WO2020067422A1 - Composition for activating t cells - Google Patents

Composition for activating t cells Download PDF

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Publication number
WO2020067422A1
WO2020067422A1 PCT/JP2019/038145 JP2019038145W WO2020067422A1 WO 2020067422 A1 WO2020067422 A1 WO 2020067422A1 JP 2019038145 W JP2019038145 W JP 2019038145W WO 2020067422 A1 WO2020067422 A1 WO 2020067422A1
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Prior art keywords
cells
composition
lactic acid
eps
present
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PCT/JP2019/038145
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French (fr)
Japanese (ja)
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啓誠 川鍋
真梨枝 中村
聖也 牧野
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株式会社明治
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a composition containing a culture of lactic acid bacteria for activating T cells.
  • the immune system is broadly divided into innate immunity and acquired immunity. Activation of acquired immunity with the help of innate immunity is important for eliminating pathogens and damaging cancer cells. This specific and powerful immune mechanism is triggered by the activation of T cells against antigens. It would be very useful if T cell activation could be easily promoted.
  • Patent Document 1 discloses a method for improving an immune function in a mammal using a Lactobacillus reuteri strain.
  • a significantly higher amount of CD4 + cells was present in ileal biopsies from subjects who had taken masticated tablets containing cells of the Lactobacillus reuteri strain for 28 days, such strains were used.
  • Patent Document 2 discloses a method for enhancing an immune response to a microbial pathogen in a subject, including a step of administering a Bacillus coagulans bacterium or a fragment thereof to the subject infected with the microbial pathogen. I do.
  • IL-8 production and IFN- ⁇ production were altered in subjects who consumed one capsule daily containing 500 million CFU of Bacillus coagulans GBI-30 and ATCC @ PTA-6086 for a viable 30 days.
  • a composition containing Bacillus coagulans bacteria to enhance an immune response against a pathogen.
  • lactic acid bacteria produce various substances during the fermentation process, one of which is exopolysaccharides (EPS).
  • EPS exopolysaccharides
  • the EPS of lactic acid bacteria has several physiological activities, for example, an autoimmune disease preventive action (Patent Document 3), an NK cell activating action (Patent Document 4), a pneumococcal infection preventive action (Patent Document 5), and an improvement in fatigue.
  • An effect (Patent Document 6) and an inhibitory effect on acquired immune function decrease by an anti-influenza drug (Patent Document 7) are known.
  • T cells Treatment of infectious diseases and cancer by activating T cells is considered to be less burdensome for patients than conventional anti-cancer drug treatment, surgical resection, and radiation treatment that exhibit cytotoxic effects. Needless to say, it is desirable to have a composition capable of activating T cells by using a substance having a dietary experience as an active ingredient. It would be more desirable if there was a method that could easily measure the activation of T cells.
  • T cells to antigens can be easily determined only by separating peripheral blood mononuclear cells (PBMC) from a blood sample and measuring the CD69 expression rate of T cells with a flow cytometer. It is common knowledge in immunology that the expression of CD69 molecule in lymph nodes is transiently increased when T cells recognize and activate antigens and can be widely used as an activation marker. The present inventors have found that the expression of the CD69 molecule of blood T cells also reflects the response to antigen in lymph nodes, and is useful as a liquid biopsy.
  • PBMC peripheral blood mononuclear cells
  • the present invention provides the following.
  • a non-medical method for reducing the risk of developing an infectious disease or cancer comprising ingesting a composition containing an effective amount of an exopolysaccharide of a lactic acid bacterium into a subject, the CD4 + T cell, and the CD8 + T Increasing the CD69 expression rate in at least one of the cells.
  • the present invention also provides the following.
  • a method for activating at least one of CD4 + T cells and CD8 + T cells comprising a step of (orally) administering (ingesting) an exopolysaccharide of a lactic acid bacterium or a composition containing the same to a subject.
  • a method for activating at least one of CD4 + T cells and CD8 + T cells in a subject comprising a step of (orally) administering (ingesting) the exopolysaccharide of a lactic acid bacterium or a composition containing the same to the subject.
  • a non-therapeutic method for improving the CD69 expression rate in a subject comprising the step of (orally) administering (ingesting) the exopolysaccharide of a lactic acid bacterium or a composition containing the same to the subject.
