JPWO2012160702A1 - Dendritic cell differentiation and culture methods - Google Patents

Abstract

[PROBLEMS] To provide a method for efficiently and reliably performing DC differentiation and culture in a short period in order to efficiently realize DC immunotherapy. It provides technologies for obtaining mature DCs, further spreads DC immunotherapy, and greatly contributes to the prevention and treatment of viral infections such as cancer and AIDS. The present invention relates to a method for differentiating and culturing human peripheral blood mononuclear cells into mature dendritic cells. Human peripheral blood mononuclear cells isolated from peripheral blood collected from humans are cultured in the presence of cytokines and then matured by stimulating with a maturation stimulator to produce mature dendritic cells with immunostimulatory activity . The cytokine to be used is preferably IL-4, GM-CSF, or IFN-β, and the maturation stimulating substances are preferably TNF-α and PGE2.

Description

  The present invention relates to a method for differentiating and culturing dendritic cells, which are a kind of antigen-presenting cells, and particularly to a method capable of efficiently differentiating and culturing dendritic cells in a short period of time outside the body.

  Traditionally, researchers have made various efforts to combat serious life-threatening diseases. Among them, a method for dealing with a disease by utilizing an immune function inherent in living organisms instead of using a drug has been researched and developed.

In such research and development, a disease treatment method using dendritic cells (hereinafter simply referred to as “DC”) has attracted attention. DC, as its name suggests, is a type of immune cell that has branched processes, and is a cell that has antigen-presenting ability.
This DC causes the adaptive strain in the body to remember the antigen by processing and presenting the antigen protein to T cells. In other words, DC degrades the protein of the captured foreign antigen in a limited manner and presents the produced peptide on the cell surface as a complex with MHC. The naive T cell in the bloodstream recognizes and activates the antigen-specific immune response.

  Thus, various methods for treating diseases using DCs that play a central role in the immune response have been studied. For example, “adoptive immunotherapy (DC immunotherapy)” using DC has been developed in the treatment of cancer, which is a serious life-threatening disease. This is also referred to as dendritic cell vaccine therapy, and is a method in which a tumor antigen or the like is added to DCs, which are cells that stimulate T cells, and administered to a patient.

  The DC immunotherapy expected to be used for cancer treatment is also expected to be used for preventive treatment of acquired immune deficiency syndrome (AIDS), a type of viral infection. AIDS is caused by infection with human immunodeficiency virus (hereinafter referred to as “HIV virus”). This is a disease that is caused by opportunistic infection with other pathogens due to the extremely low immune capacity resulting from the destruction of helper T cells. In AIDS-infected individuals, there is a decrease in DC from before onset and functional abnormalities associated with this decrease. For this reason, DC immunotherapy using DC is expected to contribute to preventive treatment of AIDS.

  Immature DC is excellent in antigen uptake ability, and becomes mature DC upon stimulation with inflammatory cytokines (eg, TNF, IL-1 etc.) and bacteria. This mature DC acquires high expression of T cell stimulating molecules and ability to migrate to regional lymphocytes. DCs that have migrated to the regional lymph nodes then contact the specific T cells that have the corresponding T cell receptor among the T cells that are colonized and circulating, and stimulate these specific T cells. . The stimulated specific T cells are rapidly activated to proliferate and differentiate. Furthermore, CD40 ligand induced on activated T cells binds to CD40 on DC and stimulates DC in reverse, and this stimulation causes various cytokines including chemokines (cell proliferation, differentiation and functional expression). The immune response is further enhanced.

  Thus, although an immune response is performed, it is known that HIV-1 virus establishes systemic infection by utilizing immature DC migration to lymph nodes and T cell stimulation ability. In other words, immature DCs support HIV-1 proliferation in the early stages of infection. Moreover, DC is infected with HIV-1 virus and produces virus, depending on conditions such as differentiation stage. Furthermore, HIV-1 envelope spike gp120 molecule binds to DC-SIGN, a protein involved in cell adhesion on the DC surface, without HIV-1 virus infection in the invading site mucosa. -1 virus allows migration to lymph nodes rich in T cells.

