KR20210026634A - Method for preparing cytotoxic CD4 T cell and use thereof - Google Patents

Abstract

The present invention relates to a method for producing cytotoxic CD4 T cells, cytotoxic CD4 T cells produced by the method, and a use thereof. The method comprises a step of stimulating abdominal CD49d CD4^+ T cells by type I interferon. When the CD49d^(high) CD4^+ T cells of the present invention are stimulated by interferon β (IFNβ), cytotoxicity is exhibited, and MHCII of B1 cells are specifically recognized. Thus, the B1 cells are killed, and thus an effect of reducing the amount of natural IgM antibodies in mouse serum is excellent. Furthermore, the CD49d^(high) CD4^+ T cells also exhibit cytotoxicity to a B1 cell-derived lymphoma cell line WEHI-231, thereby significantly inhibiting lymphoma cells. Accordingly, the CD49d^(high) CD4^+ T cells stimulated by IFNβ can be effectively used for a pharmaceutical composition, immune cell therapeutic agent, or immunosuppressant agent for preventing or treating cytotoxic CD4 T cells or immune diseases.

Description

[Method for preparing cytotoxic CD4 T cell and use thereof]

The present invention relates ^{to a method for producing cytotoxic CD4 T cells, comprising the step of stimulating peritoneal CD49d CD4 +} T cells with type I interferon, to a cytotoxic CD4 T cell prepared by the method, or a use thereof. .

Antibody production is achieved by plasma cells that are activated and differentiated when B cells meet antigens, and this process is usually aided by helper T cells. The help that helper T cells provide to B cells is an important characteristic of the immune system that regulates the development of homeostasis, high affinity memory B cells, and long-lived plasma cells. In addition, B cells can differentiate into short-lived plasma cells or low-affinity B cells outside the follicle through TLR (Toll like receptor) signals without the help of other cells. ^{Among the various cells, CD4 +} T cells, which are typical helper cells of B cells, can form active bonds with B cells. With the help of these CD4 ⁺ T cells, B cells go through one of two differentiation processes: germinal center (GC) development and outside follicle development, and B cells that develop at the seed center are high-affinity long-lived plasma cells in the seed center. I differentiate into memory B cells, and at this time, class switching may or may not occur. The seed center provides a microenvironment in which activated antigen-specific B cells can differentiate into memory cells or plasma cells through clonal selection and proliferation. Follicular helper T cells (Tfh) are ^{cells in which CD4 +} T cells are specially differentiated, and play an important role in the formation of the seed center by moving the peripheral follicular B cells to the seed center. The developmental process outside the follicle is the secretion of low-affinity antibodies by differentiating short-lived plasma cells at the periphery of the seed center of the spleen or lymph nodes, and the helper T cells for this are not well known.

B cells that are generally known and occupy most of the blood and secondary lymphoid tissues are B-2 cells, and unlike B-1 cells that develop in the early embryo, they produce natural antibodies. B-1 cells are mainly located in the abdominal cavity and are divided into B-1a and B-1b cells. The T cells in the peritoneal cavity are not well known, but a recent study revealed that many CD49d ^high CD4 ⁺ T cells exist. B-1a cells are known to spontaneously activate and secrete natural IgM antibodies that recognize not only bacteria but also autoantigens independent of T cells.

Interferons (IFN) are cytokines expressed in various cells with antiviral and growth inhibitory effects. IFN prevents viral infection and plays an important role in immune surveillance of malignant tumor cells. The IFN group is largely classified into two major groups. Type 1 IFNs (IFNα, β , ω, ε, and κ) are induced in viral infected cells to enhance the antiviral status of non-infected cells. In addition, type 1 IFN plays an important immunomodulatory function in both intrinsic and adaptive immunity.

Cytotoxic T lymphocytes (CTL) are antigen-specific reactive cells and have a function of lysing infectious cells or tumor cells. CTLs usually express a CD8 molecule that has the ability to bind to a target cell ligand (MHCI). The antigen-recognized CTL is activated through proliferation and differentiation to produce specific lysosomal vesicles. Cell killing mechanisms are largely classified into two types: one is a method of secreting a lytic protein such as perforin/granzyme out of the cell, and the other is a method of binding a receptor-ligand such as Fas/FasL. When CTL releases the soluble protein, Perforin makes a hole in the cell surface through polymerization, and granzyme enters the cytoplasm, affecting the cysteine protease of the target cell, inducing apoptosis. The CTL on the cell surface and the Fas-FasL interaction of the target cell induces the formation of DISC (death induction signal complex) in the target cell, and the target cell expressing Fas is apoptized by the continuous action of caspase.

