JP4863452B2 - Human Th1 / Th2 differentiation induction system and use thereof - Google Patents

Description

The present invention relates to a human Th1 / Th2 differentiation induction system using human naive T cells, a method for detecting differentiation-induced cells, and a screening method and screening kit for the influence (promotion or suppression, etc.) of a test compound on the differentiation induction. Etc.

Helper T cells (Th) are a group of T cells that recognize antigens, produce various cytokines collectively called helper factors, and regulate immune response induction. For example, differentiation of B cells into antibody-producing cells requires cytokines such as interleukins (IL) 4, 5 and 6 produced from helper T cells in addition to the recognition of the antigen by B cells themselves. In addition, IL-2 and interferon γ are often involved in effector induction of killer T cells. Such helper T cells are classified into a subset of Th1, which produces IL-2 and interferon γ, and Th2, which produces IL-4, 5, 6 and the like. These subsets are known to be activated by antigenic stimulation and act to suppress each other's subsets, and are important cell populations that depend on the mode of the body's immune response depending on which of them is dominant. is there.

Previously, the present inventors established a mouse Th1 / Th2 differentiation induction system, and analyzed the role of Ras / MAPK pathway and Ca ²⁺ / CN pathway in Th1 / Th2 differentiation of peripheral T cells that had not been analyzed ( Masashita Yamashita, Toshinori Nakayama, Experimental Medicine, 19: 570-575, 2001). That is, by using the mouse Th1 / Th2 differentiation induction system, the MEK / Erk cascade is important among the three cascades in the Ras / MAPK pathway (Yamashita, M. et al., Proc. Natl. Acad. Sci UST, 96: 1024-1029, 1999, Yamashita, M. et al., J Biol Chem., 280: 29409-29419, 2005), and FK506 and cyclosporine targeting calcineurin (CN) as mouse Th1 It has been shown to modulate the / Th2 differentiation induction system (Yamashita, M. et al., J. Exp. Med., 191: 1869-1879, 2000). From these studies, it is possible to evaluate the differentiation induction into Th1 or Th2 by compounds etc. under the condition that Th1 / Th2 differentiation induction system in mice, especially Ras / MAPK pathway or Ca ²⁺ / CN pathway is working It became.

Regarding human systems, JP-A-2002-186482 describes Th1 cells or Th2 cells obtained by stimulating human umbilical cord blood leukocytes. In Th1 culture, umbilical cord blood-derived leukocytes are expressed as IL-12. And a conventional medium differentiation induction method using a medium composition containing IL-4 neutralizing antibody and Th2 culture using a medium composition containing IL-4 and IL-12 neutralizing antibody. . In addition, as a method for intracellular immunofluorescence staining of cytokines, permeabilized human umbilical cord blood-derived Th1 cells or Th2 cells are stained for IL-4 or IFN-γ.

Further, a method for inducing differentiation from human naive T cells to Th1 / Th2 using IL-4 or IFNγ (Torres, KC et al., Hum Immunol. 2004 Nov; 65 (11): 1328-35), CD3 Method using monoclonal antibody (Suzuki Y, et al., J Virol. 1999 Jan; 73 (1): 316-24., Demeure, CE et al., Eur J Immunol. 1995 Sep; 25 (9): 2722 There is a report of -5). However, there is no report on the Th1 / Th2 differentiation induction system under conditions where the Ras / MAPK pathway or Ca ²⁺ / CN pathway of human peripheral T cells is working.
JP 2002-186482 A Masashita Yamashita, Toshinori Nakayama, Experimental Medicine, 19: 570-575, 2001 Yamashita, M. Et al., Proc. Natl. Acad. Sci. UST, 96: 1024-1029, 1999, Yamashita, M. Et al., J Biol Chem., 280: 29409-29419, 20 Yamashita, M. Et al., J. Exp. Med., 191: 1869-1879, 2000 Torres, KC et al., Hum Immunol. 2004 Nov; 65 (11): 1328-35 Suzuki Y, et al., J Virol. 1999 Jan; 73 (1): 316-24., Demeure, CE et al., Eur J Immunol. 1995 Sep; 25 (9): 2722-5

By the way, a compound that is positive in the Th1 / Th2 differentiation induction system of rodents such as mice does not necessarily become positive in a human system. That is, IL-4, IL-12, or IFN-γ targeting naïve CD4T cells can be mentioned as a case where species differences between rodents such as mice and humans do not react with each other. The signal transduction system related to these cytokines is also considered to correspond to a case where there is a large species difference between rodents such as mice and humans from the process of evolution.

As already described, there has been no appropriate means for evaluating the human Th1 / Th2 differentiation induction system in the past, and in the Th1 / Th2 differentiation induction system of human peripheral T cells, particularly the Ras / MAPK pathway or Ca ²⁺ / CN pathway. Establishing a human Th1 / Th2 differentiation induction system that can evaluate related drugs was essential for searching compounds that modulate human Th1 / Th2 differentiation induction by these pathways.

Accordingly, the present inventor has conducted extensive research on compositions and methods for stimulating human Th1 differentiation induction or Th2 differentiation induction using human naive T cells of peripheral T cells in vitro, respectively. The present invention was completed by successfully developing a Th1 / Th2 differentiation induction system.

That is, the present invention relates to the following aspects.
[1] Human naive CD4T cells are cultured under stimulation with an anti-CD3 antibody in a differentiation induction medium (Th1 differentiation induction conditions) containing IL-2, IL-12 and anti-IL-4 antibodies, or IL-2 Culturing under stimulation with anti-CD3 antibody in differentiation-inducing medium (Th2 differentiation-inducing conditions) containing IL-4 and anti-IFNγ antibody, and only anti-IL-4 antibody or anti-IFNγ antibody from the differentiation-inducing medium, respectively. A method for inducing Th1 and / or Th2 differentiation of human naïve CD4 T cells, further comprising culturing without stimulation with an anti-CD3 antibody under culture conditions excluding.

