JP2002069001A - Cell vaccine consisting mainly of dendritic cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new cell vaccine. SOLUTION: This cell vaccine consists mainly of dendritic cells obtained by culturing mononuclear cells, especially monocytes, derived from cord blood. The dendritic cells produced from human cord blood are different from those produced from marrow or peripheral blood, have stronger immunopotentiating action and are usable as a more effective cell vaccine.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a cell vaccine containing dendritic cells as a main component.

[0002]

2. Description of the Related Art Conventional tumor immunotherapy includes immunostimulation therapy for administering interleukin-2 (IL-2) and the like, lymphokine-activated killer cells (LAK) derived from lymphocytes in peripheral blood of a patient, and the like. Various immunization methods have been tried, such as adoptive immunotherapy in which tumor-infiltrating lymphocytes (TIL) are transfused or missile therapy using anti-tumor antigen monoclonal antibodies. Has not been obtained.

[0003] Recently, gene therapy for cancer has also been actively performed. In particular, cell vaccine therapy for inducing immunity against cancer by transplanting cancer cells into which a cytokine gene such as GM-CSF has been introduced, and for treating cancer has attracted attention. Macrophages and dendritic cells are known as typical antigen presenting cells. Macrophages present antigen to activated T cells and B cells (Sornasse et al. (1992)
J. Exp. Med. 175: 15-21). However, activation of helper T cells depends on antigen presentation by dendritic cells. Dendritic cells are thought to capture antigens in peripheral blood, migrate to lymphoid tissues, and eventually mature in the paracortical T cell portion of lymph nodes. Macrophages and dendritic cells can present antigen to already activated T cells.
Only mature dendritic cells have unactivated naＴve T
It is reported that cells can be sensitized (Mehta-D
amani et al. (1994) J. Immunology 153: 996).

[0004] Immature dendritic cells generally have a high ability to take in foreign substances serving as antigens into cells, a high ability to digest foreign substances, and a low antigen presenting ability. Conversely, more differentiated mature dendritic cells are characterized by low antigen uptake ability and high antigen presentation ability (Ebner et al. (1998) Immunobiology 19
8: 568). From this, in vivo, immature dendritic cells take up foreign substances, digest them in the cells, and mature dendritic cells differentiated by the stimulation present a part of the digested foreign substances outside the cells, It is thought to convey specific antigenic species to lymphocytes.

In vivo, dendritic cells are derived from bone marrow hematopoietic stem cells. Dendritic cell precursors and immature dendritic cells are present in blood and lymph, and fully mature dendritic cells are present in the spleen and lymph nodes. In playing a role in antigen presentation, dendritic cells overexpress MHC class I and class II proteins. In recent years, dendritic cells have become possible to prepare in large quantities in vitro
(Romani et al. (1994) J. Exp. Med. 180: 83-93). Undifferentiated CD34-positive cells derived from bone marrow cells or cord blood, or peripheral blood monocytes are progenitor cells of dendritic cells, and granulocyte-macrophage colony stimulating factor (G
M-CSF), interleukin-4 (IL-4), tumor necrosis factor (TN
F-α) stimulation is known to induce differentiation into dendritic cells.

When peripheral blood monocytes are stimulated with GM-CSF and IL-4, they can differentiate into immature dendritic cells. Add TN
Stimulation with F-α can differentiate into mature dendritic cells. Progenitor cells of dendritic cells are GM-CSF, IL-4,
TNF-α can also differentiate into mature dendritic cells by acting simultaneously.

In general, cancer cells of cancer patients are expected to be degenerated by administration of an anticancer drug, and the degenerated cancer cells are expected to be completely eliminated by immunization of the patient. However, in many cases, administration of an anticancer drug to a patient also reduces the function of its own immunocompetent cells, and as a result, phenomena such as recurrence and metastasis are often observed. In order to solve such a situation, development of adjuvant therapy for immune cells is expected. These results were obtained by the present inventors in Japanese Patent Laid-Open Publication No. 2000-143534.
It is described in.

[0008]

An object of the present invention is to provide a more effective cell vaccine containing dendritic cells as a main component, which is used in the above-mentioned treatment method.