Existing anticancer drugs or treatments for autoimmune diseases attack not only cancer cells or autoimmune cells, but also surrounding normal cells, and the side effects are very severe. To solve this problem, research on immunotherapy is being actively conducted. Immunotherapy, which is currently being developed, is mainly known as a method of treating a disease by controlling the activity of a patient's immune cells, and an immune cell therapy in which a pathogenic cell is specified and killed using an externally injected immune cell. CAR-T cells and NK cells, which are currently being studied up to the clinical stage, have side effects such as neurotoxicity or cytokine release syndrome, and the effect is not complete, so the disadvantage of having to be administered with other anticancer drugs and the problem that all patients do not see the same effect. Exists.

Korean Patent Application Publication No. 10-2011-0035633 relates to a method for inducing cytotoxic T cells in vitro using Th1 cells and dendritic cells expressing ^{CXCR3 +} CCR4 ^{- , and Th1 cells expressing CXCR3 +} CCR4 ^- are immature dendritic cells. It induces cell maturation, and the dendritic cells matured therefrom have the property of inducing antigen-specific cytotoxic T cells in vitro, and thus can be applied to adoptive T cell immunotherapy.

However, ^{no studies or descriptions have been made on whether CD49d high} CD4 ⁺ T cells are cytotoxic to B cells when stimulated with IFNβ.

Accordingly, the present inventors have made diligent efforts to provide an immune cell therapy with little side effects and excellent effect. As a result, ^{when stimulating peritoneal CD49d CD4 +} T cells with type I interferon, it exhibits cytotoxicity to B cells and It was confirmed that B cells can be specifically killed with cytotoxic substances such as perforine or granzyme, and the present invention was completed.

Accordingly, an object of the present invention is a ^{method for producing cytotoxic CD4 T cells comprising the step of stimulating peritoneal CD49d CD4 +} T cells with type I interferon, cytotoxic CD4 T cells prepared by the method It is to provide a use.

In order to achieve the above object, the present invention provides a ^{method for producing cytotoxic CD4 T cells, including the step of stimulating peritoneal CD49d CD4 +} T cells with type I interferon, to a cytotoxic CD4 T prepared by the method. Provides cells.

According to a preferred embodiment of the present invention, the CD49d CD4 ⁺ T cells may be CD49d ^high CD4 ⁺ T cells.

According to a preferred embodiment of the present invention, the type 1 interferon may be any one or more selected from the group consisting of interferon α, interferon β, interferon ω, interferon ε, and interferon κ.

According to a preferred embodiment of the present invention, the manufacturing method ^{may be stimulation with 1×10 2} to 1×10 ⁷ CD49d CD4 ⁺ T cells at a concentration of 1 ng/ml to 50 ng/ml of type 1 interferon.

According to a preferred embodiment of the present invention, the cytotoxic CD4 T cells may kill B cells.

The present invention also provides a pharmaceutical composition for preventing or treating immune diseases, an immune cell therapy agent or an immunosuppressant agent comprising the cytotoxic CD4 T cells.

According to a preferred embodiment of the present invention, the immune disease may be a humoral immune disease caused by B cells.

The present invention may also provide a composition for inducing cytotoxicity of ^{CD49d CD4 + T cells comprising interferon β.}

According to a preferred embodiment of the present invention, the CD49d CD4 ⁺ T cells may be CD49d ^high CD4 ⁺ T cells.

According to a preferred embodiment of the present invention, the cytotoxicity may be specific cytotoxicity for B cells.

^{CD49d high} of the present invention When ^{stimulated with interferon β (IFNβ), CD4 +} T cells show cytotoxicity and kill B1 cells by specifically recognizing MHCII of B1 cells, thereby reducing the amount of natural IgM antibodies in mouse serum. Furthermore, B1 cell-derived lymphoma cell line WEHI-231 also exhibits cytotoxicity and can significantly suppress lymphoma cells. Thus, the IFNβ stimulated CD49d ^high CD4 ⁺ T cells can be effectively used as cytotoxic CD4 T cells or pharmaceutical compositions for preventing or treating immune diseases, immune cell therapy agents or immunosuppressants.