[2] After differentiation induction of human Th1 / Th2 by the differentiation induction method of the present invention, intracellular staining is performed using labeled antibodies against IFNγ, IL-4, and CD4, and then T is performed using anti-CD4 antibody. A method of selecting only cells, measuring IL-4 and IFNγ intracellular staining intensity of the selected cells, and evaluating the degree of differentiation induction into Th1 or Th2 based on the intensity.

[3] Expression intensity of at least one of a total of 13 types of Th2-specific expression genes described in Tables 1 and 2 and / or at least one of the two types of Th1-specific expression genes described in Table 3 And measuring the degree of differentiation induction into Th1 or Th2 based on their strength.

[4] A method for screening the influence of a test compound on Th1 and / or Th2 differentiation induction of human naive CD4 T cells, using a differentiation induction medium to which a predetermined amount of a test compound is added, to induce differentiation of the present invention The screening method comprising evaluating the degree of differentiation induction to Th1 or Th2 after human Th1 / Th2 differentiation induction by the method.

[5] A kit used in the method of the present invention.

The method for inducing human Th1 / Th2 differentiation of the invention reacts with compounds related to the Ras / MAPK pathway or Ca ²⁺ / CN pathway. This is a novel screening system that can search for compounds that modify / Th2 differentiation induction.

In the evaluation method of the present invention, after differentiation induction, intracellular staining is performed using an anti-IFNγ antibody and an anti-IL-4 antibody, and then only T cells are selected using an anti-CD4 antibody. Only IL-4 and IFNγ intracellular staining intensity is directly measured, so it is possible to directly evaluate differentiation induction into human Th1 or Th2. Furthermore, there is provided a method for evaluating the degree of induction of differentiation of human Th1 / Th2 using the expression intensity of a gene that exhibits specific expression fluctuations in differentiation-induced human Th1 / Th2. Alternatively, a method is provided for accurately and directly evaluating the effect of an addictive compound on human Th1 / Th2 differentiation induction.

Therefore, in the present invention, using the above method, a compound related to the Ras / MAPK pathway or the Ca ²⁺ / CN pathway, or a compound that modifies human Th1 / Th2 differentiation induction under the condition that these pathways are moving, etc. Can be searched, and a novel screening system that has not existed before can be provided.

The differentiation antigen (cell surface antigen) expressed by human naive CD4 T cells used in the Th1 and / or Th2 differentiation induction method of the present invention is represented by CD4 ⁺ CD8 ⁻ CD45RO ⁻ . Such cells are usually obtained from human peripheral blood, human umbilical cord blood, human lymphoid tissue, etc. by using any method known to those skilled in the art using antibodies against these differentiation antigens, various cell separation devices, cell analysis devices, etc. And can be easily separated.

  In the differentiation induction method of the present invention, stimulation with an anti-CD3 antibody can be performed by any method known to those skilled in the art. Stimulation with anti-CD3 antibody can be performed.

  In the differentiation induction method of the present invention, the cells are cultured usually for 1 to 3 days, preferably 2 days under stimulation with an anti-CD3 antibody, and usually cultured for 4 to 6 days, preferably 5 days, without stimulation with an anti-CD3 antibody. .

When culturing under stimulation with anti-CD3 antibody, in Th1 differentiation-inducing conditions or Th2 differentiation-inducing conditions, the differentiation-inducing medium usually has IL-2 of 5 to 500 U / ml, IL-12 of 0.1 to 100 ng / ml, It is preferable that anti-IL-4 antibody is contained at a concentration of 0.5 to 50 μg / ml, IL-4 is contained at 0.1 to 100 ng / ml, and anti-IFNγ antibody is contained at a concentration of 0.5 to 50 μg / ml.

On the other hand, in the culture without stimulation with anti-CD3 antibody, the differentiation induction medium used does not contain the above-mentioned anti-IL-4 antibody or anti-IFNγ antibody, and cytokines such as IL-2, IL-12, and IL-4 included.

In addition, the efficiency of differentiation induction can be improved by continuously performing a culture cycle consisting of culture under stimulation with anti-CD3 antibody under Th2 differentiation-inducing conditions and culture without stimulation with anti-CD3 antibody under the same differentiation-inducing conditions. More enhanced.

  In the labeled antibodies against IFNγ, IL-4 and CD4 used in the evaluation method of the present invention, any compound known to those skilled in the art can be used as the labeling substance. For example, FITC (fluorescein isothiocyanate), PE (phycoerythrin), APC (allophycocyanin), etc. can be mentioned. These antibodies can be labeled by any method known to those skilled in the art, or commercially available products can be used as labeled antibodies.

Selection of T cells using anti-CD4 antibody and measurement of IL-4 and IFNγ intracellular staining intensity for the selected cells are performed by appropriate automatic cell analysis known to those skilled in the art such as fluorescence activated cell sorter (FACS). This can be done using a separation device.

  Furthermore, the degree of differentiation induction into Th1 or Th2 can be evaluated by measuring the expression intensity of a Th1 or Th2-specific expression gene. Examples of such genes include, for example, a total of 13 types of Th2-specific expression genes described in Tables 1 and 2 and two types of Th1-specific expression genes described in Table 3. .

The expression intensity of such a Th1- or Th2-specific expression gene can be determined, for example, by measuring the expression level of mRNA (cDNA) or expression product (protein) of these genes in the target cells. Such measurement can be quantitative, semi-quantitative, or qualitative depending on the measurement method and principle.

Nucleic acid molecules containing such cDNA or mRNA corresponding thereto apply any method / means known to those skilled in the art, for example, various quantitative PCR methods such as RT-PCR method, real-time PCR, and electrochemical detection method. The hybridization strength measurement method and various microarray (DNA chip) methods can be used by methods known to those skilled in the art. Detection of nucleic acid molecules amplified by the PCR method can be performed by an appropriate method such as a method for directly determining the base sequence (sequence method), an electrochemical detection method, or a combination with electrophoresis. Various primers and probes used in such a method can be appropriately designed and prepared by those skilled in the art based on the base sequence information of genes available from each database.

