JP2004166697A - Immortalized dendritic cell strain derived from marrow - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immortalized dendritic cell strain retaining functions/characteristics inherent in the dendritic cell, to provide a method for establishing the same, to provide a method for screening a useful substance, by using the immortalized dendritic cell strain, and to provide a cellular vaccine containing the immortalized dendritic cell strain as essential ingredient. <P>SOLUTION: This immortalized dendritic cell strain is established so that marrow cells of a transgenic mouse into which a large T antigen gene of a temperature-sensitive mutant tsA58 of SV40 is introduced are subjected to hemolysis treatment, lymphocytes and Ia positive cells are removed therefrom, the obtained cells are cultured in the presence of GM-CSF so as to induce the dendritic cells, subcultures thereof are repeated ten or more times, myeloid molecules and leukocyte molecules are expressed on surfaces of the cells, and therefore the cell strain has antigen incorporation potency, antigen presentation potency, and CTL activity induction potency. <P>COPYRIGHT: (C)2004,JPO

Description

  The present invention relates to an immortalized dendritic cell line derived from bone marrow, and more specifically, a tree derived from the bone marrow of a transgenic mouse (ts SV40 LT Tg mouse) into which a large T antigen gene of SV40 temperature-sensitive mutant tsA58 has been introduced. The present invention relates to an immortalized dendritic cell line that can be established by subculturing dendritic cells (DC), a production method thereof, 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 modification and enhancement of immune responses.

  Conventionally, animals have been mainly used for research and studies on the safety and efficacy of pharmaceuticals, but instead of using animals from the viewpoint of animal welfare, the efficacy and safety of pharmaceuticals in vitro using cultured cells, etc. Research is being conducted at a practical level of technology for testing and researching. For example, an animal test is performed after testing in advance by a method using a primary cultured cell collected from a living tissue or an immortalized cell (established cell) system that grows indefinitely. However, primary cells proliferate well in the initial stage, but gradually stop with subculture and eventually die (this phenomenon is called cell aging). Furthermore, it has been pointed out that primary cells change with passage in addition to the fear that their characteristics differ each time they are collected from living tissue. In particular, when the growth rate is very slow or when it is derived from a micro-organ, it is said that it is very difficult to obtain primary cells sufficient for the test.

  On the other hand, immortalized cells that have acquired the ability to proliferate indefinitely by avoiding cell aging through repeated passages of primary culture will have stable and uniform characteristics, but many of these immortalized cells Loses some or all of the forms and functions that the cells originally had in the body. Therefore, in tests using such an immortalized cell line, it has been difficult to accurately reflect the original characteristics of the tissue from which the cell line is derived. Therefore, by transforming the cells by introducing oncogenes such as ras gene and c-myc gene, adenovirus E1A gene, SV40 virus large T antigen gene, human papillomavirus HPV16 gene, etc. into the primary cells, Attempts have been made to establish immortalized cells that retain their active proliferative ability and do not lose their inherent properties by further passage. However, even in such an immortalized cell, depending on the target organ, the primary cell is prepared, and when these oncogene and large T antigen gene are introduced, some functions are already lost. It has been difficult to obtain immortalized cells in a strict sense that retains the original function. In particular, it has been extremely difficult to prepare and establish primary cells when the growth rate is very slow or when they are derived from micro-organs.

  On the other hand, instead of introducing oncogenes and large T antigen genes into individual cells using recently established gene transfer techniques to individual animals, transgenic animals in which these genes are stably integrated into the chromosome And preparing an immortalized cell by substituting a primary cell from an animal organ that already has an oncogene or a large T antigen gene in the cell at the time of development It has been reported. In particular, immortalized cells obtained from the organs of ts SV40 LT Tg mice can be manipulated by changing the temperature of their growth and expression of differentiation traits, and are therefore considered to be very effective (for example, non-patented Reference 1-8).

On the other hand, dendritic cells (DC) are cell populations having a dendritic form derived from hematopoietic stem cells and are widely distributed in the living body. The immature dendritic cells recognize and take up foreign substances such as viruses and bacteria that have invaded each tissue, generate peptides by digestion in the process of migration to the lymphoid organ T cell region, and convert them into MHC molecules. By binding and presenting it on the cell surface, it plays a role as an antigen-presenting cell that activates antigen-specific T cells to induce an immune response (see, for example, Non-Patent Documents 9 and 10). Thus, DC plays a very important role in triggering a T cell response that can elicit a T cell-dependent initial immune response (see, for example, Non-Patent Documents 11 and 12). DCs born in the bone marrow are immature and distributed with various eating and drinking effects in various tissues in the living body. The immature DCs take up the antigen, mature and migrate to secondary lymphoid organs. It accumulates in the T cell region and selectively activates antigen-specific T cells circulating in the body to drive an immune response. However, these detailed mechanisms of DC in vivo are not yet known and need to be analyzed in vitro. By using granulocyte-macrophage colony stimulating factor (GM-CSF) in the mouse in vitro system, functional DCs can be induced in the initial culture, but their lifetime is at most 2 months (eg, (Refer nonpatent literature 13.). In recent years, it has been reported that DC can be cultured for a long time (12 months or more) in a GM-CSF-dependent manner from the spleen, which is a secondary lymphoid organ of mice (D1 cells: see, for example, Non-Patent Document 14), but immature. No DC cell line has been established from primary lymphoid tissues such as bone marrow that have not taken up antigen.
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 Ann. Rev. Immunol. 9, 271-296, 1991 J. Exp. Med., 185, 2133-2141, 1997 Nature, 392, 245-252, 1998 Annu. Rev. Immunol., 18, 767-811, 2000 J. Exp. Med., 175, 1157-1167, 1992 J. Exp. Med., 185, 317-328, 1997