  In order to avoid such a situation, it is necessary to transplant mature DC instead of transplanting immature DC at the early stage of differentiation. In addition, immature DC has an antigen uptake ability, but mature DC does not have an antigen uptake ability, and therefore, it is necessary to artificially incorporate the antigen into the DC at the final stage of maturation. Furthermore, in order to perform treatment by transplanting mature DC sensitized with antigen in this manner, it is necessary to culture and adjust mature DC in large quantities. Thus, in order to efficiently realize DC immunotherapy, it is necessary to efficiently and reliably perform DC differentiation and culture.

Under such circumstances, a method for differentiating and culturing DC has been proposed. This differentiation / culture method is as shown below. A simple scheme is shown in FIG. First, human peripheral blood-derived monocytes were purified from human peripheral blood mononuclear cells (hereinafter simply referred to as “PBMC”), and the purified human peripheral blood-derived monocytes were isolated in the presence of IL-4 and GM-CSF. Incubate about a day. Next, TNF or the like is added, and mature DC is finally created in about 2 days (see, for example, Non-Patent Document 1).
In addition, as shown in Non-Patent Document 2 and Non-Patent Document 3, the inventors have proposed a method for differentiating and culturing DC. According to these methods, for example, in Non-Patent Document 3, IFN-β A method for differentiating and culturing DC in 3 days by using IL-4 has been proposed (see, for example, Non-Patent Document 2 and Non-Patent Document 3).

J. Virol., Vol. 77, 8719-8723 (2003); Atsushi Yoshida, Reiko Tanaka, Tsutomu Murakami, Yoshiaki Takahashi, Yoshio Koyanagi, Masataka Nakamura, Mamoru Ito, Naoki Yamamoto, Yuetsu Tanaka Immunological Society General Meeting / Academic Meeting Record: “Generation of mature dendritic cells with unique phenotype and function by in vitro short-term culture of human monocytes in the presence of IL-4, GM-CSF and IFN-β” Vol .39, Page.34; 2009.11.10, Yugo Tanaka Basic Research on Prevention and Treatment of HIV Infection and AIDS Development 2007 Summary / Shared Research Report: Page.15-18; 2008 “Example of 3-day culture in the presence of IFN-β and IL-4”, Yugo Tanaka

Thus, the above non-patent document discloses that a system that induces a human immune response in a mouse individual can induce a protective immune response against HIV-1 antigen using cultured mature DC. In the method, DC differentiation / culture requires about one week.
In addition, although not as much as Non-Patent Document 1, Non-Patent Document 2 and Non-Patent Document 3 also require time and labor for DC differentiation and culture. In other words, the above method has a problem that it takes time and effort to obtain a necessary amount of mature DC.

  In view of the above problems, an object of the present invention is to provide a method for efficiently and reliably performing DC differentiation and culture in a short time in order to efficiently realize DC immunotherapy. It is intended to further contribute to the prevention and treatment of viral infections such as cancer and AIDS by further providing DC immunotherapy by providing technology for obtaining the necessary amount of mature DC.

  The above-mentioned problem is a method for differentiating and culturing human peripheral blood mononuclear cells into mature dendritic cells according to the method for differentiating and culturing dendritic cells according to the present invention, which is isolated from peripheral blood collected from humans. It is solved by culturing human peripheral blood mononuclear cells in the presence of cytokines and then maturing by stimulating with a mature stimulating substance to produce mature dendritic cells with immunostimulatory activity.

Specifically, the cytokine is IL-4, GM-CSF, or IFN-β.
As a more specific combination, it is preferable that the cytokine is IL-4, GM-CSF, and IFN-β, and the maturation stimulating substance is TNF-α and PGE2.