1 shows the surface protein expression levels of peritoneal CD49d ^high CD4 ⁺ T cells, CD49d ^low CD4 ⁺ T cells, and spleen CD44 ^low primitive CD4 ⁺ T cells (spleen naive CD4 T) in terms of mRNA levels. It was confirmed that the expression levels of CD44, CD49d, CXCR3, β1-integrin, PD-1, ICOS and CD122 related to homeostasis of B-1a cells were highest in peritoneal CD49d ^high CD4 ^{+ T cells.}
(A) or (B) of Figure 2 before peritoneal cells (whole PEC cells) or the separated CD19 ⁺ B cells (sorted CD19 ⁺ B cells) and then stimulated with the IFNα or IFNβ a B-1a cell measurement, CD69 ⁺ Alternatively, ^{the ratio of CD69-} B cells is shown for each cell group. (C) shows the ratio of B-1a cell population among B lymphocytes in each culture condition, and (D) shows the ratio of activated CD69 ⁺ cells among B-1a cells in each culture condition. When all peritoneal cells were stimulated with IFNβ, it was confirmed that the proportion of B-1a cells or CD69 ^{+ cells among CD19 + B cells decreased.}
3 shows CD69 expression measured after stimulation of peritoneal CD49d ^high CD4 ⁺ T cells (red) or CD49d ^low CD4 ⁺ T cells (black) with IFNα or IFNβ, respectively. It was confirmed that CD69 expression was increased only when ^{CD49d high} CD4 ^{+ T cells were stimulated with IFNβ.}
4 shows the expression levels of perforine-granzyme-related proteins in ^{peritoneal CD49d high} CD4 ⁺ T cells according to the presence or absence of IFNβ stimulation. When CD49d ^high CD4 ⁺ T cells were stimulated with IFNβ, it was confirmed that the expression of genes related to cytotoxicity such as Gzma, Gzmb, Lamp1 or Prf1 was increased.
Of Figure 5 (A) shows the number to the ratio of live or dead CD19 ⁺ B cells in the CD19 ⁺ B cells in each culture condition. (B) shows the number of live B-1a cells in each culture condition. It was confirmed that the number of B-1a cells decreased only when ^{CD49d high} CD4 ^{+ T cells were stimulated with IFNβ.}
6A shows the expression levels of various cytotoxic molecules for peritoneal CD49d ^high CD4 ⁺ T cells (red) and CD49d ^low CD4 ^{+ T cells (black).} (B) is a bar graph showing the average fluorescence intensity of Perforin, CRTAM, LAMP-1 and Granzyme of ^{CD49d high} CD4+ T cells in the peritoneal cavity according to the presence or absence of IFNβ stimulation.
Figure 7 is a live B-1a cells in each culture ^condition represents the (CD19 ^+, viability staining). It was confirmed that peritoneal CD49d ^high CD4+ T cells exhibited B-1a apoptosis effect through MHC Ⅱ.
Figure 8 ^{shows the binding of CD49d high} CD4 ⁺ T cells and B-1a cells.
9 shows the B cell killing effect of ^{CD49d high} CD4 ⁺ T cells stimulated with IFNα or IFNβ. ^{It was confirmed that CD49d high} CD4 ⁺ T cells stimulated with IFNβ specifically inhibited B-1a cells and significantly inhibited the secretion of IgM antibodies known to be secreted by B1 cells.
^{10 shows the inhibitory effect of CD49d high} CD4 ⁺ T cells on lymphoma cell lines stimulated with IFNβ. It was confirmed that the B1 cell-derived lymphoma cell line, WEHI-231, was specifically inhibited.

Hereinafter, the present invention will be described in more detail.

'CD49d ^high CD4 ⁺ T cells (CD49d ^high CD4 T cells)' of the present invention refers to CD4 T cells whose expression level of CD49d is about 3 times higher than that of spleen primitive CD4 T cells, and correspondingly,'CD49d ^low CD4 ⁺ T cells (CD49d ^low CD4 T cells)' refer to CD4 T cells that do not correspond to ^{the CD49d high} CD4 ^{+ T cells.}

The “immune cell therapy agent” of the present invention means any agent capable of inducing, enhancing or suppressing an immune response in a subject. The subject may refer to any animal capable of developing immune-related diseases, including humans.

The'immune (cell) inhibitor' of the present invention inhibits the proliferation and related functions of the subject's immune cells and improves (reduces symptoms), treatment, and prevention of immune diseases caused by hyperimmune reactions or abnormal immune responses of the body, It means to inhibit or delay onset. The subject may refer to any animal capable of developing immune-related diseases, including humans.

As described above, conventional methods for treating cancer or autoimmune diseases using immune cells have severe side effects such as neurotoxicity or cytokine release syndrome, and have a limitation in that the effect alone is insufficient.

Among the types of peritoneal T cells, peritoneal CD49d ^high CD4 ⁺ T cells were activated only by IFNβ, and CD49d ^low CD4 ⁺ T cells were not activated by type I IFN (Fig. 3). Perforine and granzyme ^{are known as important functional elements in CD4 +} CTL. Granzyme is polymerized to gain intracellular access to target cells with the help of perforin. In this way, peritoneal CD49d ^high CD4 ⁺ T cells are able to modulate activated B cells to terminate the immune response in an IFNβ-rich environment such as various viral infections. Type 1 IFNs, such as IFNα or IFNβ, are produced in viral infected cells or activated macrophages, and are known to exist in sufficient amounts in the abdominal cavity. The abdominal cavity is a space where the intestine and liver are located close to each other and is a place where immune responses to symbiotic bacteria or pathogens frequently occur.

Peritoneal CD49d ^high CD4 ⁺ T cells expressed the proteins of granzyme (A and B), perforin and CD107a in just 2 hours when stimulated with IFNβ (Fig. 6). This shows that the IFNβ can rapidly induce CTL differentiation of CD4 ⁺ T cells, peritoneal CD49d ^high, IFNα and IFNβ acts in different ways in peritoneal CD4 ⁺ T cells CD49d ^high.