  Dendritic cells (DC) are important cells that drive the immune response. However, analysis of DC dynamics in vivo and application to cancer immunostimulation have just started, and many unclear points remain. Although DC can be cultured by preparing it from a living body, its lifetime is limited, and even if it is cultured in the presence of cytokines such as GM-CSF, it can only last for about one month at a long time and then die. Until now, it has been very difficult to prepare dendritic cells that continue to proliferate stably, and there is no simple method for establishing dendritic cells, which can be used for induction and modification of immunity, treatment using dendritic cells, etc. 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 immortalized dendritic cell line, a screening method for useful substances using such an immortalized dendritic cell line, and such an immortalized tree. An object of the present invention is to provide a cellular vaccine mainly composed of dendritic cell lines.

  The present inventors have intensively studied to solve the above problems, and after lysing bone marrow cells of ts SV40 LT Tg mice, lymphocytes and Ia positive cells were removed, and the resulting cells were treated with GM-CSF. Dendritic cells are induced by culturing in the presence, the subculture is repeated 10 times or more, and the established immortalized dendritic cell line expresses myeloid molecules and leucosite molecules on the cell surface, and the antigen uptake ability, The present invention was completed by confirming that DC had the properties inherent to it, such as the ability to present antigen and the ability to induce CTL activity.

  That is, the present invention relates to an immortalized dendritic cell line derived from bone marrow (Claim 1), a myeloid molecule and a leucosite molecule expressed on the cell surface, antigen uptake ability, antigen presentation ability, And an immortalized dendritic cell line according to claim 1 (claim 2), which is capable of growing at 33 ° C, but is inhibited at 37 ° C. The immortalized dendritic cell line according to claim 1 or 2 (claim 3) or the immortalized dendritic tree according to any one of claims 1 to 3, which has a response ability to LPS stimulation. The cell line (Claim 4) or the immortalized dendritic cell line (Claim 5) or the rodent of any one of claims 1 to 4, wherein the rodent is a mouse An immortalized dendritic cell line according to claim 5 (claim 6) or an immortalized tree Cell lines TDC (FERM BP-08527) relates to (claim 7).

In the present invention, the bone marrow cells of a transgenic mouse introduced with the large T antigen gene of SV40 temperature-sensitive mutant tsA58 are hemolyzed, lymphocytes and Ia positive cells are removed, and the resulting cells are treated with GM- Dendritic cells are induced by culturing in the presence of CSF, and subculture is repeated 10 times or more to express myeloid molecules and leucosite molecules on the cell surface, antigen uptake ability, antigen presentation ability, and CTL activity A method for producing an immortalized dendritic cell line characterized by establishing a cell line having an inducibility of the cell line (claim 8), and can be grown at 33 ° C., but growth is suppressed at 37 ° C., and LPS 9. A method for producing an immortalized dendritic cell line according to claim 8 (claim 9), wherein a cell line capable of responding to stimulation is established; Izu A method for screening a substance that promotes or suppresses maturation in dendritic cells, comprising culturing the immortalized dendritic cell line described above, and measuring and evaluating the level of expression of the maturation marker protein in the cell line (claim 10). Or the marker protein is a myeloid molecule, a leucosite molecule, IA ^b , CD86 and / or CD40, the method for screening a substance for promoting or suppressing maturation in dendritic cells according to claim 10 (claim) (11) or an immortalized dendritic cell line according to any one of (1) to (7) in the presence of a test substance, and the degree of proliferation of the cell is measured and evaluated. A method for screening a substance that promotes or suppresses cell proliferation in cells (Claim 12), or an immortalized dendritic cell line according to any one of Claims 1 to 7 in the presence of a test substance Intensity, measured IL-12 production level of the cell, a screening method of dendritic activation accelerating or inhibitory substance in cells, characterized by evaluating (claim 13).

  Furthermore, the present invention relates to a substance that promotes maturation in dendritic cells obtained by the screening method according to claim 10 or 11 (claim 14), or a substance that promotes cell growth in dendritic cells obtained by the screening method according to claim 12. 15), a dendritic cell activation promoter obtained by the screening method according to claim 13 (claim 16), or an immortalized dendritic cell line according to any one of claims 1 to 7 as a main component. A cell vaccine (Claim 17) or an immortalized dendritic cell line characterized in that it is capable of growing at 33 ° C., but is an immortalized dendritic cell line whose growth is suppressed at 37 ° C. The cell vaccine according to claim 17 (claim 18) or the immortalized dendritic cell line is an immortalized dendritic cell line incorporating an antigen or an antigen-IgG immune complex. To and claims 17 or 18, wherein the cellular vaccine (Claim 19), relating to claim 19, wherein the cell vaccine antigen is characterized in that the tumor antigen (claim 20).