Thus, in the present invention, the cytokine is IL-4, GM-CSF, IFN-β, the culture period in the presence of the cytokine is 1 day, and the subsequent maturation stimulating substance The maturity period is one day.
That is, mature DC having immunostimulatory activity can be obtained in a total of 2 days.

In the present invention, generally, a known method can differentiate and culture mature DC that takes 7 days in 2 days.
Further, it should be noted that, in the present invention, as shown in claim 1, “human peripheral blood mononuclear cells separated from peripheral blood collected from humans” can be cultured as they are. That is, usually, in a known method, monocytes are purified from peripheral blood mononuclear cells (PBMC) by techniques such as antibody magnets and plastic adhesion, and the purified monocytes are differentiated and cultured. However, the present invention can differentiate and culture mature DCs having immunostimulatory activity from unpurified monocytes, that is, peripheral blood mononuclear cells (PBMCs) themselves. In other words, it is not necessary to purify monocytes from peripheral blood mononuclear cells as in the known art. For this reason, the step for purification can be eliminated, and mature DC having immunostimulatory activity can be efficiently obtained in a shorter period of time.

  According to the method for differentiating and culturing dendritic cells according to the present invention, dendritic cells that are a kind of antigen-presenting cells can be differentiated and cultured in vitro in mature DCs that have an immunostimulatory activity efficiently in a short period of time. . That is, specifically, with known methods, it is possible to perform differentiation and culture of mature DC for 7 days in 2 days. Unlike known methods, mature DCs can be differentiated and cultured from monocytes that are not purified, that is, peripheral blood mononuclear cells (PBMCs) themselves. Therefore, it is possible to provide a technique for obtaining mature DC having a necessary amount of immunostimulatory activity from the collected PBMC in a high efficiency and in a short period of time, further spreading DC immunotherapy, and viruses such as cancer or AIDS. It can greatly contribute to preventive treatment of infectious diseases.

It is a schematic diagram which shows the usefulness of a dendritic cell. It is a schematic diagram which shows the differentiation / culture method of a dendritic cell. It is a histogram which shows the surface marker (CD11c, CD83, CD86) expression of mature DC. It is a histogram which shows the alloCD4 + T cell stimulation activity ability. It is a histogram which shows the inducibility of the CMV antigen specific CD8 + T cell by PBMC-4 (beta) G-DC. It is a histogram which shows the inducibility of influenza virus Flu-M1 (58-66) -specific CD8 + T cell of PBMC-4bGDC. It is a schematic diagram which shows the differentiation / culture method of the conventional dendritic cell.

  Hereinafter, an embodiment of the present invention will be described. Note that the individual tools described below do not limit the present invention, and various modifications can be made in accordance with the spirit of the present invention.

  Below, an experimental system and a result are demonstrated. FIG. 1 briefly shows the usefulness of dendritic cells (hereinafter simply referred to as “DC”). In DC immunotherapy, monocytes purified from PBMC collected from a patient are differentiated and cultured into DC. The antigen is sensitized, washed and collected, and then administered to the patient. This was intended to induce a CTL response and to ward off HIV and cancer.

As its name suggests, DC is an immune cell having dendrites like branches and exhibits antigen-presenting ability. Usually, in the body, DC captures a recognized foreign substance and moves to a lymphoid tissue such as a lymph node or spleen via a lymphatic vessel or blood vessel. DCs that have migrated to the lymphoid tissues have a limited degradation of proteins in the captured foreign body (foreign antigen), and the resulting peptides are complexed with MHC molecules (human: HLA molecules) to the cell surface. To present.
Naive T cells circulate throughout the body according to the bloodstream and come into contact with DCs in lymphoid tissues. Then, T cells having a specific receptor for the antigen presented on the DC are recognized and activated to become a mobile effector T cell. Among these effector T cells, CD4 + T cells activate B cells as helper T cells and are involved in antibody production. CD8 + T cells are cytotoxic T cells (killer T cells, hereinafter simply referred to as “CTL”). As such, it identifies self and non-self, kills and removes cells that are harmful to the body.
On the other hand, to combat HIV, the human immune system induces the acquired immune system following interferon and natural killer cells. In the acute infection stage, it has been confirmed that the viral load decreases with the CTL response, and it is suggested that this CTL response is an important factor for HIV countermeasures. Therefore, DC immunotherapy aims to efficiently differentiate and culture DCs, and as a result, mature DCs are sensitized to antigens and administered to patients to induce acquired immune system to counter HIV. Make it possible.