  • a method for producing a composition for activating at least one of CD4 + T cells and CD8 + T cells comprising a step of mixing an exopolysaccharide of a lactic acid bacterium with a pharmaceutically acceptable additive.
  • the present invention also provides the following.
  • activation of at least one of CD4 + T cells and CD8 + T cells can be effectively promoted. Since the activation and / or administration of at least one of CD4 + T cells and CD8 + T cells can be effectively promoted by ingestion / administration of the composition of the present invention, suppression of tumor growth and promotion of elimination of pathogenic bacteria in a subject can be expected.
  • the present invention provides the use of CD69 of blood T cells as a liquid biopsy.
  • A IFN- ⁇ production upon stimulation of lymph node T cells in mice orally administered with EPS
  • B CD69 expression rate in spleen cells of mice orally administered with EPS.
  • B Percentage of TEMRA in peripheral blood CD8 + T cells of a subject who has consumed fermented milk
  • D CD69 expression rate in peripheral blood CD8 + TEMRA of a subject who ingested fermented milk (dotted line: control group, solid line: fermented milk group).
  • the present invention relates to a composition containing an exopolysaccharide (EPS) produced by a lactic acid bacterium as an active ingredient. More specifically, the present invention relates to a composition for activating at least one of CD4 + T cells and CD8 + T cells (hereinafter, referred to as “CD4 + / CD8 + T cells”) using EPS as an active ingredient.
  • EPS exopolysaccharide
  • composition of the present invention contains lactic acid bacteria EPS as an active ingredient.
  • Lactic acid bacterium is a general term for microorganisms that utilize glucose to produce lactic acid at a yield of 50% or more in terms of sugar yield, and are physiologically gram-positive cocci or rods that have no motility and often have a sporulating ability. None (there is also a lactic acid bacterium capable of forming spores such as Bacillus coagulans) and catalase negative. Lactic acid bacteria have been eaten around the world through fermented milk and the like since ancient times, and can be said to be extremely safe microorganisms. Lactic acid bacteria are classified into several genera.
  • the EPS of the lactic acid bacterium contained in the composition of the present invention is preferably produced by a lactic acid bacterium belonging to the genus Lactobacillus classified into the genus Lactobacillus.
  • EPS used in the composition of the present invention is not particularly limited as long as it has the desired effect.
  • EPS produced by lactic acid bacteria is structurally classified into homopolysaccharides and heteropolysaccharides (for example, those composed of galactose and glucose), and is modified by phosphorylation or sulfation. In some cases, any of them can be used as an active ingredient of the composition of the present invention.
  • a preferred EPS is one containing a neutral polysaccharide and / or an acidic polysaccharide having a phosphate group added to the neutral polysaccharide.
  • Such EPS is known to be produced by Lactobacillus delbruchy subspecies bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus) or Lactococcus lactis subspecies cremoris (Lactococcus lactis subsp. Cremoris). ing.
  • the EPS used in the present invention may be one kind or a combination of two or more kinds.
  • Examples of particularly preferred EPS-producing lactic acid bacteria used in the composition of the present invention are Lactobacillus lactic acid bacteria.
  • Examples of lactic acid bacteria of the genus Lactobacillus include Bulgaricus spp., Casei spp., Acidophilus spp., Plantarum spp., And the like.
  • lactic acid bacteria classified as Bulgaricus species also referred to as Bulgaricus bacteria
  • it is more preferable that the lactic acid bacterium is classified into Lactobacillus delbrueckii subsp.
  • the lactic acid bacterium may be referred to as Lactobacillus delbrueckii subsp. Bulgaricus OLL1073 @ R-1 (accession number: FERM @ BP-10741) ("Bulgarix strain R-1").
  • Lactobacillus delbruchy subspecies Bulgaricus OLL1073 @ R-1 accession number: FERM @ BP-10741
  • the EPS of lactic acid bacteria contained in the composition of the present invention may be contained as a fermented product of lactic acid bacteria.
  • the fermented product of lactic acid bacteria includes a fermented product of lactic acid bacteria itself and a processed product thereof.
  • the fermented lactic acid bacteria itself includes, for example, fermented milk (specifically, yogurt or the like).