Peritoneal CD49d ^high CD4 ⁺ T cells must be activated by IFNβ in order to recognize B-1a cells, and B-1a cells activated by IFNβ can be selected by MHC II antigen presentation and are in the Perforin-Granzyme pathway. It can be killed by (Fig. 7). It appears that peritoneal CD49d ^high CD4 ⁺ T cells regulate overactivated B-1a cells in a severe inflammatory environment containing IFNβ, known to be produced by fibroblasts or damaged tissues, viral infections, murine cancer models, or autoimmunity. It means that in diseases such as peritoneal CD49d ^high CD4 ⁺ T cells may be cytotoxic.

That is, peritoneal CD49d ^high CD4 ⁺ T cells can act as positive and negative regulators in the regulation of B-1a cells, one is a helper function that helps the survival of B-1a cells through APRIL secretion, and the other. Is a function of preventing overexpression of B-1a by activating cytotoxicity through MHC II specifically for B-1a cells in certain inflammatory conditions including IFNβ.

Accordingly, the present invention can provide a method for producing cytotoxic CD4 T cells comprising the step of stimulating ^{CD49d CD4 + T cells with type I interferon.}

^{The CD49d CD4 +} T cells of the present invention ^{may be CD49d high} CD4 ⁺ T cells. In addition to CD49d, the CD49d ^high CD4 ⁺ T cells may have a higher expression level of CD44, CXCR3, PD-1, ICOS, CD122 or β1 integrin than that of splenic primitive CD4 T cells, especially CD49d, CD44, CXCR3, ICOS or CD122. It may be about three times higher than that of primitive CD4 T cells.

The type 1 interferon of the present invention may be any one or more selected from the group consisting of interferon α, interferon β, interferon ω, interferon ε, and interferon κ, preferably interferon β (IFNβ).

The manufacturing method of the present invention ^{may be stimulation with 1×10 2} to 1×10 ⁷ CD49d CD4 ⁺ type 1 interferon at a concentration of 1 ng/ml to 50 ng/ml per T cell, and preferably the manufacturing method is 1 ×10 ³ to 1×10 ⁶ CD49d CD4 ⁺ T cells may be stimulated with type 1 interferon at a concentration of 5 ng/ml to 40 ng/ml, and more preferably, the preparation method is 1×10 ⁴ to 1×10 It may be stimulated with type 1 interferon at a concentration of 10 ng/ml to 30 ng/ml per ⁵ CD49d CD4 ^{+ T cells.}

The above preparation method can be used in vitro.

The present invention can also provide a cytotoxic CD4 T cell prepared by the above production method.

The cytotoxic CD4 T cells of the present invention can kill cells that express MHCII higher than that of normal cells, and specifically, the cells expressing MHCII high may be B cells. The B cells are plasmablasts, plasma cells, lymphoplasmacytoid cells, memory B cells, B-1 cells, B-2 cells, and regulatory B cells (Breg cells). It is preferably any one or more selected from the group consisting of, more preferably B-1 cells, and most preferably B-1a cells.

The present invention may also provide a pharmaceutical composition for preventing or treating immune diseases comprising the cytotoxic CD4 T cells.

The immune disease of the present invention is a humoral immune disease caused by B cells, which means all immune diseases that can occur due to overactivation or overexpression of B cells, but preferably, graft versus host disease, rejection during organ transplantation. , Humoral rejection, allergic disease, irritable immune disease, autoimmune disease, cancer, parasitic infection and viral disease, preferably any one or more selected from the group consisting of, more preferably, humoral rejection, autoimmune disease , It is preferably any one or more selected from the group consisting of cancer and viral diseases. The autoimmune disease may be one selected from the group consisting of Crohn's disease, ulcerative colitis, Behcet's disease, systemic lupus erythematosus, Sjogren's syndrome, myasthenia gravis, scleroderma, polyarteritis nodosa, hypothyroidism, psoriasis and rheumatoid arthritis. have.

The cancer is a concept including a tumor, blood cancer or solid cancer, but preferably lung cancer, liver cancer, esophageal cancer, stomach cancer, colon cancer, small intestine cancer, pancreatic cancer, melanoma, breast cancer, oral cancer, brain tumor, thyroid cancer, parathyroid cancer, kidney cancer, It is preferably any one or more selected from the group consisting of cervical cancer, sarcoma, prostate cancer, urethral cancer, bladder cancer, testicular cancer, hematologic cancer, lymphoma, skin cancer, psoriasis and fibroadenoma, more preferably lymphoma. The lymphoma may be B cell lymphoma, T cell lymphoma, or NK cell lymphoma, but is preferably B cell lymphoma, and more preferably B-1 cell related lymphoma.