According to the present invention, an immortalized dendritic cell line that continues to proliferate stably can be obtained, induction and modification of immunity using the dendritic cell line, development of a therapeutic method using the dendritic cell line, etc. Can be used. In addition, according to the present invention, since the original function / property in the tissue from which the cell line is derived is retained, a useful substance screening method for dendritic cells using the same and a substance that enhances the immune response are provided. can do.

  The immortalized dendritic cell line of the present invention may be any immortalized dendritic cell line derived from bone marrow, and can grow at 33 ° C., and cells whose growth is suppressed at 37 ° C. Strains are preferred, and those having the properties inherent to DC are more preferred except for this temperature sensitivity. For example, cell lines that express myeloid molecules and leucosite molecules on the cell surface, have the ability to take up antigen, present antigen, and induce CTL activity, and maturation and activation of dendritic cells by stimulation with LPS Preferred examples include cell lines that have the ability to respond to LPS stimulation, such as IL-12 production, and cell lines that combine these properties. Moreover, an immortalized dendritic cell line TDC can be given as a specific example of such an immortalized dendritic cell line. As a transfer from FERM P-19044 deposited on September 26, 2002, deposits have been made based on 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 animals such as rodents such as mice has a pathological model rich in mice, It is preferable because it is widely used for evaluation of pharmacological action. Hereinafter, the method for producing an immortalized dendritic cell line of the present invention will be described taking a method using a mouse as an example.

Immortalized dendritic cell line of the present invention derived from mouse, for example, after a bone marrow cells ts SV40 LT Tg mice were hemolyzed with ammonium chloride, for example anti-CD4 antibody, anti-CD8 antibody, anti-I-A ^b antibody The cells from which lymphocytes and Ia positive cells were removed using anti-rat Ig antibody and rabbit complement were cultured in complete RPMI medium (5% FCS) containing 20 ng / mL mouse recombinant GM-CSF, Cells that induce dendritic cells (DCs) and are subcultured 10 times or more to express myeloid molecules and leucosite molecules on the cell surface, and have the ability to take up antigen, present antigen, and induce CTL activity It can be obtained by establishing a strain.

  A ts SV40 LT Tg mouse can be prepared as follows. Plasmid pSVtsA58 (−)-2 (OhnoT. Et al., Cytotechnology 7) in which the entire DNA of tsA58ori (−)-2 from which the SV40 replication origin (ori) has been deleted was opened with restriction oxygen BamHI and introduced into pBR322. 165-172, 1991) in a large amount in E. coli according to a conventional method, this amplified plasmid is cleaved with the restriction enzyme BamHI to remove the vector site, and a DNA fragment having the large T antigen gene of tsA58 is prepared. . The gene fragment having the large T antigen gene of SV40 temperature-sensitive mutant tsA58 is introduced into all cells by introducing the DNA fragment containing the promoter of the large T antigen gene into a totipotent mouse cell according to a conventional method. Mice, i.e. transgenic mice, can be created. Such transgenic mice express the large T antigen gene of tsA58 in all somatic cells. Specific examples of the totipotent cells include fertilized eggs and early embryos, ES cells having multipotency, and the like. In addition, as a method for introducing DNA into totipotent cells, a known gene introduction method such as a microinjection method, an electric pulse method, a liposome method, or a calcium phosphate method can be used.

  The nucleus of totipotent cells (cultured cells) of the mouse is transferred to an enucleated unfertilized egg and initialized (nuclear transfer) to introduce the large T antigen gene of the SV40 temperature-sensitive mutant tsA58 into the egg. be able to. In addition, an oocyte obtained by microinjecting the large T antigen gene of the SV40 temperature-sensitive mutant tsA58 into the male pronucleus of a pronuclear-stage fertilized egg was transplanted into an oviduct of a foster parent, and then a litter was obtained. A transgenic mouse in which the large T antigen gene of tsA58 has already been integrated into the chromosome of the cell of each tissue at the time of ontogeny, That is, transgenic mice can be produced efficiently.

  Since the immortalized dendritic cell line of the present invention has a permanent growth ability at 33 ° C., growth is suppressed at 37 ° C., and growth is stopped at 39 ° C., thus controlling the expression of cell-specific differentiation traits It has the feature that it can. In addition, this immortalized dendritic cell line shows good growth at 33 ° C. even when subcultured for 7 months or more, and retains the function as a dendritic cell. The immortalized dendritic cell line of the present invention can continue to proliferate stably, and the T cell can be activated by the antigen uptake ability and antigen presentation ability of the dendritic cell. In addition to its usefulness, it can be used for the study of immunity induction and modification, and treatment using dendritic cells. Moreover, as shown below, it can use for the screening of the useful substance with respect to a dendritic cell.

As a screening method in the present invention, the immortalized dendritic cell line of the present invention is cultured in the presence of a test substance, and myeloid molecule, leucosite molecule, IA ^b , CD86, CD40 and the like in the cell line are cultured. Screening method for a substance that promotes or suppresses maturation in dendritic cells that measures and evaluates the degree of expression of the maturation marker protein, and screening for a substance that promotes or suppresses cell growth in dendritic cells that measures and evaluates the degree of proliferation of the cell line Examples thereof include a method of screening for a dendritic cell activation promoter or inhibitor that stimulates LPS of the cell line and measures and evaluates the amount of IL-12 produced by the cell. The present invention also includes a maturation promoting substance in dendritic cells, a cell growth promoting substance in dendritic cells, and a dendritic cell activation promoting substance obtained by the screening method.