In designing the primer, the primer preferably has a number of bases capable of specific binding to the template, for example, 15-40 bases, more specifically about 15-25 bases. It is also important to have a base sequence that does not have a hairpin structure in the primer or that the sense strand and the antisense strand do not anneal with each other. For example, commercially available primer design software such as Oligo ™ (National Bioscience Inc.) can be used.

The amount of protein expression can be measured by any method known to those skilled in the art. Examples include, for example, a method using immunostaining using an appropriate antibody and various immunological specific reactions such as EIA, an amino acid sequence analysis method for peptides such as a gas phase sequencer using the Edman method, and MALDI- It can detect by mass spectrometry represented by TOF / MS, ESI Q-TOF / MS method, etc.

The antibody can be prepared by an appropriate method known to those skilled in the art using the corresponding protein, or an appropriate partial polypeptide (peptide fragment) thereof, or various derivatives or complexes thereof as an antigenic substance or immunogen. Is possible. For example, in the case of a polyclonal antibody, it can be administered to an appropriate animal such as a mouse, rat, rabbit, goat or chicken and prepared from the antiserum. Alternatively, a known monoclonal antibody production method (“monoclonal antibody”, Kamei Nagamune, Hiroaki Terada, Yodogawa Shoten, 1990; “Monoclonal Antibody” James W. Goding, third edition, Academic Press, 1996), etc. It is also possible to prepare a monoclonal antibody by a method using cell fusion.

In addition, as long as the original antibody activity is not lost, such an antibody can be obtained by genetic engineering (DNA recombination technology), for example, various antibodies derived from complete antibodies such as Fab, F (ab ′) ₂ and Fv fragments. It may be a derivative, recombinant or fragment modified into various forms known to those skilled in the art, including derivatives.

Such antibodies are appropriately labeled with various labeling substances known to those skilled in the art, such as enzymes, radioisotope fluorescent dyes, and metal atoms.

In the screening method of the present invention, human Th1 / Th2 differentiation is induced by the differentiation induction method of the present invention using a differentiation induction medium supplemented with a predetermined amount of a test compound, and then differentiation induction into Th1 or Th2 is induced. By evaluating the degree, the influence (modification) of the test compound on Th1 and / or Th2 differentiation induction of human naive CD4 T cells can be screened. The amount and timing of adding the test compound can be appropriately adjusted according to the properties of the test compound.
Usually, the test compound is added in a predetermined amount throughout both the culture step under stimulation with the anti-CD3 antibody and the subsequent culture step under non-stimulation with the anti-CD3 antibody. However, depending on the nature of the compound to be added, it is also possible to add the test compound only during the culture process under either stimulation with anti-CD3 antibody or non-stimulation with anti-CD3 antibody. .

Specifically, the degree of differentiation induction into Th1 or Th2 can be evaluated by the evaluation method of the present invention already described. As a result, it becomes possible to screen for compounds that affect Th1 and / or Th2 differentiation induction, such as compounds that promote or suppress human Th1 / Th2 differentiation induction, compounds that modulate Th1 / Th2 balance, and the like. An example of such a compound is an allergy suppressing compound.

  In each of the methods described so far, various cell culture conditions (cell culture density, medium components, culture time, culture temperature, oxygen / carbon dioxide concentration, etc.), cell culture apparatus, etc. are the purpose of each method. Those skilled in the art can appropriately select the cell or test compound depending on the type and amount thereof.

A kit (for example, a screening kit) used in various methods of the present invention can have an appropriate configuration depending on the measurement object or the measurement principle. The kit has, as its components, for example, various (labeled) antibodies, primers for amplification of mRNA (cDNA), and probes for hybridization used in DNA chips and the like (for example, about 10 to 100 consecutive base sequences). Consisting of). Further, the kit includes other elements or components known to those skilled in the art, such as various reagents, enzymes, buffers, reaction plates (containers), and the like, depending on the configuration and purpose of use.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the technical scope of this invention is limited and is not interpreted at all by description of a following example. Also, unless otherwise stated, the following examples are described in, for example, Sambrook and Maniatis, in Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, FM et al., Current Protocols. It can be performed according to standard genetic engineering and molecular biology techniques known to those skilled in the art, as described in Molecular Biology, John Wiley & Sons, New York, NY, 1995, etc. Moreover, the description content of the literature referred in this specification comprises some disclosure content of this specification.

Separation of human naive CD4 T cells Human naive CD4 T cells were separated by an automatic magnetic cell separator (autoMACS, Miltenyi Biotec GmbH) by the method described below. 50 ml of human peripheral blood was collected and diluted 1:50 with D-PBS solution (Product Number D5652, Sigma-Aldrich Inc). Next, 25 ml of the above-mentioned diluted peripheral blood was slowly layered on four centrifuge tubes into which 12.5 ml of Ficoll-Paque Plus (Catalog No. 17-1440-02, Amersham Biosciences) had been dispensed, and a centrifuge equipped with a horizontal rotor. Centrifugation was performed at 950 G for 30 minutes at room temperature. After centrifugation, lymphocytes present in the intermediate layer between Ficoll-Paque and the supernatant were collected, 3% fetal calf serum (FCS: Cat No. 2103-500M, JRH Biosiences), 2mMEDTA (Cat No 345-01865 , Dojindo Inc) in a D-PBS solution (hereinafter referred to as “MACS buffer”), and centrifuged at 500 G for 5 minutes at 4 ° C. using a centrifuge to precipitate lymphocytes. . The sedimented lymphocyte cell pellet was suspended again in MACS buffer and centrifuged at 500 G for 5 minutes to sediment lymphocytes, and a washed lymphocyte cell pellet was obtained. This cell pellet is suspended again in MACS buffer at 1x10 ⁸ cells / ml, and FITC-labeled anti-human CD8 antibody (Cat. No 555366, BD BioScience Phamingen) 2 μL against 1x10 ⁷ lymphocytes, FITC-labeled Add 2 μL each of anti-human CD45RO antibody (Cat. No. 555492, BD BioScience Phamingen), react on ice for 30 minutes to bind each antibody, add MACS buffer, centrifuge and wash twice at 4 ° C did. Next, 10 μL of anti-FITC microbeads (Cat No. 120-000-293, Miltenyi Biotec GmbH) were added to 1 × 10 ⁷ lymphocytes to which each antibody was bound, and reacted on ice for 15 minutes. The buffer was added, centrifuged, and washed twice at 4 ° C. The obtained cell pellet was prepared with MACS buffer to a cell concentration of 5 × 10 ⁷ / ml, passed through a nylon mesh (# 200 Abe Kagaku), and then an automatic magnetic cell separator (AutoMACS, Miltenyi Biotec GmbH) was used. By collecting the CD8 and CD45RO negative fractions, human naive CD4T cells were isolated and subjected to Th1 differentiation induction or Th2 differentiation induction experiments.