The pharmaceutical composition of the present invention may be in various oral or parenteral dosage forms. When formulating the composition, one or more buffers (e.g., saline or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, bulking agents, binders, adjuvants (e.g., Aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient in one or more compounds, such as starch (corn starch, wheat starch, rice starch, potato Starch), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl -It is prepared by mixing cellulose or gelatin. For example, tablets or dragees can be obtained by blending the active ingredient with a solid excipient, pulverizing it, adding a suitable auxiliary, and processing it into a granule mixture.

In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions or syrups, and various excipients, such as wetting agents, sweeteners, fragrances or preservatives, are included in addition to water and liquid paraffin, which are commonly used simple diluents. I can. In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, and an anti-aggregating agent, a lubricant, a wetting agent, a fragrance, an emulsifying agent and a preservative, etc. may be additionally included. .

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations or suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, and the like may be used.

The composition of the present invention may be administered orally or parenterally, and for parenteral administration, the composition may be used externally to the skin; Intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine dura mater or intracerebrovascular injection; Transdermal administration; Alternatively, it may be formulated in the form of a nasal inhalant according to a method known in the art.

In the case of the injection, it must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for injections include, but are not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), mixtures thereof, and/or solvents or dispersion media containing vegetable oils. I can. More preferably, suitable carriers include isotonic solutions such as Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose. Etc. can be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, and the like may be additionally included. In addition, the injection may further contain an isotonic agent such as sugar or sodium chloride in most cases.

In the case of transdermal administration, ointments, creams, lotions, gels, external solutions, pasta, liniment, and air rolls are included. In the above, transdermal administration means that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin by topically administering the pharmaceutical composition to the skin.

In the case of inhalation dosages, the compounds used according to the invention can be used in a pressurized pack or with a suitable propellant, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be conveniently delivered from a nebulizer in the form of an aerosol spray. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

The composition of the present invention is administered in a pharmaceutically effective amount. A pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of disease, severity, drug activity, drug sensitivity, administration time of the patient. , Route of administration and rate of excretion, duration of treatment, factors including concurrent drugs and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. That is, the total effective amount of the composition of the present invention may be administered to a patient in a single dose, and may be administered by a fractionated treatment protocol that is administered for a long time in multiple doses. . It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.

The dosage of the pharmaceutical composition of the present invention varies according to the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. As a daily dose, when parenterally administered, it is preferably administered in an amount of 0.01 to 50 mg, more preferably 0.1 to 30 mg per kg of body weight per day based on ^{CD49d CD4 + T cells. Based on the CD49d CD4 +} T cells of the present invention, per 1 kg of body weight per day, preferably 0.01 to 100 mg, more preferably 0.01 to 10 mg may be administered in divided doses from 1 to several times. However, since it may increase or decrease depending on the route of administration, the severity of obesity, sex, weight, age, etc., the dosage amount does not limit the scope of the invention in any way.

The composition of the present invention may be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods using biological response modifiers.

The pharmaceutical composition of the present invention can also be provided in the form of an external preparation containing ^{CD49d CD4 + T cells as an active ingredient.} When using the pharmaceutical composition for preventing and treating immune diseases of the present invention as an external preparation for skin, additionally fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents ), fragrances, surfactants, water, ionic emulsifiers, nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic activators, lipophilic activators Or it may contain adjuvants commonly used in the field of dermatology such as any other ingredients commonly used in skin external preparations such as lipid vesicles. In addition, the above ingredients may be introduced in an amount generally used in the field of dermatology.

When the pharmaceutical composition for preventing and treating immune diseases of the present invention is provided as an external preparation for skin, it may be a formulation such as an ointment, a patch, a gel, a cream, or a spray, but is not limited thereto.

In addition, the present invention can provide an immune cell therapy or immunosuppressant comprising the cytotoxic CD4 T cells.

The description of the formulation of the immune cell therapy or immunosuppressive agent is replaced by the description of the pharmaceutical composition.

In addition, the present invention can provide a composition for inducing cytotoxicity of ^{CD49d CD4 + T cells comprising interferon β.}

The interferon β, CD49d CD4 ⁺ T cells, and cytotoxicity are the same concepts as those used in the method for producing cytotoxic CD4 T cells, so the description is replaced by the above description.

The composition can be used in vitro.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it is apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

mouse

The 8-week-old C57BL/6 was purchased from Orient Bio (Sungnam, Korea). CD1d knockout mice and SAP (sh2d1a) knockout mice were provided by L. Van Kaer (Vanderbilt University School of Medicine, Nashville, TN) and C. Terhorst (Beth Israel Deaconess Medical Center, Boston, MA), respectively. OTII transgenic mice were purchased from Jackson Laboratory (Bar Harbor, ME). All mice used in the experiment were maintained and used according to the institutional management guidelines of the SPF facility of the Laboratory Animal Center of Sungkyunkwan University School of Medicine. 7-week-old C57BL/6 mice were bred under aseptic conditions and provided by Dr. Yoo Jeong (Postech, Pohang, Korea). This experiment was reviewed and approved by IACUC (Institutional Animal Breeding and Use Committee) of Sungkyunkwan University School of Medicine.