  • the processed product includes, for example, a crude filtrate, a culture filtrate or a culture supernatant obtained by removing bacteria by filtration, centrifugation, or membrane separation, and a concentrate obtained by concentrating the culture filtrate or culture supernatant. , And dried concentrates.
  • EPS of lactic acid bacteria is prepared as a fermented product of lactic acid bacteria
  • lactic acid bacteria producing EPS are added to the raw milk as a starter, fermented, and EPS is produced in the fermented product, whereby fermented milk containing EPS is produced.
  • Conditions for fermentation such as raw milk, fermentation temperature, and fermentation time, are not particularly limited as long as the lactic acid bacteria used can produce EPS, and those skilled in the art can appropriately set them.
  • the composition of the present invention can be used to activate at least one of CD4 + T cells and CD8 + T cells (CD4 + / CD8 + T cells).
  • CD4 + / CD8 + T cells when promoting the activation of CD4 + / CD8 + T cells, it refers to promoting the activation of antigen-specific CD4 + / CD8 + T cells, unless otherwise specified.
  • Enhancing the activation of CD4 + / CD8 + T cells includes increasing CD69 expression in CD4 + / CD8 + T cells and promoting the progression of maturation from CD8 + TEM to CD8 + TEMRA.
  • the composition of the present invention can increase the CD69 expression rate in CD4 + / CD8 + T cells. Since CD69 increases its expression within several hours when T cells or B cells are stimulated with an antigen, it serves as an early activation marker molecule and serves as an indicator of lymphocyte activation.
  • the expression ratio of CD69 refers to the ratio of cells expressing CD69 to all the cells. Detection by a flow cytometer is performed using a fluorescently labeled anti-CD69 antibody, which can be measured as the ratio of labeled cells in all the cells.
  • CD3 + and CD4 + mononuclear cells can be CD4 + T cells, and CD3 + and CD8 + mononuclear cells are CD8 + T cells. Can be.
  • the CD69 expression rate can be a value based on the analysis value of peripheral blood T cells derived from the subject.
  • the analysis refers to measuring any of the presence or absence and, if present, the degree (amount) of the molecule to be analyzed.
  • ingestion of the EPS according to the present invention had the effect of increasing IFN- ⁇ production of T cells in lymph nodes of mice and improving the expression rate of CD69 in the spleen.
  • Ingestion of EPS also had the effect of increasing the expression rate of CD69 on T cells in peripheral blood in humans.
  • measuring the CD69 expression rate of blood T cells can be achieved by replacing a conventional biopsy (biopsy) of collecting tissue using an endoscope or a needle with a body fluid (eg, blood) that is easier to collect. It is also useful as a technique (liquid biopsy) for diagnosis and treatment effect prediction.
  • the composition of the present invention can promote the maturation of CD8 + T cells from TEM to TEMRA. That is, the existence ratio of CD8 + TEMRA can be increased.
  • Human CD8 + T cells can be further classified by surface marker and function (cytokine production or cytotoxicity). Specifically, naive T cells (TN) (CCR7 + CD45RA +), central memory T cells (TCM) (CCR7 + CD45RA-), effector memory T cells (TEM) (CCR7-CD45RA-), CD45RA + effector memory T cells (TEMRA) (CCR7) -CD45RA +).
  • TN naive T cells
  • TCM central memory T cells
  • TEM effector memory T cells
  • TEMRA CD45RA + effector memory T cells
  • TEMRA CD45RA + effector memory T cells
  • CD45RA and CD45RO are isoforms, and CD45RA can be replaced with CD45RO in some cases.
  • Peripheral blood T cell lymphocytes can be divided into two different populations, CD45RA-CD45RO + and CD45RA + CD45RO-.
  • CD45RA is also expressed on medullary thymocytes and naive / resting peripheral CD4 + lymphocytes.
  • CD45RO is expressed on cortical thymocytes, CD4 + memory T cell subsets, and activated T cells. CD45RO is also weakly expressed on monocytes and granulocytes.
  • CD8 + TEM refers to CD8 + T cells that are CD45RO positive CCR7 negative.
  • CD8 + TEMRA refers to CD8 + T cells that are CD45RO negative CCR7 negative.
  • the CD8 + TEM presence ratio and CD8 + TEMRA presence ratio can be defined as the ratio of TEM cells (CD45RO-positive CCR7-negative) and TEMRA cells (CD45RO-negative CCR7-negative) in all CD8 + T cells (CD3 + CD8 +). The same applies to CD4 + T cells.