Next, for the purpose of examining gene expression after Th1 differentiation induction or Th2 differentiation induction, anti-human CD4 antibody (CD4 PE) labeled with phycoerythrin was used to isolate human naive CD4 T cells isolated above. Cells were stained to examine the content of target naive CD4 cells. Human naive CD4 T cells were suspended at 1 × 10 ⁸ cells / ml, 2 μL of CD4 PE was added to 1 × 10 ⁷ cells, reacted on ice for 30 minutes to bind the antibody, and then 2 times with MACS buffer. Washed twice. The following results were obtained by measurement by a conventional method using a cell analyzer (FACScalibur, Becton Dickinson). When the cells expressing human CD4 in the prepared cell group were examined, the content of cells expressing only the target human CD4 was 36.3% (FIG. 1-A), but human CD8 / CD45RO-expressing cells were 0%, and CD8 T cells and CD45RO positive memory T cells could be completely removed. The content of other cells (mostly non-T cells) was 63.5% (FIG. 1-B). This confirmed that the isolation of human naive CD4 T cells was good. A part of the naive T cells (Th0 cells) obtained here was used in an experiment for measuring the gene expression intensity.

Induction of Th1 / Th2 differentiation from human peripheral blood CD4 T cells Using the naive T cells isolated and prepared from human peripheral blood in Example 1, the differentiation induction method of the present invention was carried out to produce human Th1 induced cells and humans. Th2-induced cells were obtained. That is, naive CD4 T cells isolated from human peripheral blood were seeded so as to be ⁵ × 10 ⁵ cells / 0.5 ml / well under Th1 differentiation conditions, and stimulated and subsequently cultured under the following conditions. Th1 stimulation and subsequent culture or Th2 stimulation and subsequent culture were performed at 37 ° C. in an air containing 5% CO 2 saturated with water vapor using a CO 2 incubator.

Under Th1 differentiation conditions, 10% fetal calf serum (FCS: Cat. No., 2103-500M JRH, Biosiences), 10 mM HEPES (Cat. No. 15630-130, GIBCO), 100 μM Non-Essential Amino Acids (Cat. No. .
11140-076, GIBCO), 1mMSodiumPyruvate (Cat.No. 11360-070, GIBCO), RPMI-1640 medium (Cat. No. 2006-06, containing 55 μM 2-Mercaptethanol (Cat.No. 21985-023, GIBCO) SIGMA) (hereinafter referred to as “differentiation induction medium”), IL-2 (Immunes, Yoshishi Shiono) to a concentration of 50 U / ml, IL-12 (Cat.No.) to a concentration of 1 ng / ml 200-12, Peprotech) and anti-IL-4 antibody (Cat. No BD-554481, Pharmingen) was added to a concentration of 5 μg / ml. The culture was carried out for 2 days using a flat bottom 48-well tissue culture plate (code, 3548, Costor) solidified with 20 μg / ml of anti-CD3 antibody (orthoclone OKT3, Janssen Pharma). Furthermore, the cells were transferred to a non-layered plate containing no anti-CD3 antibody, and further cultured for 5 days in a culture condition of Th1 differentiation conditions that did not contain anti-IL-4 antibody but contained IL-2 and IL-12. did.

In addition, naive T cells were seeded at 5 × 10 ⁵ cells / 0.5 ml / well under Th2 differentiation conditions and stimulated under the following conditions. That is, IL-2 (Immunes, Yoshishi Shiono) added to a concentration of 50 U / ml, IL-4 (Cat. No. 204-IL-050, R & D Systems added to a concentration of 1 ng / ml) ) And 5 μg / ml of anti-IFNγ antibody (Cat. No. BD-5545471, Pharmingen) containing an anti-CD3 antibody solidified plate under culture conditions using a differentiation-inducing medium. The cells were transferred to a non-solidified plate containing no anti-CD3 antibody, and the cells were transferred to a non-layered plate containing no anti-CD3 antibody. Cultured. In the case of Th2 cell differentiation, good results were obtained for the first time by the method of the present invention in which the culture for the purpose of differentiation induction for 5 days following 2 days was continued for 2 cycles. Th1 differentiation-induced cells (Th1 cells) and Th2 differentiation-induced cells (Th2 cells) were subjected to experiments for measuring gene expression intensity.

