respectively. was added to 0 t L, 9 to 6 well plates 7. plated cells at ^{5 X 1 0 3/1 0} 0 n L / well, MT T solution was added 1 0 x L / well at the time of measurement, over time A measurement was made. The results are shown in Figure 3. As a result, the growth was best at the concentration of 20 ng / mL used for DC induction in normal primary culture. The immortalized cells were found to be cells that proliferate in a GM-CSF-dependent manner. Example 6 (Examination of proteins expressed on the cell surface)

ts SV40 LTT g mouse (SV40 TB6) and B6 mouse (primary culture B6). The expression of certain myeloid and leucosite molecules was analyzed by FACS. The results are shown in FIGS. As a result, the expression levels of myeloid molecules and leucosite molecules in DCs derived from the above two lines did not change. Example 7 (Comparison of antigen uptake ability)

ts SV 40 LT T g mice (SV 40 TB 6) and B 6 mice (primary culture B 6) were used as DCs, and 10 / g / mL antigen (OVA-FI On the second day after addition of TC (Molecule Probes), the uptake capacity was compared by FACS. Fig. 6 shows the results. As a result, DC of tsSV40LTTg showed stronger uptake ability than DC of B6 mouse. Example 8 (Comparative study of maturation of dendritic cells to LPS stimulation)

For each DC originating from two lines, ts SV 40 LT T g mouse (SV 40 TB 6) and B 6 mouse (primary culture B 6), add 2 HgZmL LPS to the culture wells of each DC. Te, and analyzed a maturity markers one on cell surface after 24 hours I one a ^b, the expression level of CD 8 6 and CD 4 0 at FACS. Fig. 7 shows the results. As a result, it was found that upregulation of the maturity marker in DC of tsSV40LTTg, that is, maturity occurred as in DC of B6 mouse (primary culture B6). Example 9 (Comparative study of activation of dendritic cells to LPS stimulation)

ts SV 40 LTT g mouse (S V40 TB 6) and B 6 mouse (primary culture B 6). p70 production was measured by ELISA. Fig. 8 shows the results. As a result, 2 g / mL of LPS was added to each DC culture well, and 24 hours later, the amount of IL-12p70 produced in the supernatant did not change between the two strains.

8 Example 10 (Maturation of dendritic cells when antigen is incorporated)

ts SV 40 LT T g mice (SV 40 TB 6) and B 6 mice (primary culture B 6) originated from 10 strains of DCs, respectively. After incorporation, their maturity was analyzed by FACS two days later. The results are shown in FIG. As a result, DC of B6 mouse (primary culture 6) showed stronger maturity. Example 11 1 (Comparative study of the ability of dendritic cells to present antigen to OVA-T cells) Using OVA-specific CD4 T cells previously established by the present inventors, ts SV40 LTT g mice (SV The antigen presenting ability of each DC derived from two strains, 40 TB 6) and B 6 mouse (primary culture B 6) was measured. The IL-14 production and proliferation of T cells were measured as presentation ability. X-ray irradiated ^{DC (5 X 1 0 3 /} well) for 1 X 1 0 ⁵ of OVA-specific T cells with various concentrations OVA or OVA-I g G immune complexes (IC) 9 6-well in the presence Co-culture was performed on the plate. It is said that highly efficient antigen presentation occurs when an immune complex incorporates an antigen through the Fcγ receptor (J. Immunol., 161, 6059-6067, 1998, J. Exp. Med. , 189, 371-380, 1999, Eur. J. Immunol., 30, 848-857, 2000, J. Exp. Med., 195, F1-F3, '2002). The OVA-IgG immune complex (IC) was prepared by mixing ovalbumin (OVA; Sigma) with Egret Anti-OVAI gG (BioDesign) in a weight ratio of 1:10, It was prepared for one hour with a computer. Twenty-four hours later, the culture supernatant was collected, and the amount of IL-14 produced by the T cells was measured by ELISA. T cell proliferation was determined by measuring [ ³ H] —T dR incorporation after 48 hours of co-culture. The results are shown in FIG. As a result, each of the DCs originating from the two lines both had the same leprosy T cell proliferation and IL-14 production. However, IL-1-4 production smell When the OVA added at 1 agZmL was used, when OVA-IgG immune complex (IC) was used as an antigen, the amount of IL-14 produced by DC of ts SV 40 LTT g was increased by B6 mice (primary culture B 6) It was less than DC. Example 1 2 (Timely anti-OVA antibody titer)

ts SV 40 LT T g mice (SV 40 TB 6) and B6 mice (primary culture B 6) were loaded with OVA or OVA A-IgG immune complex on each DC. Then, the anti-OVA antibody titer over time after the transfer was examined. In the in vivo experiment, mature DCs with antigen were recovered two days after replacement of the culture medium with fresh medium containing OVA or OVA_IgG immune complex at 10 βg / mL. PBS (-) was washed with and populate the recipient to become B 6 mice DC 1 X 1 0 ⁶ cells per animal into the tail vein. After immunization, blood was collected from the fundus, and the anti-OVA antibody titer over time was measured by ELISA. The results are shown in FIG. As a result, in the OVA-IgG immune complex (IC), effective antibody production was observed in both IgGI, IgG2a and IgG2b. When OVA was used as an antigen, antibody production by DC of tsSV 40 LTT g was significantly higher in IgG 2a (after 2 weeks). Example 13 (Immunohistochemical staining of spleen tissue with anti-GL-7 antibody)