  • the effects of CD4 + / CD8 + T cell activation include, for example, reducing the risk of infection, reducing the risk of onset, suppressing the growth of pathogens in infected subjects, reducing the risk of infection by pathogens, Kill cells infected by pathogens, reduce the risk of developing cancer, suppress cancer cell growth, kill cancer cells, cure cancer, prevent cancer from metastasis and recurrence Prevention is included.
  • pathogens are various viruses, bacteria, fungi, mycoplasmas, parasites and the like.
  • cancer are malignant melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, stomach cancer, urothelial cancer, Merkel cell carcinoma and the like.
  • the compositions of the present invention can be used for activation of CD4 + / CD8 + T cells against any of these.
  • compositions of the present invention can be used even when CD4 + / CD8 + T cell activation is performed in conjunction with other therapies for the treatment of infectious diseases and cancer.
  • Such other therapies include drug therapy, surgery, radiation therapy, other immunotherapy (eg, cytokine therapy, administration of immunostimulants, immune cell transplantation, etc.), ingestion of health foods and supplements, exercise therapy Etc.
  • a cellular immunity activating composition comprising EPS as an active ingredient, which is based on promoting the production of interferon- ⁇ (IFN- ⁇ ) when spleen cells immunized with an antigen are cultured in a medium containing the antigen and EPS; -A composition for promoting IFN- ⁇ production by at least one cell selected from the group consisting of CD4 ⁇ T cells, CD8 ⁇ T cells, ⁇ T cells, Th17 cells, and ILC3 cells.
  • IFN- ⁇ interferon- ⁇
  • composition of the present invention can be a food composition or a pharmaceutical composition.
  • Foods and pharmaceuticals include not only those for humans but also those for non-human animals, unless otherwise indicated.
  • foods include general foods, functional foods, and nutritional compositions. Cooked on the basis of food), dietary meals, ingredient-adjusted meals, nursing care meals, and treatment support foods.
  • the food includes not only solids but also liquids such as beverages, drinks, liquid foods, and soups, unless otherwise specified.
  • Functional foods refer to foods that can impart predetermined functionality to living organisms, and include, for example, health foods including specified health foods (including conditional Tokuho [specified health foods]), functionally labeled foods, and nutritionally functional foods.
  • Functional foods special use foods, dietary supplements, health supplements, supplements (for example, tablets, coated tablets, dragees, capsules, various types of liquids such as liquids), beauty foods (for example, diet foods), etc. Includes a whole range of health foods.
  • the “functional food” includes a health food to which a health claim based on a food standard of the Codex (FAO / WHO Joint Food Standards Committee) is applied.
  • the composition of the present invention is suitable for ingestion / administration of a subject in which activation of CD4 + / CD8 + T cells is preferred.
  • Such subjects include infants, children, adults (aged 15 and over), middle-aged and elderly, elderly (aged 65 and over), persons ill and ill, pregnant women, maternal women, men, and women.
  • the composition of the present invention is particularly suitable for elderly people who are important to build a body that is less susceptible to illness in daily life because the active ingredient is EPS with a long eating experience and is suitable for long-term ingestion. .
  • composition of the present invention may be administered parenterally, for example, transluminally (gastrostomy, enterostomy), nasally, or orally. Oral administration is preferred.
  • the EPS content of the lactic acid bacterium in the composition of the present invention may be any amount as long as the desired effect is exhibited.
  • the composition, the age of the subject, weight, taking into account various factors such as symptoms, the dose or intake can be appropriately set, the amount of EPS of lactic acid bacteria per daily dose, For example, it can be 0.1 mg or more, preferably 0.6 mg or more, more preferably 1 mg or more, and particularly preferably 3 mg or more.
  • the upper limit of the amount of EPS per day, regardless of the lower limit, can be 500 mg or less, preferably 300 mg or less, and particularly preferably 250 mg or less. preferable.
  • the amount of EPS of lactic acid bacteria per administration or per serving, that is, per dose can be, for example, 0.03 mg or more, preferably 0.2 mg or more, and more preferably 1 mg or more.