Intracellular staining of Th1 differentiation-inducing cells or Th2 differentiation-inducing cells Th1 differentiation-induced cells (Th1 cells) and Th2 differentiation-induced cells (Th2 cells) in Example 2 were treated with Monensin (Cat No. M-5273 Sigma). ) In the presence of PMA (Phorbol-12-Myristate-13-Acetate, Produc No. P1585 Siguma-Aldrich), medium containing Ionomycin (Cat. No 407952, Calbiochem) was used for stimulation for 4 hours. After stimulation, intracellular staining was evaluated by analysis using a cell analyzer (FACScalibur, Becton Dickinson) for the degree of Th1 differentiation induction or Th2 differentiation induction by the following method. Specifically, Th1-induced cells or Th2-induced cells were seeded in a flat-bottomed 48-well tissue culture plate to give 5-10x10 ⁵ cells per well, 10 ng / ml PMA, 1 μM in the presence of 2 μM Monensin. Ionomycin 1 was added to a concentration, and stimulation was carried out by culturing at 37 ° C. in air containing saturated water vapor containing 5% CO ₂ for 4 hours using a CO ₂ incubator. In the presence of Monensin (Cat No. M-5273, Sigma), PMA, Ionomycin-stimulated Th1 or Th2 differentiation-induced cells are labeled with PE-labeled anti-human IL-4 antibody (IL-4 PE) and FITC The amount of cytokine of human IL-4 or human IFNγ accumulated in the cells using the anti-human IFNγ antibody (IFNγFITC) (Cat No. 340456 Becton-Dickinson) was evaluated by intracellular staining. At the same time, intracellular staining was performed with an APC-labeled anti-human CD4 (CD4 APC (CatNo. 555349 BD Biosciences) antibody, and measurement was performed using a cell analyzer (FACScalibur, Becton Dickinson) .The analysis was performed only on T cells expressing CD4. I went over the gate.

As a result, in Th1 differentiation induction, the CD-4 positive T lymphocyte fraction was 67.3% (FIG. 2-A1), and among the CD-4 positive T lymphocyte fraction, Th1 differentiation-inducing cells (Th1 Cells), that is, 54.3% of the cells that do not produce IL-4 and produce IFNγ, and 3.1% of the cells that produce both IL-4 and IFNγ were confirmed to be good Th1 differentiation-inducing cells (Fig. 2-A2). In Th2 differentiation induction, the CD-4 positive T lymphocyte fraction was 57.4% (Fig. 2-B1). Of the CD-4 positive T lymphocyte fraction, IFNγ was not produced and IL- The percentage of cells producing 4 (Th2 differentiation-inducing cells (Th2 cells)) was 23.0%, and cells producing both IL-4 and IFNγ were confirmed to be good as Th2 differentiation-inducing cells at 0.8% (Fig. 2-B2). The Th1 differentiation-induced cells (Th1 cells) and Th2 differentiation-induced cells (Th2 cells) obtained here were subjected to experiments for measuring gene expression intensity. In addition, using the differentiation induction conditions shown in Example 2, the cells were also subjected to experiments for examining the effects of the compounds under the conditions of compound addition.

Modification of human Th1 / Th2 differentiation inducing system of the present invention using naive T cells with FK506 or hydrocortisone Using naive T cells isolated from human peripheral blood by the method of Example 1, anti-CD3 antibody The human Th1 / Th2 differentiation induction system shown in Example 2 was used except that FK506 (F4679-5, Sigma) was added to the differentiation induction medium to 0.01 μM or 0.1 μM as a test compound during culture under stimulation. Each was used to induce human Th1 differentiation or Th2 differentiation, and whether the added compound modifies human Th1 induction or human Th2 induction was evaluated by intracellular staining by the method described in Example 3. . In addition, hydrocortisone (H0396, Sigma) as a steroid compound is added to the differentiation induction medium to 0.1 μM or 1.0 μM, or in parallel with FK506 using physiological saline as a negative control. Modifications by various compounds in the human Th1 / Th2 differentiation induction system were investigated. In addition, modification by the above compound addition in Th1 differentiation induction or Th2 differentiation induction uses an anti-CD3 antibody solidified plate for 2 days of culture, and then transfers cells to a non-solidified plate that does not contain anti-CD3 antibody. Instead, the influence was examined under the compound addition conditions throughout the whole culture process of both culture processes for 5 days.

As a result, under the Th1 differentiation induction condition, the ratio of Th1 cells (IL-4 negative and IFNγ positive cells) in the addition of physiological saline used as a negative control was 61.1%. In contrast, in the presence of 0.1 μM FK506, the ratio of Th1 cells (IL-4 negative and IFNγ positive cells) was 26.5%, which was significantly lower than the negative control. In the presence of 0.01 μM FK506, the ratio of Th1 cells (IL-4 negative and IFNγ positive cells) was 28.3%, which was similarly decreased. In the presence of 1.0 μM or 0.1 μM hydrocortisone (Hydrocortisone), the ratio of Th1 cells (IL-4 negative and IFNγ positive cells) was 66.0% and 65.5%, respectively. . (The upper part of FIG. 3).

Under Th2 differentiation-inducing conditions, the ratio of Th2 cells (IL-4 positive and IFNγ negative cells) was 30.2% when physiological saline used as a negative control was added. However, in the presence of 0.1 μM and 0.01 μM FK506, the concentration of the compound was too high to damage the cells and the effect could not be measured. From this result, it was found that it was necessary to further reduce the concentration in order to confirm the effect on FK506. Regarding the effect of hydrocortisone (Hidrocortisone), in the presence of 1.0 μM or 0.1 μM hydrocortisone (Hydrocortisone), the ratio of Th2 cells (IL-4 positive and IFNγ negative cells) was 18.0% and 22.1%, respectively. Suppression was observed. FIG. 3 bottom).

As a result, in the human Th1 / Th2 differentiation induction system of the present invention, modification is recognized by the immunosuppressant FK506 and steroid (hydrocortisone), and the effect of Th2 differentiation induction in FK506 needs to be added at a lower concentration There was found. It was shown that the human Th1 / Th2 differentiation induction system of the present invention is useful.

Modification with cyclosporin A or low concentration FK506 in human Th1 / Th2 differentiation induction system In the method described in Example 4, cyclosporin A (CyA) (0.01 μM and 0.1 μM), which is known to inhibit the Ca ²⁺ / CN pathway, is used. μM, Cyclosporin A; C3662, Sigma) and FK506 (F4679-5, Sigma) at lower concentrations than Example 4 (0.001 μM and 0.01 μM) were used for the test to evaluate Th1 / Th2 differentiation induction modification. did. In addition, modification by the above compound addition in Th1 differentiation induction or Th2 differentiation induction uses an anti-CD3 antibody solidified plate for 2 days of culture, and then transfers cells to a non-solidified plate that does not contain anti-CD3 antibody. The influence was examined under the compound addition conditions throughout the whole culture process of both of the five-day cultures.