[0009]

As a method for preparing a large amount of dendritic cells, which are antigen-presenting cells, differentiation is induced by culturing from hematopoietic undifferentiated cells of bone marrow or cord blood or peripheral blood monocytes. Coming technologies have been reported. Efficient antigen presentation of dendritic cells depends on the major histocompatibility complex (MH) of the presenting dendritic cells.
It is known that C) and MHC such as T lymphocytes to be presented need to be the same. Therefore, attempts have been made to isolate dendritic cells of the same type, which are autologous or have substantially the same MHC, from the blood and pulse antigen peptides or the like to present the target antigen.

In order to obtain dendritic cells having a higher immune activating function, the present inventors examined various sources and induced differentiation under various conditions to obtain more desirable dendritic cells. As a result of our hard work, we found the following.

That is, it has been found that cord blood-derived mononuclear cells, in particular, dendritic cells that have been induced to differentiate from monocytes in vitro have a strong antigen-presenting ability, and are cell preparations that can be used as effective cell vaccines. Was found. According to the present invention, immature dendritic cells obtained by culturing cord blood-derived monocytes with the addition of GM-CSF and IL-4 were similarly induced from peripheral blood-derived monocyte cells. The cells were found to have stronger antigen-presenting ability than immature dendritic cells. Further, according to the present invention, monocytes derived from cord blood are GM-CSF, IL-4
And simultaneously stimulated and cultured with TNF-α from the beginning to induce differentiation into mature dendritic cells, but similarly, peripheral blood-derived monocytes, GM-CSF, IL-4 and TNF-α It is possible to produce dendritic cells having higher antigen-presenting ability than mature dendritic cells that have been added, cultured and differentiated.

Conventionally, GM-CSF and IL were obtained from monocytes derived from peripheral blood.
Immature dendritic cells obtained by culturing with the addition of
DR (low), CD1a (high), CD14 (-), CD25 (-), CD83
(-), Shows the expression pattern of cell surface markers such as CD86 (-), mature dendritic cells, extending the projections characteristic of dendritic cells, exhibit dendritic, HLA-DR (high), CD1a (low),
CD14 (-), CD25 (+), CD83 (+), shows the expression pattern of surface markers such as CD86 (high) (Yanagihara et al. (1998)
J. Immunol. 161, 3096).

However, the immature dendritic cells obtained by culturing monocytes derived from cord blood with addition of GM-CSF and IL-4 are HLA-DR (high), CD86. (high) and the like.

The cell vaccine of the present invention containing dendritic cells as an active ingredient activates autologous lymphocytes specifically in MHC and induces lymphocytes capable of specifically attacking cancer cells. Furthermore, the cell vaccine containing the dendritic cells of the present invention as an active ingredient was found to activate lymphocytes non-specifically to MHC, and was found to be usable not only as an autologous vaccine but also as a homologous vaccine. . The vaccine can also be administered directly into the body, but in vitro,
It can also be used to generate antitumor lymphocytes for cancer cells. In this case, it has been found that lymphocytes can be derived from autologous cells, but can be efficiently used even from the same species.

The present invention provides a method for culturing cells composed of cells derived from cord blood in the presence of a differentiation inducer.
A cell vaccine mainly composed of dendritic cells characterized by high expression of MHC class II and CD86 molecules obtained by differentiation, and a differentiation inducer comprising granulocyte macrophage colony stimulating factor (GM-CSF), Leukin-4 (IL-4)
Or GM-CSF, IL-4, tumor necrosis factor (TN
F-α).

In the present invention, the high expression means CD86, umbilical cord blood monocyte-derived cells as shown in Examples and FIG.
This means that HLA-DR antigen is expressed at the same level as mature dendritic cells. That is, high expression is equivalent to expression at the same level as mature dendritic cells. As another rule, a high expression is defined as a fluorescence intensity detected under the conditions shown in the examples that is 50 times or more that of the control. More preferably, it means high expression of 100 times or more.

The mononuclear cell generally refers to a nucleated cell having a nucleus in blood cells other than multinucleated cells such as granulocytes and megakaryocytes. For example, it is composed of lymphocytes, monocytes, undifferentiated cells and the like.