  • the upper limit of the amount of EPS per dose can be 200 mg or less, preferably 100 mg or less, and more preferably 70 mg or less, regardless of the lower limit. It is particularly preferable that the content be 30 mg or less.
  • the daily dose as the composition can be, for example, 30 mg or more, and preferably 50 mg or more, It is more preferably at least 60 ⁇ g, particularly preferably at least 100 ⁇ g.
  • the upper limit of the daily amount as fermented milk in any case, the lower limit can be, for example, 1500 g or less, preferably 1200 g or less, more preferably 900 g or less More preferably, it is more preferably 600 mg or less.
  • a single dose of the composition can be, for example, 10 ⁇ g or more, preferably 20 ⁇ g or more, and more preferably 30 ⁇ g or more.
  • the upper limit of the single dose of the composition can be, for example, 500 g or less, preferably 400 g or less, more preferably 200 g or less. It is particularly preferable that the content be 125 mg or less.
  • the composition may be administered and taken once a day, or may be administered several times a day, for example, three times for each meal.
  • the composition contains, as an active ingredient, EPS of a lactic acid bacterium with a good dietary experience. Therefore, the composition of the present invention may be taken repeatedly or over a long period, for example, continuously administered for 3 days or more, preferably 1 week or more, more preferably 4 weeks or more, particularly preferably 1 month or more. Can be taken.
  • compositions of the present invention may include other active or nutritional ingredients that are food or pharmaceutically acceptable.
  • active or nutritional ingredients include amino acids (eg, lysine, arginine, glycine, alanine, glutamic acid, leucine, isoleucine, valine), carbohydrates (glucose, sucrose, fructose, maltose, trehalose, erythritol, maltitol, palatinose).
  • electrolytes eg, sodium, potassium, calcium, magnesium
  • vitamins eg, vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, Biotin, folic acid, pantothenic acid and nicotinic acids
  • minerals eg, copper, zinc, iron, cobalt, manganese
  • antibiotics eg, dietary fiber, proteins, lipids and the like.
  • the composition may further include a food or a pharmaceutically acceptable additive.
  • additives are inert carriers (solid or liquid carriers), excipients, surfactants, binders, disintegrants, lubricants, solubilizers, suspending agents, coatings, coloring.
  • water other aqueous solvents, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, water-soluble dextrin, sodium carboxymethyl starch, Pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, sucralose, stevia, aspartame, acesulfame potassium, Citric acid, lactic acid, malic acid, tartaric acid, phosphoric acid, acetic acid, fruit juice, vegetable juice and the like.
  • the pharmaceutical composition of the present invention is suitable for oral administration, such as tablets, granules, powders, pills, capsules and other solid preparations, liquid preparations such as liquid preparations, suspensions and syrups, gel preparations, aerosol preparations and the like. In any dosage form.
  • the food composition of the present invention may be prepared in any form such as solid, liquid, mixture, suspension, powder, granule, paste, jelly, gel, capsule and the like. Further, the food composition according to the present invention can be in any form such as dairy products, supplements, confectionery, beverages, drinks, seasonings, processed foods, prepared dishes, soups, and the like. More specifically, the composition of the present invention comprises milk drink, soft drink, fermented milk, yogurt, ice cream, tablet, cheese, bread, biscuit, cracker, pizza crust, powdered milk, liquid food, food for the sick , Nutritional foods, frozen foods, processed foods, and the like, and granules, powders, pastes, concentrated liquids, and the like for mixing and ingesting into beverages and foods.
  • the stage of compounding the lactic acid bacteria with EPS can be appropriately selected.
  • the blending step is not particularly limited as long as the EPS characteristics of the lactic acid bacteria are not significantly impaired.
  • it can be mixed and blended with raw materials at an early stage of production.
  • a lactic acid bacterium that produces EPS is added to the raw milk as a starter, fermented, and EPS is produced, whereby fermented milk containing EPS can be produced.
  • composition of the present invention can indicate that it can be used for activating CD4 + / CD8 + T cells, and can indicate that it is recommended to be taken to a specific subject.
  • Labeling can be direct or indirect, examples of direct labeling are descriptions on tangible objects such as products themselves, packages, containers, labels, tags, etc.
  • Examples of indirect labeling are: Includes advertising and promotion activities by location or means, such as websites, storefronts, brochures, exhibitions, seminars such as media seminars, books, newspapers, magazines, televisions, radios, mailings, emails, voices, etc.