The result of adding CyA was as follows. That is, under the Th1 differentiation induction condition, the ratio of Th1 cells (IL-4 negative and IFNγ positive cells) was 62.8% when physiological saline used as a negative control was added. On the other hand, the ratio of Th1 cells (IL-4 negative and IFNγ positive cells) in the presence of 0.1 μM CyA was 30.4%, which was significantly lower than the negative control. Further, in the presence of 0.01 μM CyA, the ratio of Th1 cells (IL-4 negative and IFNγ positive cells) was 57.9%, which was slightly decreased (the upper left in FIG. 4).

Under the Th2 differentiation induction condition, the ratio of Th2 cells (IL-4 positive and IFNγ negative cells) was 22.1% when physiological saline used as a negative control was added. In contrast, in the presence of 0.1 μM CyA, the ratio of Th2 cells (IL-4 positive and IFNγ negative cells) was greatly reduced to 10.5% compared to the negative control. Further, in the presence of 0.01 μMCyA, the ratio of Th2 cells (IL-4 positive and IFNγ negative cells) was 26.6%, and no inhibition was observed (upper right of FIG. 4).

From these results, when CyA was added, strong Th1 differentiation suppression was observed at 0.1 μM under Th1 differentiation induction conditions, and strong Th2 differentiation induction suppression was also observed under Th2 differentiation induction conditions. These results suggest that CyA is a factor that acts suppressively in Th1 and Th2 differentiation of cells.

Next, the result of adding FK506 to 0.001 μM or 0.01 μM was as follows. Under Th1 differentiation induction conditions, the ratio of Th1 cells (IL-4 negative and IFNγ positive cells) was 62.2% when physiological saline used as a negative control was added. In contrast, in the presence of 0.01 μM FK506, the ratio of Th1 cells (IL-4 negative and IFNγ positive cells) was 29.3%, which was significantly lower than the negative control. In the presence of 0.001 μM FK506, the ratio of Th1 cells (IL-4 negative and IFNγ positive cells) was 62.4%, which was not changed compared to the control (lower left in FIG. 4).

Under Th2 differentiation-inducing conditions, the ratio of Th2 cells (IL-4 positive and IFNγ negative cells) was 21.9% when physiological saline used as a negative control was added. On the other hand, in the presence of 0.01 μM FK506, the ratio of Th2 cells (IL-4 positive and IFNγ negative cells) was significantly reduced to 7.1% compared to the negative control. Further, in the presence of 0.001 μM FK506, the ratio of Th2 cells (IL-4 positive and IFNγ negative cells) was 16.6%, which was slightly reduced (right in the lower row of FIG. 4).

From these results, when FK506 was added, strong differentiation induction suppression was observed under Th1 differentiation induction conditions, and further strong differentiation induction suppression was observed under Th2 differentiation induction conditions. These results suggest that FK506 is a factor that acts more strongly and suppressively on Th1 and Th2 differentiation than CyA.

Modification by PD98509 in human Th1 / Th2 differentiation induction system
In the method described in Example 4, PD98509 (3 μM and 30 μM, No. 513000, Calbiochem) was used as a map kinase inhibitor known to inhibit the Ras / MAPK pathway to induce Th1 / Th2 differentiation. It was subjected to a test to evaluate the modification.

  As a result, under the Th1 differentiation induction condition, the ratio of Th1 cells (IL-4 negative and IFNγ positive cells) was 62.9% when physiological saline used as a negative control was added. In contrast, in the presence of 30 μM PD98509, the ratio of Th1 cells (IL-4 negative and IFNγ positive cells) increased to some extent as compared to the negative control, 73.3%, and even in the presence of 3 μM PD98509, Th1 cells (IL-4 The ratio of negative and IFNγ positive cells) was 68.4%, which was increased to some extent compared to the control.

Under the Th2 differentiation inducing condition, the ratio of Th2 cells (IL-4 positive and IFNγ negative cells) was 24.9% when physiological saline was used as a negative control. In contrast, in the presence of 30 μM and 3 μM PD98509, the ratio of Th2 cells (IL-4 positive and IFNγ negative cells) was 20.3% and 17.8%, which were suppressed to some extent compared to the negative control. Furthermore, the ratio of Th1 cells (IL-4 negative and IFNγ positive cells) was slightly increasing to 18,4%, 22.1%.

From the above results, it is clear that PD98509 has a tendency to slightly increase Th1 cells under Th1 differentiation-inducing conditions, and further has a tendency to slightly suppress Th2 cells under Th2 differentiation-inducing conditions. PD98509 has a tendency to slightly inhibit Th2 differentiation. It was suggested that Th1 induction was slightly increased and suppressed (FIG. 5).

Analysis of specific gene expression in human Th1 / Th2 differentiation induction system of the present invention using naive T cells
5x10 ⁶ cells naive T prior to the differentiation induced Th1 differentiation induction or Th2 differentiation induction (Th0 cells), 5x10 ⁶ cells (Th1 cells) obtained by induction Th1 differentiation and 5x10 ⁶ cells obtained by inducing Th2 differentiation ( Total RNA was prepared using Trizol solution (Invitrogen's TRIsol reagent) and RNase-free water (Invitrogen's DNase / RNase Free) using Th2 cells as starting materials. Specifically, each cell is dissolved in 1 ml Trizol solution in a 1.5 mL polypropylene centrifuge tube, 200 μL of chloroform (Trizol solution: chloroform = 1: 4 ratio) is added, and a vortex mixer may be used. After shaking, the mixture was left at room temperature for 3 minutes and centrifuged at 15,000 rpm for 15 minutes at 4 ° C. using a small cooling centrifuge. Collect the upper layer (aqueous solution layer) separated by centrifugation into another 1.5 mL polypropylene centrifuge tube, add 1 μL of glycogen saturated aqueous solution and 250 μL of isopropanol, and shake well using a vortex mixer for 10 minutes at room temperature. After standing, it was centrifuged at 15,000 rpm for 15 minutes at room temperature. After centrifugation, the supernatant was removed, 1 ml of cold 70% ethanol was added, and the mixture was centrifuged at 15,000 rpm for 10 minutes at 4 ° C. After centrifugation, the supernatant was removed again, and the total RNA precipitated on the bottom of the 1.5 mL centrifuge tube was air-dried at room temperature. 20 μL of RNase-free water was added to the dried total RNA, the total RNA was dissolved by pipetting, and the mixture was incubated for 5 minutes in a water bath kept at 60 ° C. and then placed on ice.