DC were transferred to the mouse, and about 3 weeks later, the spleen of the mouse was collected, and the expression of GL-7, an indicator of activated germinal centers, was examined by immunohistochemical staining. The results are shown in Fig. 12 (Reference Photo 2). As a result, when the immune complex (IC) was used as an antigen for both the B6 mouse and the ts SV40 LTT g mouse, the formation was effective. There was no difference between these two lines. Example 14 (in vivo CTL activity)

The in vivo CTL activity by DC transfer into mice was compared. Spleen cells of mice after transfection 7 days were taken to remove the adherent cells by incubating for 30 minutes at C_〇 ₂ incubator 3 7 ° C, and the T cell-rich. This nonadherent cells ^{1 X 1 0 7, E. G} 7 stopping the X-ray irradiation proliferate - the OVA 1 X 1 0 ⁶ were co-cultured in 24-well plates. E. G 7 -0 VA (CRL2113; ATCC) is a transfection of OVA cDNA into EL-14, a thymoma derived from B6, and the OVA peptide is always loaded on its MHC class I. Have been. On day 5 culture, E G 7 -. And the O VA over N a ₂ ^{5 1} C r 〇 ₄ (Amer sham Pharmacia Biotech Co.) at 1 hour to label. And ^{5 1} C r label viable splenocytes and its various concentrations E. G 7 - 0 and VA 1 X 1 0 ⁴ were co-cultured in 9 6-well U bottom plate, in the supernatant after 4 hours ⁵¹ The release of Cr was measured with an automatic gamma system (Aloka). The results are shown in FIG. As a result, when OVA was added to ts SV40 LTT g DC, when compared with DC of B6 mouse (Primary), considerably stronger CTL activity was obtained at the same level as when the immune complex was incorporated. Induced. Example 15 (Expression of MHC class IZ OVA peptide complex in dendritic cells)

In Example 14 above, when OVA was added to DC of ts SV 40 LTT g, compared to DC of B6 mouse (primary culture B6), it was almost the same as when the immune complex was incorporated. The reason for the induction of the rather strong CTL activity was that more antigen-derived peptides were presented on the DC MHC class I molecule, and the specificity of the MHC class I / OVA peptide complex Site using typical monoclonal antibodies

Two The analysis was performed by the measurement. In the presence of 50 wg / m 1 OVA, DCs originating from two strains of ts SV 40 LTT g mouse (SV 40 TB 6) and B 6 mouse (primary culture B 6) were treated for 48 hours. Cultured. Then, after blocking the Fc receptor with an anti-Fcr RIIZIII antibody, the anti-CD11c—PE antibody and anti-MHC I—FITC antibody, or the anti-CD11c—PE antibody and anti-MHC IZ〇VA Staining was performed using a peptide antibody. In the case of anti-MHC I / OVA peptide antibody staining, staining was performed with a secondary antibody (anti-mouse IgG1_FITC antibody). After staining, measurement was performed by flow cytometry (BDLSR), and the data was analyzed by BD CellQuest. FIG. 14 shows the result of histogram showing the amount of MHC I or MHC I / 〇VA peptide cell surface expression of dendritic cells (CD1lc positive cells). As a result, the expression level of MHC class I in DC of ts SV40 LTT g was almost the same as that of wild-type B6 mouse (primary culture B6), but the expression of MHC class I ZOVA peptide complex Was dramatically higher in DCs with ts SV 40 LTT g. In other words, it was found that ts SV 40 LTT g DC was a cell capable of efficiently presenting antigen to CTL by efficiently displaying ΔVA peptide to MHC class I. Example 16 (antitumor activity in in vivo)

To specifically evaluate the enhanced CTL response of ts SV 40 LTT g DC in vivo, anti-tumor experiments were performed. Ts SV40 LTT g mice (SV40T Β6) and Β6 mice (primary culture Β6), which were given OVA stimulation (10 g / m 1, 48 hours) Each DC (5 × 10 ⁵ / mouse) or saline (200 1) was administered through the tail vein of naive mice (7 to 8), and after 7 days, DC or After administration of saline, tumor cells expressing OVA (E. G 7) planting in 1 X 1 0 ⁵ Nomausu the left thigh, the day tumor formation was additionally connexion observed, those tumor diameter of more than 5 mm is determined that the tumor is formed. The results are shown in FIG. The tumor suppression rate in FIG. 15 is the percentage of mice in which no tumor was formed, expressed as a percentage. Mice to which ts SV40 LTT g DC had been transferred had slower tumor formation than mice to which wild-type B6 mouse DC had been transferred, and efficiently suppressed tumor formation. That is, it was found that ts SV40 LTT g DCs are cells that induce antitumor activity more efficiently in vivo than wild-type DCs. (Discussion)