By culturing monocyte cells derived from cord blood, which are precursor cells of dendritic cells, functional dendritic cells are
It is provided in large quantities as an active ingredient of a cancer vaccine. Differentiated dendritic cells can be used as a source of antigen presenting cells for use in in vitro assays and immunomodulatory treatments, particularly for sensitizing naive T cells.

As a source of dendritic cell precursor cells, monocytes derived from cord blood are used. Umbilical cord blood can be obtained with a syringe. It is desirable that monocytes derived from cord blood, which are precursor cells of dendritic cells, be separated and purified from the collected blood. Any method of separating nucleated cells from red blood cells can be employed. Ficoll fractionation or Ficoll-
Methods utilizing Ficoll-Paque density gradients or elution are commonly used. Alternatively, blood cells may be dissolved in a solution that selectively lyses adult red blood cells, such as ammonium chloride-potassium (ammonium chloride-pota).
ssium), ammonium oxalate
Etc. and can be separated.

[0020] Further separations can be performed utilizing appropriate surface antigens. For example, an antibody against the CD14 antigen can be used. Separation methods include magnetic beads, antibody-immobilized beads,
It can be carried out using a column and a flow cytometer packed with these.

For the culture for inducing differentiation of dendritic cells, RPMI-164 was used.
A suitable commercially available medium such as a 0 medium, Dulbecco's modified Eagle's medium (DMEM), or Iscove's medium (IMDM) can be used. Clinically, X-VIVO15 medium (Biowittaker) and the like are more preferable. Serum is about 5-20% bovine serum, fetal calf serum,
Alternatively, human serum or the like can be used. It is preferably used without serum, but if necessary, bovine albumin (BSA), human albumin (HSA) and the like can be added. Antibiotics, antibodies, pyruvate (0.1-5mM), glutamine as needed
(0.5-5 mM) and 2-mercaptoethanol (10-100 μM). A differentiation inducer is added to these media, and the cells are cultured at about 37 ° C. in an atmosphere of about 5% carbon dioxide for about 5 to 21 days. Culture for about 7 to 14 days is desirable. Culture temperature (34 to 38 ° C), gas mixture ratio (carbon dioxide 2 to 10
%, Or nitrogen gas or oxygen gas can be appropriately mixed. ) Can be performed by setting appropriate conditions.

In order to induce the differentiation of dendritic cells into dendritic cells from a cell group containing progenitor cells, it is necessary to use an appropriate inducer. The inducer can be selected from cytokines and used. Appropriate cytokines may be used, but especially GM-CSF, IL-4, stem cell factor (SCF), interleukin-13 (IL-13), TNF-α in the concentration range of about 1 to 1000 ng / ml. , Flt3-Ligand, etc. can be used efficiently. Interleukin-1 (IL-1), Interleukin-2 (IL-2), Interleukin-3 (IL-3)
Additions such as can also be used. Desirably, a combination of GM-CSF and IL-4, a combination of GM-CSF, IL-4, and TNF-α is more efficient. In a culture system using these combinations, antigen-presenting cells can be efficiently produced.

As the cytokine used in the culture, there may be used a factor derived from a heterologous animal such as a mouse, but a human-derived factor is preferable. Many of these cytokines are commercially available from a number of companies such as Boehringer and PeproTech (treated by Immune Biological Laboratories), and they can be used equally. The factors used may be of natural origin or synthetic, for example those prepared by genetic recombination. Mononuclear cells, lymphocytes, or culture supernatants of certain cell lines can also be used as a source of cytokines.

Various methods for detecting the antigen-presenting activity of dendritic cells are known, and these can be used. For example, as a mixed lymphocyte reaction test, the ability of cells to be measured by the activity of promoting the proliferation of isolated T cells stimulated with a sub-optimal concentration of anti-CD3 antibody can be measured (Thomas (1994). ) J. Immunol. 153:
4016-4028).

The group of dendritic cells during or after culture is
It can be used as an antigen presenting cell to stimulate T cells in vivo or in vitro. The protein antigen or its peptide is incorporated into cells and displayed on the cell surface after modification, or is directly displayed on the cell surface. Antigenic peptides usually consist of about 6 to 20 amino acids in length, more usually about 10 to 18 amino acids.
This peptide has sequences derived from a wide variety of different proteins. In many cases, it is desirable to use a peptide that acts as a T cell antigenic determinant, usually the major antigenic determinant.