  • EPS Extracellular Polysaccharide
  • Example 1 EPS in a culture obtained by culturing Lactobacillus delbrueckii subsp. Bulgaricus OLL1073R-1 in a 10% by mass skim milk medium was purified. That is, the denatured proteins were removed by adding trichloroacetic acid to a final concentration of 10% by mass to the culture cultured at 37 ° C. for 18 hours, and cold ethanol was added thereto, and the mixture was allowed to stand at 4 ° C. for 2 hours to precipitate EPS. I got something.
  • Example 2 Lactobacillus delbrueckii subsp. BulgaricusOLL1073R-1 and a thermophilus bacterium were added as a starter to a mixture containing raw milk, skim milk powder, cream, sugar, and stevia, and fermented to produce fermented milk.
  • an acidic milk beverage adjusted to be indistinguishable from fermented milk by adjusting pH and acidity was used.
  • the composition is 25 ⁇ l of TiterMax Gold (TiterMax), 5 ⁇ l of H-2D b human gp100 peptide (sequence KVPRNQDWL, MBL), 5 ⁇ l of IA b HBc helper peptide (sequence TPPAYRPPNAPIL, MBL), and 15 ⁇ l of PBS, a total of 50 ⁇ l / animal. It was used after emulsification. One week after the administration of the immune composition, all mice were euthanized, and the swollen right popliteal lymph node and spleen were collected. A cell suspension obtained by treating each organ was used in the test to compare the two groups.
  • the H-2D b human gp100 peptide can stimulate antigen-specific stimuli to cytotoxic T cells (CTLs), it can be used to confirm cytokine production, cell surface antigen expression, and cytotoxic activity. It is a reagent that can be used for stimulating DNA, inducing antigen-specific CTL, and the like.
  • the IA b HBc helper peptide is a reagent that can be used for production of cytokines, assistance for induction of antigen-specific CTL, and the like, because it can stimulate CD4-positive T cells with antigen-specific stimulation.
  • Antibody MIX containing anti-mouse CD3 antibody, anti-mouse CD4 antibody, anti-mouse CD8 antibody, anti-mouse CD69 antibody (all of which are BD Japan) fluorescently labeled spleen cells adjusted to 1 ⁇ 10 6 cells / animal The cells were stained, and CD69 expression rates of CD4 + T cells and CD8 + T cells were measured using a flow cytometer (FACS Verse, BD Japan).
  • BD vacutainer CPT blood collection tube for mononuclear cell separation
  • PBMC adjusted to a cell count of 1 ⁇ 10 6 cells / sample was subjected to antibody MIX containing anti-human CD3 antibody, anti-human CD8 antibody, anti-human CD45RO antibody, anti-human CCR7 antibody, and anti-human CD69 antibody (all BD Japan).
  • Stain and measure CD4 + TEM and CD4 + TEMRA abundance ratio, CD8 + TEM and CD8 + TEMRA abundance ratio, and CD69 expression ratio of CD4 + TEM and CD8 + TEMRA using flow cytometer (FACS Verse, Japan BD) did.
  • the progression of maturation from TEM to TEMRA was observed in the peripheral blood CD8 + T cells of the fermented milk group as compared with the control group (FIGS.
  • the CD4 + TEM presence ratio and CD4 + TEMRA presence ratio were defined as the ratio of TEM cells (CD45RO-positive CCR7-negative) and TEMRA cells (CD45RO-negative CCR7-negative) in all CD4 + T cells (CD3 + CD4 +). The same applies to CD8 + T cells.
  • the CD69 expression rate of CD4 + TEM was defined as the ratio of CD69-positive cells in all CD4 + TEM cells (CD3-positive CD4-positive and CD45RO-positive CCR7-negative). The same was applied to the CD69 expression rate of CD8 + TEMRA.
  • EPS contained in the fermented milk used in this test had the effect of promoting IFN- ⁇ production in lymph nodes (Example 1), and that it was specifically induced to antigens. Since the obtained TEM cells differentiated into more mature TEMRA cells (Example 2), the expression of CD69 on blood T cells in this test is considered to reflect the reactivity to antigen. Normally, it is difficult to examine CD69 expression of T cells in lymph nodes in humans. However, from the results of Examples 1 and 2, it is only necessary to examine the expression of CD69 on blood T cells (liquid biopsy) to obtain T cells against antigen. It was found that the reaction could be examined.