  Screening for substances that promote or suppress maturation in the above dendritic cells involves culturing an immortalized dendritic cell line in the presence of various concentrations of the test substance, and detecting and measuring the amount of marker protein expressed after incubation for a certain period of time. Then, it is performed by 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 immunochemically detecting each using a specific antibody. Moreover, it can also measure by detecting the expression level of mRNA corresponding to these by a conventional method. Screening for substances that promote or suppress cell growth in the above dendritic cells involves culturing an immortalized dendritic cell line in the presence of various concentrations of the test substance, measuring the number of cells and cell morphology after culturing for a certain period of time. The analysis is performed by comparing and evaluating with a control cultured in the absence of the test substance. In addition, the screening for dendritic cell activation promoting or inhibiting substances involves culturing immortalized dendritic cell lines in the presence of various concentrations of test substances, and increasing IL-12 production after culturing for a certain period of time. This is carried out by measuring and comparing with 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 is mainly composed of the immortalized dendritic cell line of the present invention, but the immortalized dendritic cell line is immortalized from humans. Dendritic cell lines are preferred, such human-derived immortalized dendritic cell lines are isolated from human peripheral blood or bone marrow and introduced with the large T antigen gene of the SV40 temperature-sensitive mutant tsA58, The large T antigen gene of SV40 temperature-sensitive mutant tsA58 is introduced into human embryonic stem cells (ES cells) by repeated subculture, and differentiated into a dendritic cell line in vitro. It can be established by repeating the culture. Further, as an immortalized dendritic cell line, it can grow at 33 ° C., but its growth is suppressed at 37 ° C., or an immortalized dendritic cell line is an antigen such as a tumor antigen or an antigen-IgG immune complex. What incorporated the body is preferable. The cell vaccine of the present invention can be used as an antigen-presenting cell for stimulating T cells, which incorporates an antigen in vivo or in vitro and presents an antigenic peptide on the cell surface after modification. For example, the cell vaccine of the present invention comprising a suspension of the immortalized dendritic cell line of the present invention is inoculated as a therapeutic vaccine in the human body, but growth is suppressed at 37 ° C. High safety. Usually, heat treatment, radiation treatment, mitomycin C treatment, or the like is required to enhance safety as a cell vaccine, but the immortalized dendritic cell line of the present invention does not require such cell inactivation treatment. In addition, it can grow at 33 ° C., but since growth is suppressed at 37 ° C., it can be said to be a highly safe vaccine.

  The cell vaccine of the present invention, particularly a cell vaccine mainly composed of a human-derived immortalized dendritic cell line, is a cell vaccine that can be transferred to humans, and includes various tumors such as leukemia, liver cancer, lung cancer, stomach cancer, and colon cancer, and It can be advantageously used against infectious diseases caused by various viruses and bacteria. The dose of the cell vaccine of the present invention varies depending on the patient's age, weight, sex, cancer type, cancer progression, symptoms, etc., and cannot be determined in general, but is injected with the current cell vaccine therapy A similar amount of can be administered to a patient. Although the cellular vaccine of the present invention can be used for the patient himself, it can be administered to many patients of the same type who are MHC compatible by the development of bone marrow bank and cord blood bank.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

(Creation of transgenic mice)
Transgenic mice into which the DNA of SV40 temperature-sensitive mutant tsA58 was introduced were produced by the following procedure.

(Preparation of transgene)
For microinjection, a genomic DNA of SV40 temperature-sensitive mutant tsA58 was modified by genetic engineering techniques. The genomic DNA of tsA58 was opened with the restriction enzyme BamHI, introduced into the BamHI site of pBR322, the SfiI sequence was converted to SacII, and the DNA clone pSVtsA58ori, which had the SV40 replication origin (ori) deleted as ori (-) ( -)-2 (Ohno T. et al., Cytotechnology, 165-172, 1991), DNA for introduction was prepared according to a conventional method. That is, the plasmid DNA pSVtsA58ori (−)-2 obtained by amplifying in large quantities in E. coli was digested with the restriction enzyme BamHI (Takara Shuzo), and then agarose electrophoresis (1% gel; Boehringer) Then, the gel was dissolved, followed by phenol / chloroform treatment and ethanol precipitation treatment to recover DNA. The recovered purified DNA was dissolved in TE buffer (10 mM Tris-HCl containing 1 mM EDTA; pH 7.6) to obtain a solution containing 170 μg / ml purified DNA. This DNA solution was diluted with an injection buffer (10 mM Tris-HCl containing 0.1 mM EDTA; pH 7.6) to 5 μg / ml to prepare an injection DNA solution. The prepared DNA solution was stored at −20 ° C. until the injection operation.