Since the cells containing dendritic cells, which are the active ingredients of the vaccine produced according to the present invention, are inoculated as a therapeutic vaccine into the human body, it is safer if the cell proliferation is eliminated. It is known that cord blood-derived monocytes have extremely reduced proliferative capacity by inducing differentiation, but as a cell vaccine, heat treatment, radiation treatment, or mitomycin C treatment for safer use. The treatment can be performed under such a condition that the protein of the cancer cells is denatured, while retaining the function as a vaccine. For example, when using X-ray irradiation, a flask containing dendritic cells is placed under the tube of the X-ray irradiator, and the total radiation dose is 10
Irradiate at 00-3300 Rad. Mitomycin C treatment method,
For example, dendritic cells are suspended at a density of 1-3 × 10 ⁷ cells / ml, and mitomycin C25-50 μm / ml of cell suspension is used.
g at 37 ° C for 30-60 minutes. The method of treating cells by heat is, for example, to reduce the concentration of living cells to 1 × 10 ⁷
The centrifuge tube containing the cell suspension prepared at 50/65 ° C.
For 20 minutes.

[0027] The cell vaccine of the present invention comprises leukemia, liver cancer,
It can be used for various cancers such as lung cancer, stomach cancer, and colon cancer, and infectious diseases caused by various viruses and bacteria. The dose of the cell vaccine of the present invention varies depending on the age, weight, sex, type of cancer, degree of progression of the cancer, symptoms, etc. of the patient, and cannot be unconditionally determined. The same amount as described above may be administered to the patient. For example, dosing once a week, 1 x 10 per patient
A protocol is to administer ⁷ cells over 10 weeks. The dendritic cell vaccine according to the present invention can be administered to a large number of MHC-compatible patients of the same type by the development of a cord blood bank.

In preparing dendritic cells from dendritic cell progenitor cells, an antigen peptide may be additionally added and used as presenting cells for the antigen. The addition may be performed at the time of culture for inducing differentiation, or may be performed after differentiation into dendritic cells. Cancer patients can be protected against viruses and bacteria, such as cytomegalovirus, because their immunity has been reduced by anticancer drugs. Dendritic cells are stimulated with the antigen by placing the cells in a physiologically acceptable buffer containing the antigen at a concentration of at least about 0.1 μM to 1 mM. Peptide antigens can usually be effective at lower concentrations than whole protein antigens or cell lysates. Antigenic peptides usually consist of about 6 to 20 amino acids in length, more usually about 10 to 18 amino acids.
Consists of amino acids. The dendritic cells are typically 37
Incubation with the antigen at a temperature sufficient for the antigen to bind to the cells can be performed. Peptide antigens are usually incubated for at least about 1 hour, and 6 hours or more. For intact protein antigens, it is usually desirable to incubate for at least about 3 hours, and for about 12 hours or more.

The dendritic cells stimulated with the antigen or the membrane components thereof may be used as an immunoadsorbent for obtaining antigen-specific T cells. It is desirable that the cell vaccine of the present invention be contained in a container effective for moving and storing cells.
For example, a cryovial, a blood bag for freezing, a blood bag for blood transfusion, and the like are useful. By adding dimethyl sulfoxide or the like and preparing a preservation solution for freezing, it is also possible to freeze and preserve. By using a blood bag capable of gas phase exchange for culturing, from closed culture, washing,
It can be efficiently prepared and used as a cell preparation such as recovery and storage.

[0030]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 Preparation of dendritic cells from mononuclear cells in cord blood
A method for preparing dendritic cells from mononuclear cells in cord blood is described below. Umbilical cord blood was collected from the placenta of a healthy subject enucleated after normal delivery, and the mononuclear cell layer was collected by Ficoll. The mononuclear cells obtained by Ficoll were further collected as monocytes using a culture dish (Falcon). The monocytes obtained are 37
10% fetal bovine serum (FBS: In
tergen) and antimycotic-antibiotics (Gibco BRL)
7 days using RPMI-1640 medium supplemented with
The cells were cultured for 11 days and differentiated into immature or mature dendritic cells. 10 ng / ml GM-CSF and 50 ng / m for 7 days after the start of culture
After addition of lIL-4, 50 ng / ml TNF-α for culture after 7 days
Was added. The medium was replaced as needed. In this way, dendritic cells were prepared from monocytes derived from cord blood of healthy subjects.