The amounts of total RNA derived from Th0 cells, Th1 cells, and Th2 cells thus obtained were 8.9 μg (16 μL at a concentration of 557 μg / mL), 12.7 μg (concentration of 16 μL at 794 μg / mL), and 11.94 μg (746 μg / mL, respectively). Concentration of 16 μL). The RNA integrity numbers (RIA) of the total RNA derived from Th0 cells, Th1 cells, and Th2 cells obtained were 9.4, 9.7, and 9.7, respectively, satisfying the RNA purity recommended by Affymetrix. The RNA content was calculated with an absorbance 1 at 260 nm of 40 μg / mL.

GeneChip One-Cycle c DNA Synthesis Kit (One-Cycle Target Labeling and Control Reagents, # 900493) supplied by Affymetrix with 2.5 μg of total RNA derived from Th0 cells, total RNA derived from Th1 cells, or total RNA derived from Th2 cells Using the T7-Oligo (dT) primer (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG- (dT) 24-3 ') and reverse transcriptase (SuperScript II Reveres transcriptase, Invitrogen) using the method described by Affymetrix A single strand was made. Next, using First Cycle, Second-strand Master Mix supplied by Affymetrix, the first strand of the prepared Th0 cell-derived cDNA, Th1 cell-derived cDNA or Th2 cell-derived cDNA was used as a template, respectively, RNaseH, E. coli The cDNA second strand was synthesized by a second strand extension reaction using DNA polymerase and E. coli DNA ligase. The purity of the double-stranded cDNA synthesized was increased by using a purification column for double-stranded cDNA (cDNA Cleanup Spin Column, Affymetrix).

The biotin-labeled cRNA used as a probe was synthesized by using the GeneChip IVT Labeling Kit supplied by Affymetrix, using Th0 cell-derived cDNA, Th1 cell-derived cDNA or Th2 cell-derived cDNA as starting materials (template cDNA), respectively, and T7 RNA. It was prepared by a cRNA elongation reaction with polypolymerase. Each of the prepared biotin-labeled cRNAs increased the purity of the biotin-labeled cRNA by using a purification column (GeneChip IVT cRNA Cleanup Spin Column, Affymetrix) for biotin-labeled cRNA supplied by Affymetrix. The concentrations of the Th0 cell-derived labeled cRNA, Th1 fine-derived labeled cRNA, or Th2 cell-derived labeled cRNA were 502 ng / μL, 644 ng / μL, and 574 ng / μL, respectively.

Using the synthesized biotin-labeled cRNA, fragmentation was performed by the method shown by Affymetrix. The concentrations of the fragmented labeled cRNA derived from Th0 cells, the fragmented labeled cRNA derived from Th1 cells, or the fragmented labeled cRNA derived from Th2 cells were 95 ng / μL, 115 ng / μL, and 134 ng / μL, respectively. Next, hybridization was performed by the method shown by Affymetrix using GeneChip® oligonucleotide μarray (GeneChipTM, Human Genome U133 Plus 2.0 Array, # 900466) supplied by Affymetrix. As the hybridization oven, Affymetrix product number 640 (Hybridization Oven 640, P / N800138, Affymetrix) was used. For each array chip after hybridization and washing, biotinylated anti-goat-strepteptavidin antibody (P / N BA-0500, Vector Laboratories), R-Phycoerythrin Streptavidin (P / NS- 866, Molecular Probes) were used to perform the processing necessary for fluorescence development.

The fluorescence intensity was measured and analyzed using a DNA chip analyzer for each array chip that had undergone the processing necessary for fluorescent color development. In other words, fluorescence using a DNA chip analysis fluorescence scanning device (Array Scanner 428, Affymetrix) using confocal laser microscope technology supplied by Affymetrix, and expression data analysis software (GeneChip Operating Softoware, Affymetrix) The intensity was measured and analyzed for significant gene expression.

When the expression intensity of the mRNA level in the obtained Th0 cells, Th1 cells or Th2 cells was analyzed, a plurality of gene spots whose expression intensity was 100 times or more higher in Th1 cells than in Th0 cells were observed (FIG. 6). -1A). In addition, a plurality of gene spots whose expression intensity was 100 times or more higher in Th2 cells than in Th0 cells were observed (FIG. 6-1B). Furthermore, 34 types of genes whose expression intensity was 40 times or more higher in Th2 cells than that in Th1 cells were found, and 2 genes whose expression intensity was 100 times or more higher were found. Forty-seven genes were found that had 50-fold higher expression intensity in Th1 cells compared to Th2 cells, and 9 genes were found that had 100-fold higher expression intensity (Fig. 6). 2).