(Creation of transgenic mice)
Microinjection of the prepared DNA solution for injection into mouse pronuclear fertilized eggs was performed as follows. Sexually mature 8-week-old Wistar mice are bred at a light / dark cycle of 12 hours (4: 0 to 16:00 light time), at a temperature of 23 ± 2 ° C. and a humidity of 55 ± 5%. Observed and selected the day of hormone treatment. First, 150 IU / kg pregnant horse serum gonadotropin (manufactured by Zenyak Co., Ltd .; PMS gonadotropin: PMSG) was intraperitoneally administered to female mice, and 75 IU / kg human chorionic property 48 hours later. After gonadotropin (manufactured by Sankyo Organs Co., Ltd .; human chorionic gonadotropin: hCG) was administered to perform superovulation, mating was carried out by cohabitation with males. Pronuclear fertilized eggs were collected by oviduct perfusion 32 hours after hCG administration. MKRB solution (Toyoda Y. and Chang MC, J. Reprod. Fertil., 36, 9-22, 1974) was used for oviduct perfusion and egg culture. The collected fertilized eggs were subjected to enzyme treatment at 37 ° C. for 5 minutes in mKRB solution containing 0.1% hyaluronidase (Sigma; Hyaluronidase Typel-S) to remove cumulus cells, and then 3 times with mKRB solution. It washed to remove the enzyme, CO ₂ until DNA injection operation - incubator (5% CO ₂ -95% of the Air, 37 ° C., saturated humidity) and stored in. The DNA solution was injected into the male pronucleus of the mouse fertilized egg thus prepared. The injected 228 eggs were transplanted to 9 foster parents to give birth and 80 offspring were obtained. For introduction of the injected DNA into the mouse, DNA prepared from the tail obtained by cleaving immediately after weaning was assayed by PCR [Primer used: tsA58-1A, 5′-TCTCATAATGTGCAGTCAGGTG-3 ′ (from 1365 to 1384 sites). Corresponding: SEQ ID NO: 1), tsA58-1B, 5′-ATGACGAGCTTTGGCACTTG-3 ′ (corresponding to 1571 to 1590 sites: SEQ ID NO: 2)]. As a result, 11 lines of transgenic mice (6 males, 8 females, 6 gender unknown) pups that survived to the age of 12 weeks after the sexual maturation period (20 males, 8 females, 6 gender unknown). Male lines: # 07-2, # 07-5, # 09-6, # 12-3, # 19-5, female lines: # 09-7, # 11-6, # 12-5, # 12-7 , # 18-5, # 19-8). Crossed these G ₀ generation of transgenic mice and Wistar mouse, 2 line of male founders (# 07-2, # 07-5) and female founders 3 line (# 09-7, # 11-6, # In 19-8), gene transfer to the next generation and later was confirmed.

(Separation and adjustment of DC from mouse bone marrow)
Two mice, C57BL / 6 (B6 mouse) and temperature-sensitive SV40T antigen transgenic mouse (ts SV40 LT Tg mouse; B6 background) were used. All of these mice were females aged 6 to 8 weeks. Mouse bone marrow cells were erythroid lysis treated with 0.144M ammonium chloride, anti-CD4 antibody, anti-CD8 antibody, anti-I-A ^b antibody, anti-rat Ig antibody (respectively TIB207,211,154,216: Amerikan Type Culture Collection ) And rabbit complement (Cedarlane) were used to remove lymphocytes and Ia positive cells. This cell 1 × 10 ⁶ / well was cultured in a 24-well plate using complete RPMI medium (5% FCS) containing 20 ng / mL mouse recombinant GM-CSF (manufactured by Peprotech), and after 6 days dendritic cells ( DC) was induced. DCs from ts SV40 LT Tg mice were induced under conditions of 33 ° C. and 5% CO ₂ , and passage was repeated 10 times or more at 5 × 10 ⁵ / mL / well (medium exchange every 4 to 5 days, 3 weeks And subcultured for 7 months or more. DC from B6 mice were isolated from bone marrow cells by the above-described method, induced, and cultured under conditions of 37 ° C. and 5% CO ₂ , and used for analysis at that time.

(Morphological observation by light Giemsa staining)
The DC obtained in Example 2 was stained with Wright-Giemsa. Each DC originating from two strains of B6 mice and ts SV40 LT Tg mice was adhered to a slide glass by cytospin, stained with the Light Giemsa method (Light stain solution and Giemsa stain solution, both from Merck), and visualized. did. The results are shown in FIG. As a result, the cell size of ts SV40 LT Tg DC (SV40T B6) was larger than that of B6 mouse DC (primary culture B6).

(Proliferation ability at different temperatures)
MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazo-lium bromide) is cleaved by dehydrogenase etc. of the inner mitochondrial membrane to produce magenta MTT-formazan. The proliferative ability of ts SV40 LT Tg DC at different temperatures (33 ° C., 37 ° C., 39 ° C.) was measured by MTT assay based on this color reaction being proportional to the proliferative ability of the cells. In the MTT assay, 10 μL of a 5 mg / mL MTT (Sigma) solution was added to 100 μL of a cell suspension containing the 20 ng / mL mouse recombinant GM-CSF (Peprotech), and 7.5 μl in a 96-well plate. × plated cells at 10 ³ / 100μL / well, MTT solution was added 10 [mu] L / well at the time of measurement, was measured over time. The results are shown in FIG. As a result, the proliferation ability was highest at 33 ° C. where the SV40T antigen was expressed.