Abstract

The present invention addresses the problem of providing a composition for activating CD4+ T cells and/or CD8+ T cells. Provided is a composition for activating CD4+ T cells and/or CD8+ T cells, the composition including an extracellular polysaccharide of a lactic acid bacterium. An example of a preferred lactic acid bacterium is a bacterium of the genus Lactobacillus (Lactobacillus delbrueckii subsp. bulgaricus OLL1073 R-1).

Description

Ｔ細胞を活性化するための組成物Compositions for activating T cells
　本発明は、Ｔ細胞を活性化するための、乳酸菌の培養物を含有する組成物に関する。 The present invention relates to a composition containing a culture of lactic acid bacteria for activating T cells.
　免疫系は自然免疫と獲得免疫に大別され、自然免疫の手助けによる獲得免疫の活性化が、病原体の排除やがん細胞に対する傷害などには重要である。この特異的で強力な免疫機構は、抗原に対してＴ細胞が活性化することで引き起こされる。Ｔ細胞の活性化を簡便に促進することができれば、非常に有用である。 The immune system is broadly divided into innate immunity and acquired immunity. Activation of acquired immunity with the help of innate immunity is important for eliminating pathogens and damaging cancer cells. This specific and powerful immune mechanism is triggered by the activation of T cells against antigens. It would be very useful if T cell activation could be easily promoted.
　特許文献１は、ラクトバチルス・リューテリ（Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｒｅｕｔｅｒｉ）株を用いる哺乳類における免疫機能の改善方法を開示する。詳細には、ラクトバチルス・リューテリ株の細胞を含むそしゃく錠剤を２８日間摂取した被験者からの回腸生検材料において、有意に高い量のＣＤ４＋細胞が存在したとの実験結果に基づき、そのような菌株の細胞を含有する製品を提案する。また特許文献２は、バシラス・コアギュランス（Ｂａｃｉｌｌｕｓ　ｃｏａｇｕｌａｎｓ）細菌や、該細菌の断片等を微生物病原体に感染した被験体へ投与する工程を含む、被験体において微生物病原体に対する免疫応答を増強する方法を開示する。詳細には、５億ＣＦＵのバシラス・コアギュランスＧＢＩ－３０、ＡＴＣＣ　ＰＴＡ－６０８６を含有する１カプセルを生存可能な３０日間毎日消費した被検体において、ＩＬ－８産生やＩＦＮ－γ産生が変化したこと等に基づき、バシラス・コアギュランス細菌を含む組成物を、病原体に対する免疫応答を増強するために用いることを提案している。 Patent Document 1 discloses a method for improving an immune function in a mammal using a Lactobacillus reuteri strain. In particular, based on the experimental results that a significantly higher amount of CD4 + cells was present in ileal biopsies from subjects who had taken masticated tablets containing cells of the Lactobacillus reuteri strain for 28 days, such strains were used. We propose a product containing cells. Patent Document 2 discloses a method for enhancing an immune response to a microbial pathogen in a subject, including a step of administering a Bacillus coagulans bacterium or a fragment thereof to the subject infected with the microbial pathogen. I do. Specifically, IL-8 production and IFN-γ production were altered in subjects who consumed one capsule daily containing 500 million CFU of Bacillus coagulans GBI-30 and ATCC @ PTA-6086 for a viable 30 days. On the basis of the above, it has been proposed to use a composition containing Bacillus coagulans bacteria to enhance an immune response against a pathogen.
　一方、乳酸菌は、その醗酵過程で種々の物質を産生するが、その一つが菌体外多糖（Exopolysaccharides; ＥＰＳ）である。乳酸菌のＥＰＳにはいくつかの生理活性、例えば、自己免疫疾患予防作用（特許文献３）、ＮＫ細胞活性化作用（特許文献４）、肺炎球菌感染予防作用（特許文献５）、疲労感の改善作用（特許文献６）、抗インフルエンザ薬による獲得免疫機能低下抑制作用（特許文献７）が知られている。 乳酸 On the other hand, lactic acid bacteria produce various substances during the fermentation process, one of which is exopolysaccharides (EPS). The EPS of lactic acid bacteria has several physiological activities, for example, an autoimmune disease preventive action (Patent Document 3), an NK cell activating action (Patent Document 4), a pneumococcal infection preventive action (Patent Document 5), and an improvement in fatigue. An effect (Patent Document 6) and an inhibitory effect on acquired immune function decrease by an anti-influenza drug (Patent Document 7) are known.