CD49d ^high CD4 ⁺ Whether T cells affect B-1a cell homeostasis

We tried to confirm the expression level of proteins related to homeostasis in B-1a cells at the mRNA level.

Specifically, in 8-week-old CD1d knockout mice, the abdominal cavity was washed with PBS and intraperitoneal CD49d ^high. CD4 ⁺ T cells and CD49d ^low CD4 ⁺ T cells were isolated. The single cell suspension was prepared by physically disrupting the spleen and homogenizing it using a nylon membrane, and then the spleen CD44 ^low raw CD4 ⁺ T cells were isolated. The total RNA (total RNA) of each of the isolated cells was isolated and purified, and mRNA transcriptome sequencing was performed at LAS Inc. (Gimpo, Korea) to determine the expression level of the surface protein of each cell at the mRNA level. Confirmed. The heat map analysis showed the results of the analysis of each cell group as a fold change (log10) based on the results ^{of the original CD4 + T cells in the spleen.} The number in each column represents the FPKM result of the gene. Color standard values are presented at the top left of the heat map.

As a result, as shown in Fig. 1, the expression levels of CD44, CD49d, CXCR3, β1-integrin, PD-1, ICOS and CD122 on the cell surface of ^{CD49d high CD4+ T cells are CD49d low} CD4+ T cells or spleen. (spleen) It ^{was found to be higher than that of primitive CD4 +} T cells, and in the case of PD-1, it ^{was confirmed that CD49d high} CD4 + T cells were expressed about 163 times higher than ^{that of spleen primitive CD4 + T cells.} On the other hand, it was confirmed that the markers of primitive T cells such as CD62L or CCR7 ^{were lower in CD49d high} CD4 ⁺ T cells than in the other two cell groups.

The effect of IFNβ on reducing activated peritoneal B-1a cells

The purpose of this study was to determine whether peritoneal B cells and CD4+ T cells were activated by type 1 IFN and whether the decrease in the number of B cells by IFNβ stimulation was due to internal or external factors of B cells.

Specifically, whole peritoneal cells (whole PEC cells, 4×10 ⁶ ~ 6×10 ⁶ ) were obtained from 8-week-old CD1d knockout C57BL/6 mice, and 20 ng/ml of IFNα or IFNβ was added, followed by in vitro culture for 18 hours. Stained with anti-CD19, CD11b, CD5 or CD69 immunoantibodies. In addition, the isolated CD19 ⁺ B cells were cultured in the same manner as described above.

As a result, when stimulating anterior peritoneal cells with IFNα, ^{the level of CD69 +} increases to about twice that of peritoneal B-1a cells (Fig. 2A), but when stimulating anterior peritoneal cells with IFNβ, CD69 ⁺ B-1a cells It was confirmed that the number was decreased (Fig. 2C, 6D). In addition, when only isolated CD19 ⁺ B cells were stimulated with IFNβ, ^{the number of CD69 +} B-1a cells did not decrease (FIG. 2B). Overall, the reduction of peritoneal B cells by IFN-β stimulation was expected to be caused by extra B cells, and the extrinsic agents were T cells, macrophages, or other peritoneal cells.

Celiac CD49d ^high CD4 ⁺ IFN stimulation effect on T cells

To determine whether type 1 IFN ^{activates peritoneal CD49d high} CD4 ⁺ T cells and whether IFNβ-stimulated peritoneal CD49d ^high CD4 ⁺ T cells acquire cytotoxic function and kill B-1a cells or activated B cells. I did. CD69 is a cell surface glycoprotein and is known to increase its expression when lymphocytes are activated.

Specifically, FACS separation was performed to isolate only ^{CD49d high} CD4 ^{+ T cells from T cells.} Whole peritoneal cells were obtained from 8-week-old CD1d knockout C57BL/6 mice, in vitro cultured for 18 hours with the addition of 20 ng/ml of IFNα or IFNβ, and stained with anti-CD4, CD49d, and CD69 immunobodies. Staining was performed for 20 minutes on ice by adding an appropriate combination of fluorescent antibodies to FACS buffer (5% NBCS and 0.05% sodium azide in PBS). After washing with FACS buffer, the stained cells were analyzed using FACS Canto II (BD Biosciences). The experimental results were analyzed using FlowJo software (Tree Star, San Carlos, CA). Cells used in the in vitro test were isolated using FACS Aria III (BD Biosciences). Peritoneal CD49d ^high or CD49d ^low CD4+ T cells were stained and isolated using anti-CD4 or CD49d antibodies.