(GM-CSF requirement)
In order to examine granulocyte macrophage colony stimulating factor (GM-CSF) requirement for ts SV40 LT Tg DC, MTT assay was performed under the same conditions as in Example 4. That is, 10 μL of a 5 mg / mL MTT (Sigma) solution was added to 100 μL of a cell suspension containing mouse recombinant GM-CSF (Peprotech) at concentrations of 20, 10, 2, and 0 ng / mL, respectively. plates seeded cells with 7.5 × 10 ³ / 100μL / well, the MTT solution was added 10 [mu] L / well at the time of measurement, was measured over time. The results are shown in FIG. As a result, the growth was the best at the concentration of 20 ng / mL used for DC induction of normal primary culture. This immortalized cell was found to be a cell that proliferates in a GM-CSF-dependent manner.

(Examination of proteins expressed on the cell surface)
Using respective DCs originating from two strains of ts SV40 LT Tg mice (SV40T B6) and B6 mice (primary culture B6), myeloid molecules and leucosite molecules that are representative proteins expressed on the cell surface Expression was analyzed by FACS. The results are shown in FIGS. As a result, the expression levels of myeloid molecules and leucosite molecules in DCs originating from the two strains were not changed.

(Comparison study of antigen uptake)
After adding 10 μg / mL of antigen (OVA-FITC; manufactured by Molecule Probes) using DCs originating from two strains of ts SV40 LT Tg mouse (SV40T B6) and B6 mouse (primary culture B6), 2 On the day, the uptake ability was compared with FACS. The results are shown in FIG. As a result, DC of ts SV40 LT Tg showed stronger uptake ability than that of B6 mice.

(Comparison study of maturation of dendritic cells in response to LPS stimulation)
For each DC originating from two strains of ts SV40 LT Tg mice (SV40T B6) and B6 mice (primary culture B6), 2 μg / mL of LPS was added to each DC culture well, and cells 24 hours later the expression level of maturity, a marker I-a ^b, CD86 and CD40 on the surface was analyzed by FACS. The results are shown in FIG. As a result, it was found that up-regulation of the maturity marker in ts SV40 LT Tg DC, that is, the maturity occurred in the same manner as DC of B6 mice (primary culture B6).

(Comparative study of dendritic cell activation in response to LPS stimulation)
IL-12p70 production was measured by ELISA as activation of DC in response to LPS stimulation in each DC originating from two strains of ts SV40 LT Tg mice (SV40T B6) and B6 mice (primary culture B6). The results are shown in FIG. As a result, 2 μg / mL of LPS was added to each DC culture well, and the amount of IL-12p70 produced in the supernatant after 24 hours did not change between the two strains.

(Maturation of dendritic cells when antigen is incorporated)
DCs originating from two strains of ts SV40 LT Tg mouse (SV40T B6) and B6 mouse (primary culture B6) were incorporated with 10 μg / mL of OVA-FITC as an antigen, and their maturity two days later Was analyzed by FACS. The results are shown in FIG. As a result, DC of B6 mice (primary culture B6) showed stronger maturity.

(Comparison study of antigen-presenting ability to OVA-T cells by dendritic cells)
Using the OVA-specific CD4 T cells established previously by the present inventors, the antigen-presenting ability of each DC originating from two strains of ts SV40 LT Tg mice (SV40T B6) and B6 mice (primary culture B6) is measured. did. IL-4 production and proliferation of T cells were measured as the presentation ability. X-irradiated DC (5 × 10 ³ / well) were co-cultured in 96-well plates in the presence of 1 × 10 ⁵ OVA-specific T cells and various concentrations of OVA or OVA-IgG immune complex (IC). did. It is said that when an immune complex incorporates an antigen via an Fcγ receptor, highly efficient antigen presentation occurs (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 egg white albumin (OVA; manufactured by Sigma) with rabbit anti-OVA IgG (manufactured by BioDesign) at a weight ratio of 1:10 and incubating at 37 ° C. for 1 hour. Produced. After 24 hours, the culture supernatant was collected, and the IL-4 production amount of T cells was measured by ELISA. T cell proliferation was measured by [ ³ H] -TdR incorporation after 48 hours of co-culture. The results are shown in FIG. As a result, each DC originating from the two lines caused the same amount of T cell proliferation and IL-4 production. However, when OVA added in IL-4 production is 1 μg / mL, when OVA-IgG immune complex (IC) is used as an antigen, IL-4 production by DC of ts SV40 LT Tg is B6 mice ( Less than the DC of primary culture B6).

(Anti-OVA antibody titer over time)
Anti-OVA over time after loading and transfecting DCs originating from two strains of ts SV40 LT Tg mice (SV40T B6) and B6 mice (primary culture B6) with OVA or OVA-IgG immune complex The antibody titer was examined. In an in vivo experiment, on the second day after replacement with a fresh medium containing 10 μg / mL of OVA or OVA-IgG immune complex in wells in which DC is cultured, the mature DC incorporating the antigen was collected and PBS (−) After washing, DC 1 × 10 ⁶ cells per recipient B6 mouse were transferred to 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 any of the IgG1, IgG2a, and IgG2b in both strains. When OVA was used as the antigen, antibody production by ts SV40 LT Tg DC was significantly higher in IgG2a (after 2 weeks).

(Immunohistochemical staining of spleen tissue with anti-GL-7 antibody)
DCs were transferred to the mice, and after about 3 weeks, the spleens of the mice were collected, and the expression of GL-7, an indicator of the activated germinal center, was examined by immunohistochemical staining. The results are shown in FIG. As a result, formation of B6 mice and ts SV40 LT Tg mice was effective when the immune complex (IC) was used as an antigen. There was no difference between these two systems.