DCs derived from bone marrow cells of ts SV 40 LTT g mice, passaged at least 10 times, and subjected to long-term culture for at least 7 months at 33 were slightly larger in size than those in the initial culture, and Although the ability was high, it was found that the ability to present Bobara in the mouth of the mouth had the same function as that via MHC class II. From this, it is considered that the cell line is useful for use in the analysis of DC in vitro. In addition, when used as a vaccine in vivo, it induced CTLs particularly strongly via MHC class I. This is consistent with the report that high-efficiency MHC class I-mediated presentation depends on high antigen uptake (Annu. Rev. Immunol., 19, 47-64, 2001). From this, DC of tsSV40LTTg can efficiently induce a vaccine effect against cancers and viruses in vivo.

Industrial applicability

According to the present invention, it is possible to obtain an immortalized 榭 -shaped cell line that keeps growing stably. The dendritic cell line can be used for induction and modification of immunity, development of a therapeutic method using the dendritic cell line, and the like. Further, according to the present invention, a method for screening a useful substance against dendritic cells and a substance that enhances an immune response using the cell line are provided, since the cell line retains its original function and characteristics in a tissue derived from the cell line. can do.

Claims

The scope of the claims

1. An immortalized dendritic cell line characterized by being derived from bone marrow.

2. The immortalized dendritic cell line according to claim 1, wherein the immortal dendritic cell line expresses a myeloid molecule and a leucosite molecule on a cell surface and has an antigen uptake ability, an antigen presenting ability, and a CTL activity inducing ability. .

3. The immortalized dendritic cell line according to claim 1, which is capable of growing at 33 ° C., but is suppressed at 37 ° C.

4. The immortalized dendritic cell line according to any one of claims 1 to 3, which has an ability to respond to LPS stimulation.

5. The immortalized dendritic cell line according to any one of claims 1 to 4, which is of rodent origin.

6. The immortalized dendritic cell line according to claim 5, wherein the rodent is a mouse.

7. Immortalized dendritic cell line TDC (FERMBP-085-27).

8. After hemolyzing the bone marrow cells of the transgenic mouse into which the large T antigen gene of the SV40 temperature-sensitive mutant tsA58 was introduced, the lymphocytes and Ia-positive cells were removed. Cultured cells in the presence of GM-CSF to induce dendritic cells, repeat subculture 10 times or more, express myeloid molecules and leucosite molecules on the cell surface, and A method for producing an immortalized dendritic cell line, comprising establishing a cell line having an antigen-presenting ability and an ability to induce CTL activity.

9. The immortalized bacterium according to claim 8, wherein the cell is capable of growing at 33 ° C, but the growth is suppressed at 37 ° C, and a cell line capable of responding to LPS stimulation is established. A method for producing a cell line.

10. An immortalized dendritic cell according to any one of claims 1 to 7, in the presence of a test substance. A method for screening a substance that promotes or suppresses maturation in dendritic cells, comprising culturing a cell line and measuring and evaluating the expression level of a maturation marker protein in the cell line.

1 1. Marker protein, Mieroi de molecules, leukocyte molecule, I one A ^b, accelerated maturation or suppressed in dendritic cells of claim 1 0, wherein it is a CD 8 6 and Z or CD 4 0 A method for screening substances. • 12. The dendritic cell characterized by culturing the immortalized dendritic cell strain according to any one of claims 1 to 7 in the presence of a test substance, and measuring and evaluating the degree of proliferation of the cell. A method for screening a cell growth promoting or inhibiting substance in a cell. 13.In the presence of a test substance, the immortalized dendritic cell strain according to any one of claims 1 to 7 is stimulated with LPS, and the amount of IL-11 produced by the cells is measured and evaluated. A method for screening a substance that promotes or suppresses dendritic cell activation. 14. A substance for promoting maturation in dendritic cells obtained by the screening method according to claim 10 or 11.

15. A cell growth promoting substance in dendritic cells obtained by the screening method according to claim 12.

16. A substance for promoting dendritic cell activation obtained by the screening method according to claim 13.

17. A cell vaccine comprising the immortalized dendritic cell line according to any one of claims 1 to 7 as a main component.

18. The immortalized dendritic cell line, wherein the immortalized dendritic cell line is an immortalized dendritic cell line that can proliferate at 33: but is inhibited from growing at 37 ° C. Cell vaccine.

19. The cell according to claim 17 or 18, wherein the immortalized dendritic cell line is an immortalized dendritic cell line that has not taken up an antigen or an antigen-IgG immune complex. vaccine.

20. The cellular actin according to claim 19, wherein the antigen is a tumor antigen.