Th2 / Th2 ratio is 0.025 or less (40-fold difference), Th0 / Th2 ratio is about 0.1 or less (10-fold difference), and Th2raw Nine genes were selected by narrowing down the fluorescence intensity shown in FIG. Next, as a Th2-specific expression KIAA gene with a relatively high expression level, the Th1 / Th2 ratio is 0.1 or less (10-fold difference), and the Th0 / Th2 ratio is about 0.14 or less (about 7-fold difference) And 4 genes were selected by limiting the fluorescence intensity shown in Th2raw to 100 or more (Table 2). Here, the KIAA gene is a name indicating a cDNA clone discovered and produced at Kazusa DNA Laboratory.
Furthermore, as a Th1-specific expression gene with a relatively high expression level, the Th1 / Th2 ratio is 50 or more (with a 50-fold difference), the Th0 / Th1 ratio is 0.25 or less (with a 4-fold difference), and Th1raw Two genes were selected by narrowing down the fluorescence intensity shown in FIG.

The human Th1 / Th2 differentiation induction system of the present invention is a compound screening method for reducing pollen allergy, which is said to cause symptoms when the Th1 / Th2 balance is tilted to Th2, such as drugs that modulate Th1 / Th2 balance. Useful because it provides

The result of having measured the surface antigen of the human naive CD4T cell isolate | separated with the automatic magnetic cell separator (AutoMACS) with the cell analyzer (FACScalibur) is shown. The result of having analyzed the amount of cytokine accumulate | stored in the cell of the Th1 differentiation-inducing cell or Th2 differentiation-inducing cell from the human peripheral blood naive T cell using the cell analysis apparatus (FACScalibur) is shown. The result of the modification by FK506 or hydrocortisone in the human Th1 / Th2 differentiation induction system is shown. The upper row shows the results of examining the effects of various compounds in cells obtained under Th1 induction conditions, and the upper row shows the results of examining the effects of various compounds in cells obtained under Th2 induction conditions. The left column is a negative control using physiological saline instead of the compound, the middle two columns and the right two columns are the conditions of adding FK506 and hydrocortisone, respectively, to examine the effect of the compound on the Th1 induction condition and Th2 induction condition, respectively. The results are shown. The result of the modification by low concentration FK506 or cyclosporin A in the human Th1 / Th2 differentiation induction system is shown. The left side shows the results of examining the effects of various compounds in cells obtained under Th1 induction conditions, and the right side shows the results of examining the effects of various compounds in cells obtained under Th2 induction conditions. Control indicates a negative control using physiological saline instead of the compound. The result by the modification by the low concentration PD98509 in a human Th1 / Th2 differentiation induction system is shown. Control indicates a negative control using physiological saline instead of the compound. Genes expressed in human peripheral blood naive T cells (Th0 cells) and naive T cells in Th1 differentiation-inducing cells (Th1 cells) or Th2 differentiation-inducing cells (Th2 cells) Affimetrix's GeneChipTM, Human Genome U133A Plus 2.0 Array The results of analysis using are shown in a distribution map.

Claims (13)

Human naive CD4T cells are cultured under stimulation with an anti-CD3 antibody in a differentiation induction medium (Th1 differentiation induction conditions) containing IL-2, IL-12 and anti-IL-4 antibodies, or IL-2, IL- 4 and a differentiation-inducing medium (Th2 differentiation-inducing conditions) containing 4 and anti-IFNγ antibody, cultured under stimulation with anti-CD3 antibody, and further, only the anti-IL-4 antibody or anti-IFNγ antibody is removed from the differentiation-inducing medium, respectively. A method for inducing Th1 and / or Th2 differentiation of human naïve CD4 T cells, which further comprises culturing without stimulation with an anti-CD3 antibody under the culture conditions. The method according to claim 1, wherein the human naive CD4T cells are isolated from human peripheral blood. Cultivation under stimulation with anti-CD3 antibody is carried out with an anti-CD3 antibody solidified plate, and culture without stimulation with anti-CD3 antibody is carried out with a non-solidified plate without anti-CD3 antibody, Item 3. The method according to Item 1 or 2. The method according to any one of claims 1 to 3, wherein the cells are cultured for 1 to 3 days under stimulation with an anti-CD3 antibody and cultured for 4 to 6 days without stimulation with an anti-CD3 antibody. The method according to claim 4, wherein the cells are cultured for 2 days under stimulation with an anti-CD3 antibody and cultured for 5 days without stimulation with an anti-CD3 antibody. In differentiation induction medium, IL-2 is 5 to 500 U / ml, IL-12 is 0.1 to 100 ng / ml, anti-IL-4 antibody is 0.5 to 50 μg / ml, IL-4 is 0.1 to 100 ng / ml, and The method according to any one of claims 1 to 5, wherein the IFNγ antibody is contained at a concentration of 0.5 to 50 µg / ml. In differentiation induction medium, IL-2 is 50 U / ml, IL-12 is 1 ng / ml, anti-IL-4 antibody is 5 μg / ml, IL-4 is 1 ng / ml, and anti-IFNγ antibody is 5 μg / ml. The method of claim 6, comprising: The culture cycle comprising a culture under stimulation with an anti-CD3 antibody under Th2 differentiation-inducing conditions and a culture without stimulation with an anti-CD3 antibody under the differentiation-inducing conditions is continuously performed a plurality of times. The method according to any one of 7 above. After induction of human Th1 / Th2 differentiation by the method according to any one of claims 1 to 8, intracellular staining is performed using labeled antibodies against IFNγ, IL-4 and CD4, and then anti-CD4 A method of selecting only T cells using an antibody, measuring IL-4 and IFNγ intracellular staining intensities of the selected cells, and evaluating the degree of differentiation induction to Th1 or Th2 based on those intensities. The evaluation method according to claim 9, wherein the antibody labeling substance is FITC (fluorescein isothiocyanate), PE (phycoerythrin), or APC (allophycocyanin). A method for screening the influence of a test compound on Th1 and / or Th2 differentiation induction of human naive CD4 T cells, wherein the differentiation induction medium to which a predetermined amount of test compound is added is used. The screening method comprising evaluating the degree of differentiation induction to Th1 or Th2 after human Th1 / Th2 differentiation induction by the method according to one item. The screening method according to claim 11 , wherein the evaluation method according to claim 9 or 10 is used. A method for searching for an allergy suppressing compound using the screening method according to claim 11 or 12 .

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