(In vivo CTL activity)
The in vivo CTL activity by DC transfer to mice was compared and examined. Seven days after transfer, spleen cells of mice were collected and incubated for 30 minutes in a 37 ° C. CO ₂ incubator to remove adherent cells and make them rich in T cells. The non-adherent cells (1 × 10 ⁷⁾ and the E. coli cells that stopped proliferation by X-ray irradiation G7-OVA 1 × 10 ⁶ was co-cultured in a 24-well plate. E. G7-OVA (CRL2113; ATCC) is obtained by transfecting OVA cDNA into EL-4, which is a B6-derived thymoma, and an OVA peptide is always loaded on its MHC class I. On day 5 of culture, G7-OVA was labeled with Na ₂ ⁵¹ CrO ₄ (Amersham Pharmacia Biotech) for 1 hour. Various concentrations of live splenocytes and their ⁵¹ Cr labeled E. coli. G7-OVA 1 × 10 ⁴ was co-cultured in a 96-well U bottom plate, and after 4 hours, the release of ⁵¹ Cr in the supernatant was measured with an autowell gamma system (manufactured by Aloka). The results are shown in FIG. As a result, when OVA was added to DCs of ts SV40 LT Tg, compared with DCs of B6 mice, a considerably strong CTL activity was induced to the same extent as when immune complexes were incorporated.

(Expression of MHC class I / OVA peptide complex in dendritic cells)
In Example 14 above, when OVA was added to DCs of ts SV40 LT Tg, compared with DCs of B6 mice (primary culture B6), it was considerably more powerful CTL, which was comparable to that when immune complexes were incorporated. The reason why the activity was induced is that many antigen-derived peptides are presented on the MHC class I molecule of DC, and the flow site was obtained using a monoclonal antibody specific for the MHC class I / OVA peptide complex. Analyzed by measurement. In the presence of 50 μg / ml of OVA, each DC originating from two strains of ts SV40 LT Tg mouse (SV40T B6) and B6 mouse (primary culture B6) was cultured for 48 hours. Next, the Fc receptor was blocked with an anti-FcγRII / III antibody, and then stained with an anti-CD11c-PE antibody and an anti-MHC I-FITC antibody, or an anti-CD11c-PE antibody and an anti-MHC I / OVA 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 data was analyzed by BD CellQuest. FIG. 14 shows the results of the expression of dendritic cells (CD11c positive cells) on the surface of MHC I or MHC I / OVA peptide cells as a histogram. As a result, the expression level of MHC class I of DC of ts SV40 LT Tg was almost the same as that of wild type B6 mouse (primary culture B6), but the expression of MHC class I / OVA peptide complex was ts SV40 LT Tg. The DC was dramatically higher. That is, it was found that the DC of ts SV40 LT Tg is a cell that can efficiently present an antigen against CTL by efficiently presenting an OVA peptide to MHC class I.

(Anti-tumor activity in vivo)
To specifically evaluate the enhanced CTL response of ts SV40 LT Tg DC in vivo, anti-tumor experiments were performed. Each DC (5 × 10 ⁵ / mouse) originating from two strains of ts SV40 LT Tg mice (SV40T B6) and B6 mice (primary culture B6) given OVA stimulation (10 μg / ml, 48 hours), or Saline (200 μl) was administered from the tail vein of naive mice (7-8 mice), DC or saline was administered again 7 days later, and tumor cells (E) expressing OVA 7 days later. .G7) was planted in the left thigh at 1 × 10 ⁵ / mouse, and tumor formation was observed day by day, and it was determined that tumors were formed if the diameter of the tumor was 5 mm or more. The results are shown in FIG. The tumor suppression rate in FIG. 15 represents the percentage of mice in which tumors are not formed in%. Mice transfected with DCs of ts SV40 LT Tg were slower in tumor formation than mice transfected with DCs of wild type B6 mice and efficiently suppressed tumor formation. In other words, it was found that ts SV40 LT Tg DCs are cells that induce antitumor activity more efficiently than wild type DCs in vivo.

(Discussion)
DCs derived from bone marrow cells of ts SV40 LT Tg mice, passaged more than 10 times, and repeated long-term culture at 33 ° C. for 7 months or more have a slightly larger size and higher uptake capacity than those of the initial culture. In addition, it was found that the antigen presentation ability in vitro has a function that does not change when mediated by MHC class II. From this, it is considered that the cell line is useful for use in analysis of DC in vitro. In addition, when used as a vaccine in vivo, CTL was strongly induced, particularly via MHC class I. This is consistent with a report (Annu. Rev. Immunol., 19, 47-64, 2001) that the high-efficiency MHC class I presentation ability depends on high antigen uptake. From this, DC of ts SV40 LT Tg can efficiently cause a vaccine effect against cancer, virus and the like in vivo.