[0031]

Example 2 Analysis of Surface Antigen Detection of the surface antigen of the dendritic cells prepared in Example 1 was performed using a flow cytometer. Cells before and after differentiation were measured. The cells to be measured were analyzed using mouse normal serum (DAKO
Add the desired antibody to phosphate saline containing
Stained at ℃ for 30 minutes. The stained antibody is PE-labeled, or
FITC-labeled CD1a (Coulter), CD4, CD11c, CD13,
CD14, CD19, CD33, CD34 (HPCA-1), CD45, HLA-DR (Beckton-Dickinson), CD40, CD86 (Pharmingen
And CD83 (Immunotech). Isotype matched antibodies were used as negative control antibodies. After washing the stained cells, use FACScalibur (Beckt
on-Dickinson). FIG. 1 shows the results of surface antigen analysis using a flow cytometer. In each figure, the vertical axis indicates the number of cells, and the horizontal axis indicates the amount of each antigen detected by the fluorescently labeled antibody. Immature dendritic cells cultured in IL-4 and GM-CSF have higher expression of CD86 and HLA-DR surface markers than immature dendritic cells derived from conventional peripheral blood-derived monocytes, Mature dendritic cells induced to differentiate with IL-4, GM-CSF, and TNF-α have CD86,
It was confirmed that HLA-DR was a high-expressing cell population.

[0032]

Example 3 Dendritic Cell Function Analysis 1 The antigen-presenting ability of immature dendritic cells derived from cord blood monocytes prepared in Example 1 was measured and evaluated in vitro by a mixed lymphocyte reaction test. On the other hand, a mononuclear cell layer was collected from peripheral blood of a healthy subject by Ficoll and cultured in a culture flask (Falc
(on company) to remove adherent cells.
T lymphocytes were isolated by the E rosette method. 1 × 10 ⁵ isolated T lymphocytes and 10 ³ , 5 × 10 ³ , 10 ⁴ immature dendritic cells irradiated with 15 to 30 Gy were separated by 96
It was spread on a well plate and cultured for 4 days. Thereafter, tritium-labeled thymidine was incorporated, and the proliferation of lymphocytes was measured. The measurement of tritium-labeled thymidine is performed using the uptake culture18.
After hours, Micro Beta TRILUX1450β-scintilation coun
ter (Wallac). FIG. 2 is a line graph showing the antigen-presenting ability of immature dendritic cells of cord blood-derived monocytes in a lymphocyte mixing test. The vertical axis indicates radioactivity as the amount of tritium-labeled thymidine incorporated. The horizontal axis indicates the number of immature dendritic cells. As a control group, monocytes derived from peripheral blood
Immature dendritic cells obtained by culturing with IL-4 and GM-CSF for 7 days and T
The values of uptake of tritium-labeled thymidine by mixed culture of lymphocytes are shown.

As a result of the main culture reaction test, immature dendritic cells obtained by inducing differentiation from cord blood-derived monocytes were significantly more lymphocytes than non-HLA-matched lymphocytes. Promoted sphere proliferation. It was confirmed that the cell vaccine obtained according to the method of the present invention exhibited higher antigen-presenting ability than immature dendritic cells derived from peripheral blood-derived monocytes. TN
The immature dendritic cells to which F-α was not added may have properties as mature dendritic cells based on the analysis of the surface markers performed in Example 2, and the efficiency of antigen uptake is high. Can be considered as an effective cell vaccine. From this, the dendritic cell vaccine according to the present invention,
The conventional conditions for inducing the differentiation of immature dendritic cells of peripheral blood monocytes showed higher efficacy as a cell vaccine. Furthermore, it was shown that not only autologous cells but also dendritic cells with inconsistent MHC activate lymphocytes.