As the antigen to be sensitized, inactive HIV-1 particles and HIV-1 peptides are used for HIV-1 infection. In addition, when the disease is cancer, a cancer cell extract, a cancer-related antigen, a cancer virus antigen, or the like is used.
Conventionally, monocytes purified from PBMCs collected from patients were differentiated and cultured into DCs, so it took considerable time for DC differentiation and culture. In the present invention, monocytes were collected from patients. Since DC can be differentiated and cultured directly from the PBMC, DC can be efficiently differentiated and cultured in a short time.
Hereinafter, DC differentiation / culture methods and effects will be described.

1. Differentiation / Culture Method FIG. 2 shows a DC differentiation / culture method according to the present invention. PBMCs separated by specific gravity centrifugation were obtained from fresh peripheral blood. This PBMC is composed of IL-4 (Interleukin-4) 20 ng / ml, GM-CSF (Granulocyte Macrophage colony-stimulating Factor) 50 ng / ml, IFN-β (interferon-β : Interferon β) 1000 U / ml was added to the medium and cultured for 1 day. As the medium, RPMI-1640 medium was used. Subsequently, TNF-α (Tumor Necrosis Factor-α: tumor necrosis factor) 20 ng / ml and PGE2 (Prostaglandin 2: Prostaglandin 2) 1 mM were added to the medium cultured for 1 day, and further cultured for 1 day.
That is, the culture was performed for 2 days in total.
The mature DC thus obtained is hereinafter simply referred to as “PBMC-4βG-DC”. In addition, mature DC (hereinafter simply referred to as “Mo-G4-LPS DC”) that has been differentiated and matured by a known method for comparison is obtained by using GM- After culturing in a medium supplemented with CSF + IL-4 for 5 days, LPS, poly I: C, IFN-b and cocktails (TNF-α, IL-1b, IL-6) etc. were added for another 2 days, A total of 7 days of culture was obtained.

2. Verification and Results (1) Expression system of mature myeloid DC The results in flow cytometry are shown in FIG. As shown in FIG. 3, DC cultured with IL-4, GM-CSF, and IFN-β added to the medium showed expression of CD11c, which is a surface marker of myeloid mature DC.
We also showed the expression of CD83 and CD86, which are expression systems of myeloid mature DC.
CD83 + and CD86 + cells exceeded 80% in both the TNF-α and PGE2 stimulated and non-stimulated groups, and the particularly stimulated group showed over 90% high expression. Thus, the group stimulated with TNF-α and PGE2 showed higher expression of these phenotypes than the group without stimulation.

(2) Allo CD4 + T cell stimulation activity
Naive T cells with different HLA types were labeled with CSFE and mixed with DCs obtained from other donors at a DC: T cell ratio of 1:20. One week later, the degree of CFSE dilution was analyzed by flow cytometry. The results are shown in FIG. Since CSFE becomes 1/2 when the cell divides once, the cells with weak CSFE are proliferating cells. Mo-G4-LPS DC differentiated and purified by a conventional method was differentiated from purified monocytes in 7 days. PBMC-4βG-DC was differentiated in 2 days.
As shown in FIG. 4, the growth of proliferating cells was shown in both Mo-G4-LPS DC and PBMC-4βG-DC, and the proportion of PBMC-4βG-DC was higher than that in Mo-G4-LPS DC. It was higher. Both IFN-γ in the cell culture supernatant were high, and IL-4 and IL-10 were below detection. That is, it is clear that both are not Th2-induced, but Th1-induced.
From the above, it was verified that both Mo-G4-LPS DC and PBMC-4βG-DC induce Th1-type cell proliferation.