In addition, ^{by stimulating peritoneal CD49d high} CD4 ⁺ T cells with IFNβ or without stimulation, whole RNA was isolated and purified, and mRNA transcript sequencing was performed in the same manner as in <Example 2>.

As a result, as shown in [Fig. 3], ^{when stimulation of peritoneal CD49d high} CD4 ⁺ T cells with IFNβ increased CD69 expression (Fig. 3, blue line), but intraperitoneal CD49d ^low CD4 ⁺ T cells did not change (Fig. 3, red line).

In addition, as shown in [Fig. 4], the peritoneal CD49d ^high CD4 ⁺ T cells stimulated with IFNβ are known as markers ^{of CD4 +} CTL Gzma, Gzmb , Lamp1 or It was confirmed that genes related to cytotoxic T cells such as Prf1 were expressed. Gzmk is known as a marker for CD8 ⁺ CTL and natural killer cells.

Intraperitoneal CD49d stimulated with IFNβ ^high Correlation between CD4+ T cells and Perforin-Granzyme

The purpose of this study was to confirm the ability of ^{peritoneal CD49d high} CD4 ⁺ T cells to kill B-1a cells through IFNβ stimulation. In addition, changes in expression of various cytotoxic proteins were analyzed to find proteins with increased expression in peritoneal CD49d ^high CD4 ^{+ cells stimulated with IFNβ.}

Specifically, each of the isolated B-1a cells (2.5×10 ⁴ ) and the isolated peritoneal CD49d ^high CD4 ⁺ T cells, CD49d ^low CD4 ⁺ T cells or CD8 ⁺ T cells (2×10 ⁴ ) contains 10% FBS. 20ng/ml IFNβ was added to the resulting RPMI 1640 for cultivation, and fluorescence staining was performed using anti-CD19 and viable chromosomal antibodies.

In addition, whole peritoneal cells were obtained from 8-week-old CD1d knockout C57BL/6 mice and cultured in vitro for 18 hours with the addition of 20 ng/ml IFNβ, followed by anti-CD4, CD49d, Perforin, CRTAM, LAMP-1, and Granzyme B immunity. Stained with antibody.

As a result, as shown in [Fig. 5], even with IFNβ stimulation, most of the B-1a cells cultured with ^{CD49d low} CD4 ⁺ T cells or CD8 ⁺ T cells survived, and B- cultured with ^{CD49d high} CD4 ^{+ T cells.} The number of 70% of 1a cells decreased.

^{In addition, stimulation of peritoneal CD49d high} CD4 ⁺ T cells with IFNβ increases the expression levels of Perforin, Granzyme B, and CD107a proteins as shown in the mRNA expression result of <Example 4>, but MHCI of target cells is increased in some CD4+ CTLs. CRTAM, which is known to have a cognitive function, was not expressed (Fig. 6). Overall, it was determined that IFNβ-stimulated CD49d ^high CD4 ⁺ T cells kill target B cells through perforin-granzyme.

Intraperitoneal CD49d stimulated with IFNβ ^high CD4 ⁺ Relationship between T cells and MHCⅡ

In general, ^{it has been reported that CD4 +} CTL can kill target cells by recognizing MHCII, an antigen presenting molecule. Therefore, an experiment was conducted to confirm how IFNβ-stimulated CD49d ^high CD4 ⁺ T cells recognize activated B-1a cells as targets.

Specifically, in ^{order to evaluate the cytotoxic activity of CD49d high} CD4+ T cells, only peritoneal B-1a cells (2.5×10 ⁴ or 5×10 ⁴ ) in 8-week-old CD1d knockout mice and 8-week-old SAP ( sh2d1a) knockout mice. Separate the abdominal cavity CD49d ^high CD4 ⁺ T cells (5×10 ³ ) were cultured with or without IFNβ (20 ng/ml) in RPMI culture. Anti-MHCI antibody, anti-MHCII antibody, and anti-LFA1 antibody were used to prevent the action of various cell-cell interaction molecules. After 18 hours of incubation, cells were stained with anti-CD4, CD19, and viable chromosomal antibodies and analyzed by FACS CantoII. And the number of live CD19 ⁺ B cells was measured.

In addition, to visualize that IFNβ-stimulated CD49d ^high CD4 ⁺ T cells kill activated B-1a cells, isolated peritoneal CD49d ^high CD4 ⁺ T cells (5×10 ³ ) and B-1a cells (5×10 ⁴ ) After in vitro culture for 2 hours or 6 hours under conditions to which IFNβ (20 ng/ml) was added together, the cultured cells were stained with anti-CD4, CD19, TCRβ, and MHCII fluorescent antibodies. CD19+ B cells and CD4+ T cells bound from the peritoneal cells were separated using FACS AriaII, used on a silane-coated glass slide, attached by a cell centrifuge at 800 rpm for 5 minutes, and covered with a cover glass. Immunofluorescence staining pictures were obtained using a Zeiss Axioplan optical microscope and a Zeiss LSM510 (Zeiss, Hamburg, Germany). Photos were corrected using Workstation.