It is a figure which shows the result of carrying out the light Giemsa dyeing | staining of the dendritic cell of the immortalized dendritic cell strain of this invention, and the dendritic cell of a B6 mouse | mouth. It is a figure which shows the result of having measured the growth ability about the immortalized dendritic cell strain of this invention by changing temperature conditions by MTT assay. It is a figure which shows the result of having measured the requirement of GM-CSF by the MTT assay in the proliferation of the immortalized dendritic cell line of this invention. It is a figure which shows the result of having analyzed the expression level of the typical myeloid molecule | numerator on the immortalized dendritic cell strain of this invention, and the dendritic cell of a B6 mouse | mouth by FACS. It is a figure which shows the result of having analyzed the expression level of the typical leucosite molecule | numerator on the dendritic cell line of the immortalized dendritic cell strain of this invention, and the dendritic cell of B6 mouse | mouth by FACS. It is a figure which shows the result of having analyzed the antigen uptake | capture ability of the immortal dendritic cell strain of this invention, and the dendritic cell of a B6 mouse | mouth by FACS. It is a figure which shows the result of having analyzed the maturity of the dendritic cell by FACS with respect to LPS stimulation of the immortalized dendritic cell line of this invention and the dendritic cell of a B6 mouse | mouth. It is a figure which shows the result of having measured the IL-12p70 production amount of the dendritic cell with respect to LPS stimulation of the immortalized dendritic cell line of this invention and the dendritic cell of B6 mouse | mouth by ELISA method. It is a figure which shows the result of having taken OVA into the immortalized dendritic cell line of this invention, and the dendritic cell of B6 mouse | mouth, and having analyzed the maturity two days later by FACS. It is a figure which shows the result of the antigen presentation ability to the OVA-T cell of the immortalized dendritic cell line of this invention and the dendritic cell of a B6 mouse | mouth. It is a figure which shows the anti- OVA antibody titer over time by transferring OVA to the immortalized dendritic cell line of the present invention and the dendritic cells of B6 mice. It is a figure which shows the result of the immunohistochemical staining (anti-GL-7 antibody) of the spleen germinal center about 3 weeks after transferring the dendritic cell of the dendritic cell line of this invention. It is a figure which shows the result of having examined comparatively the in-vivo CTL activity by having transferred the dendritic cell of the dendritic cell strain of this invention. It is a figure which shows the result of having compared the expression level of the MHC class I / OVA peptide complex in the dendritic cell line of the present invention and the dendritic cell of B6 mouse. It is a figure which shows the result of the enhanced antitumor activity in the living body by having transferred the immortalized dendritic cell line of this invention.

Claims (20)

An immortalized dendritic cell line characterized by being derived from bone marrow. 2. The immortalized dendritic cell line according to claim 1, which expresses a myeloid molecule and a leucosite molecule on the cell surface, and has an ability to take up an antigen, an ability to present an antigen, and an ability to induce CTL activity. 3. The immortalized dendritic cell line according to claim 1 or 2, wherein the immortalized dendritic cell line can grow at 33 ° C but is inhibited at 37 ° C. The immortalized dendritic cell line according to any one of claims 1 to 3, which has an ability to respond to LPS stimulation. The immortalized dendritic cell line according to any one of claims 1 to 4, which is of rodent origin. The immortalized dendritic cell line according to claim 5, wherein the rodent is a mouse. Immortalized dendritic cell line TDC (FERM BP-08527). After lysing bone marrow cells of transgenic mice introduced with the large T antigen gene of SV40 temperature-sensitive mutant tsA58, lymphocytes and Ia positive cells were removed, and the resulting cells were cultured in the presence of GM-CSF. Dendritic cells are induced, and subculture is repeated 10 times or more, and myeloid molecules and leucosite molecules are expressed on the cell surface, and have the ability to take up antigen, present antigen, and induce CTL activity. A method for producing an immortalized dendritic cell line, comprising establishing a cell line. 9. The method for producing an immortalized dendritic cell line according to claim 8, wherein a cell line capable of growing at 33 ° C. but having growth inhibited at 37 ° C. and capable of responding to LPS stimulation is established. A dendritic tree characterized by culturing the immortalized dendritic cell line according to any one of claims 1 to 7 in the presence of a test substance, and measuring and evaluating the degree of expression of a mature marker protein in the cell line. A method for screening a substance for promoting or suppressing maturation in a cell. Marker protein, myeloid molecules, leukocyte molecule, I-A ^b, CD86 and / or screening method of the mature promoting or inhibiting substances in dendritic cells of claim 10, characterized in that the CD40. Cell growth promotion in dendritic cells characterized by culturing the immortalized dendritic cell line 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 cells Or screening method of inhibitory substance. An immortalized dendritic cell line according to any one of claims 1 to 7 is subjected to LPS stimulation in the presence of a test substance, and the amount of IL-12 production of the cell is measured and evaluated. A method for screening an activation promoting or inhibiting substance. A maturation promoting substance in dendritic cells obtained by the screening method according to claim 10 or 11. A cell growth promoting substance in dendritic cells obtained by the screening method according to claim 12. A dendritic cell activation promoter obtained by the screening method according to claim 13. A cell vaccine comprising the immortalized dendritic cell line according to any one of claims 1 to 7 as a main component. 18. The cell vaccine according to claim 17, wherein the immortalized dendritic cell line is an immortalized dendritic cell line that can grow at 33 ° C., but the growth is suppressed at 37 ° C. 19. The cell vaccine according to claim 17 or 18, wherein the immortalized dendritic cell line is an immortalized dendritic cell line into which an antigen or antigen-IgG immune complex has been incorporated. The cellular vaccine according to claim 19, wherein the antigen is a tumor antigen.

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