(3) Inducibility of CMV antigen-specific CD8 + T cells by PBMC-4βG-DC (in vitro)
HLA-A2402 donor PBMC and another donor (cytomegalovirus (CMV) antibody positive) PBMC were mixed and cultured for one week with autologous PBMC-4βG DC cultured in the presence of CMV peptide for 2 days. The activation state of CD8 positive CTL was tested by a tetramer binding test. The results are shown in FIG. As shown in FIG. 5, it can be seen that PBMC-4βG DC proliferates CM8-specific CD8 + CTL from PBMC.

(4) Inducibility of influenza virus Flu-M1 (58-66) -specific CD8 + T cells of PBMC-4bG DC Similar to the verification in (3) above, the inducibility of influenza-specific CLT cells was compared. did. The results are shown in FIG. As shown in FIG. 6, it is clear that HLA-A2 donors can induce specific CLT cells by mixed culture with autologous PBMC-4βG DC and influenza peptides. The required ratio of DC to T cells is 1:80 or less.

  As described above, in this case, mature DC having immunostimulatory activity can be efficiently differentiated and cultured from PBMC in a short period of time. That is, it is possible to provide a method for differentiation culture of DC from unpurified monocytes, in other words, peripheral blood mononuclear cells (PBMC) itself. Specifically, GM-CSF, IL-4, and IFN-β were added to the medium and cultured for 1 day, and TNF-α (tumor necrosis factor) and PGE2 (prostaglandin 2) were added as maturation stimuli. In this method, the cells are further cultured for 1 day. According to this method, the differentiation / culture of mature DC is completed in 2 days.

  Various culture methods have been developed for functional human DC differentiation methods in vitro. At present, the DC progenitor cells that are generally used are monocytes that account for 5 to 10% of PBMC. Monocytes are purified from PBMC by the plastic attachment method or the antibody-coupled magnet method. A widely used cytokine is a combination of GM-CSF and IL-4, and LPS, TNF-a, CD40L or IFN-b is used for maturation. According to this combination, a total of 7 days is required for culture and maturation. However, in this case, the differentiation / culture of mature DC having immunostimulatory activity is completed in 2 days.

Mature DCs with immunostimulatory activity can be used for immunotherapy of cancer and infectious diseases. In particular, they can be used not only for the current treatment of cancer but also for the treatment of viral infections such as AIDS. Be expected. The present invention provides a method for efficiently differentiating and culturing mature DC having such useful immunostimulatory activity in a short period of time.
As described above, the present invention provides useful knowledge in the treatment of intractable diseases, and contributes to future medical care.

Claims (4)

A method of differentiating and culturing human peripheral blood mononuclear cells into mature dendritic cells,
Human peripheral blood mononuclear cells isolated from peripheral blood collected from humans are cultured in the presence of cytokines, and then matured by stimulation with a mature stimulating substance to generate mature dendritic cells having immunostimulatory activity A method for differentiating and culturing dendritic cells.   The method for differentiating and culturing dendritic cells according to claim 1, wherein the cytokine is IL-4, GM-CSF, or IFN-β.   The differentiation / culture of dendritic cells according to claim 1, wherein the cytokine is IL-4, GM-CSF, or IFN-β, and the maturation stimulating substances are TNF-α and PGE2. Method. The cytokine is IL-4, GM-CSF, IFN-β,
The method for differentiating and culturing dendritic cells according to claim 1, wherein the culture period in the presence of the cytokine is 1 day, and the subsequent maturation period by the maturation stimulating substance is 1 day.

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