As a result, as shown in [Fig. 7], IFNβ-stimulated B-1a cells did not decrease in the condition of adding the MHCII inhibitory antibody, but decreased in MHCI or ICAM. In addition, SAP knockout ( sh2d1a ^-/- ) mice ^{obtained peritoneal CD49d high} CD4 ⁺ T cells and observed the killing ability by IFNβ, and the number of B1 cells was decreased.

Also, MHCⅡ ⁺ B cells (green) and TCRβ ⁺ CD49d ^high BT binding to which CD4 ⁺ T cells (red) are bound is as shown in [Fig. 8]. Stimulation with IFNβ for more than 6 hours ^{activates the cytotoxicity of peritoneal CD49d high} CD4 ⁺ T cells, killing MHCⅡ+ B-1a cells. Taken together, it was determined that peritoneal CD49d ^high CD4 ⁺ T cells recognized cells presenting antigens to MHCII through IFNβ stimulation and showed cytotoxicity with CD107a, Perforin, and Granzyme.

Cytotoxic CD49d ^high B1 apoptosis effect of CD4 T cells

The purpose of this study was to confirm the B1 cell-specific killing function of ^{CD49d high} CD4 T cells, which became cytotoxic by stimulation of IFNβ, in vivo.

^{Specifically, CD49d high} CD4 T cells were stimulated with IFNα or IFNβ (20 ng/ml) for 18 hours, and then injected into normal C57BL/6 mice 5 times at 3 day intervals by intraperitoneal injection. 15 days after the first injection, the B1 cell population and its number were analyzed by FACS (FIG. 9A). In addition, the serum was collected from the mice at intervals of 3 days, and the collected serum was analyzed for the amount of IgM and IgG antibodies using ELISA.

As a result, as shown in [Fig. 9], ^{B1 cells were not reduced by IFNα-stimulated CD49d high} CD4 T cells, whereas B1 cells were differentiated and injected into cytotoxic CD4 T cells by stimulation with IFNα. Was decreased, the amount of IgM in the serum was rapidly decreased, and there was no change in the amount of IgG.

Cytotoxic CD49d ^high Effects of CD4 T cells on B-cell lymphoma

Since one of the causative cells of B-cell lymphoma is known as B1 cells ^{, we tried to determine whether CD49d high} CD4 T cells can suppress B-cell lymphoma.

Specifically, by co-culturing the lymphoma cell line WEHI-231 or BAL17 and CD49d ^high CD4 T cells (2×10 ⁴ ) stimulated with IFNβ (20 ng/ml), the survival of each cell line was confirmed by FACS using viability dye. Analyzed. WEHI-231 is a representative B1 cell-derived lymphoma cell line, and BAL17, a lymphoma cell line having properties similar to those of other types of B cells, B2 cells, was used as a control.

As a result, as shown in Fig. 10, it was confirmed that BAL17 was ^{not killed by cytotoxic CD49d high} CD4 T cells, but only WEHI-231, a lymphoma cell line derived from B1 cells, was killed.

Claims (14)

Cytotoxic CD4 T cell production method comprising the step of stimulating CD49d CD4 ⁺ T cells with type 1 interferon (type I interferon).
The method of claim 1, wherein the CD49d CD4 ⁺ T cells are CD49d ^high CD4 ⁺ T cells.
The method of claim 1, wherein the type 1 interferon is at least one selected from the group consisting of interferon α, interferon β, interferon ω, interferon ε, and interferon κ.
The method of claim 1, wherein the preparation method is ^{characterized in that stimulation with 1×10 2} to 1×10 ⁷ CD49d CD4 ⁺ T cells at a concentration of 1 ng/ml to 50 ng/ml of type 1 interferon.
Cytotoxic CD4 T cells prepared by the method of claim 1.
The cytotoxic CD4 T cell according to claim 5, wherein the cytotoxic CD4 T cell kills B cells.
A pharmaceutical composition for preventing or treating immune diseases comprising the cytotoxic CD4 T cells of claim 5.
The pharmaceutical composition according to claim 7, wherein the immune disease is a humoral immune disease caused by B cells.
An immune cell therapeutic agent comprising the cytotoxic CD4 T cells of claim 5.
An immunosuppressant comprising the cytotoxic CD4 T cells of claim 5.
11. The immunosuppressant according to claim 10, wherein the immunosuppressant is a humoral immunosuppressant.
A composition for inducing cytotoxicity of ^{CD49d CD4 +} T cells, including interferon β.
The composition for inducing cytotoxicity according to claim 12, wherein the CD49d CD4 ⁺ T cells are CD49d ^high CD4 ^{+ T cells.}
The composition for inducing cytotoxicity according to claim 12, wherein the cytotoxicity is specific cytotoxicity to B cells.

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