[0034]

Example 4 The ability of mature dendritic cells derived from cord blood monocytes prepared in Example 1 to present antigen was measured and evaluated in vitro by a lymphocyte mixed culture reaction test. A mononuclear cell layer is collected from peripheral blood of a healthy subject by Ficoll, and
(Falcon) to remove adherent cells by culturing. T lymphocytes were isolated by the E rosette method. 1 × 10 ⁵ isolated T lymphocytes and 10 ³ , 5 × 10 ³ , and 10 ⁴ mature dendritic cells irradiated with 15 to 30 Gy were spread on a 96-well plate and cultured for 4 days. Thereafter, tritium-labeled thymidine was incorporated, and the proliferation of lymphocytes was measured. Tritium-labeled thymidine was measured 18 hours after uptake of the culture using Micro Beta TRILUX1450β-scintilation.
The counter (Wallac) was used. FIG. 3 is a line graph showing the antigen-presenting ability of mature dendritic cells of cord blood-derived monocytes in a lymphocyte mixing test. The vertical axis indicates radioactivity as the amount of tritium-labeled thymidine incorporated. The horizontal axis indicates the number of mature dendritic cells. The control group consisted of mature dendritic cells obtained by culturing monocytes derived from peripheral blood with IL-4 and GM-CSF for 7 days and culturing only TNF-α after 7 days of culture, as in Example 1. The values of tritium-labeled thymidine incorporation by mixed culture of T lymphocytes are shown.

As a result of the main culture reaction test, the mature dendritic cells obtained by inducing the differentiation significantly promoted the proliferation of the lymphocytes even with respect to the lymphocytes inconsistent with HLA as compared with the control group. From this, the dendritic cell vaccine according to the present invention,
Under the conventional conditions for inducing differentiation of mature dendritic cells of peripheral blood monocytes, it was shown that cell vaccines are more effective. Furthermore, it was shown that not only autologous cells but also dendritic cells with inconsistent MHC activate lymphocytes.

[0036]

Example 5 The cord blood-derived dendritic cells prepared in the above example were placed in a blood pack for blood transfusion, irradiated with X-rays, and treated to prevent cell proliferation. X-ray irradiation was performed at 2000 Rad. By performing such treatment, a cell vaccine was finally produced.

[0037]

According to the present invention, dendritic cells having high antigen-presenting ability can be prepared from mononuclear cells, particularly monocytes, present in cord blood. Since these dendritic cells can efficiently activate lymphocytes, they are effective as an excellent cell vaccine for treating and preventing cancer and infectious diseases.

[Brief description of the drawings]

FIG. 1 is a histogram showing the results of analysis of surface antigens on dendritic cells by flow cytometry in Example 2. In each figure, the vertical axis indicates the number of cells, and the horizontal axis indicates the amount of each antigen detected by the fluorescently labeled antibody. In the figure, the thin line is a control to which no antibody was added, and the thick line is a control to which an anti-CD86 antibody or an anti-HLA-DR antibody was added.

FIG. 2 is a line graph showing the antigen-presenting ability of immature dendritic cells by the lymphocyte mixing test of Example 3. The vertical axis indicates radioactivity as the amount of tritium-labeled thymidine incorporated. The horizontal axis indicates the number of immature dendritic cells.

FIG. 3 is a line graph showing the antigen presenting ability of mature dendritic cells according to the lymphocyte mixing test of Example 4. The vertical axis indicates radioactivity as the amount of tritium-labeled thymidine incorporated. The horizontal axis indicates the number of mature dendritic cells.

Continued on the front page F term (reference) 4B065 AA94X BB19 BB34 CA45 4C085 AA03 BB01 CC03 CC26 DD23 EE01 4C087 AA01 AA02 AA03 BB34 BB59 CA04 DA31 NA14 ZB26

Claims (7)

[Claims] 1. A cell vaccine mainly comprising dendritic cells obtained by culturing cord blood-derived mononuclear cells in the presence of a differentiation-inducing agent. 2. The cell vaccine according to claim 1, wherein the mononuclear cells are monocytes. 3. The cell vaccine according to claim 1, wherein the dendritic cells are immature dendritic cells. 4. The cell vaccine according to claim 1, wherein the differentiation inducer is a combination of granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). 5. The cell vaccine according to claim 3, wherein the dendritic cells highly express MHC class II and CD86 molecules. 6. The method according to claim 1, wherein the dendritic cells are mature dendritic cells.
Or the cell vaccine according to 2. 7. The differentiation inducer is a combination of granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor (TNF-α).
7. The cell vaccine according to 1, 2, or 6.