特表2006-506371号公報JP 2006-506371 A 特表2012-525428号公報JP 2012-525428 A 特開2000-247895号公報JP 2000-247895 A 特開2005-194259号公報JP 2005-194259 A 国際公開WO2015/133638International Publication WO2015 / 133638 国際公開WO2017/183595International Publication WO2017 / 183595 国際公開WO2011/065300International Publication WO2011 / 065300
　Ｔ細胞の活性化による感染症やがんの治療は、従来の細胞毒性作用を示す抗癌剤治療、外科的切除、放射線治療と比較して患者の負担は少ないと考えられる。そして食経験のある物質を有効成分とし、Ｔ細胞を活性化することができる組成物があれば望ましいことはいうまでもない。またＴ細胞が活性化されたことを、簡易に測定できる方法があれば、一層望ましい。 治療 Treatment of infectious diseases and cancer by activating T cells is considered to be less burdensome for patients than conventional anti-cancer drug treatment, surgical resection, and radiation treatment that exhibit cytotoxic effects. Needless to say, it is desirable to have a composition capable of activating T cells by using a substance having a dietary experience as an active ingredient. It would be more desirable if there was a method that could easily measure the activation of T cells.
　本発明者らは、Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1で発酵したヨーグルト、または該乳酸菌により産生される菌体外多糖（Exopolysaccharide：EPS）により、次の知見を得た。
・抗原反応を模したモデルマウスで、ＥＰＳの摂取により、脾臓内のＣＤ４＋Ｔ細胞およびＣＤ８＋Ｔ細胞の活性化率（ＣＤ６９発現率）がいずれも増加した。さらに、腫脹したリンパ節内のＴ細胞を刺激した際に、産生されるＩＦＮ－γが顕著に増加した。
・健常高齢者を対象にした臨床試験で、ヨーグルトの摂取により、末梢血中のＣＤ４＋ＴＥＭ細胞およびＣＤ８＋ＴＥＭＲＡ細胞のいずれも活性化率（ＣＤ６９発現率）が向上した。また、ＣＤ８＋Ｔ細胞についてはＴＥＭ細胞からＴＥＭＲＡ細胞への成熟も認められた。 Bulgaricus OLL1073R-1 or the yogurt fermented with Lactobacillus delbrueckii subsp. Bulgaricus or the extracellular polysaccharide (Exopolysaccharide: EPS) produced by the lactic acid bacterium has obtained the following knowledge.
In a model mouse simulating an antigen reaction, the ingestion of EPS increased the activation rate (CD69 expression rate) of CD4 + T cells and CD8 + T cells in the spleen. Furthermore, when stimulating T cells in swollen lymph nodes, the IFN-γ produced was significantly increased.
-In a clinical test for healthy elderly people, ingestion of yogurt improved the activation rate (CD69 expression rate) of both CD4 + TEM cells and CD8 + TEMRA cells in peripheral blood. In addition, maturation of CD8 + T cells from TEM cells to TEMRA cells was also observed.
　また、血液検体から末梢血単核細胞（ＰＢＭＣ）を分離してフローサイトメーターでＴ細胞のＣＤ６９発現率を測定するだけで、抗原に対するＴ細胞の活性化を簡便に判定できることを見出した。リンパ節におけるＣＤ６９分子の発現は、Ｔ細胞が抗原を認識して活性化する際に一過性に上昇し、活性化マーカーとして広く用いることができる、というのが免疫学の常識であるが、本発明者らは、血液中Ｔ細胞のＣＤ６９分子の発現も、リンパ節における抗原への反応を反映しており、リキッドバイオプシーとして有用であることを見出した。 Further, it has been found that activation of T cells to antigens can be easily determined only by separating peripheral blood mononuclear cells (PBMC) from a blood sample and measuring the CD69 expression rate of T cells with a flow cytometer. It is common knowledge in immunology that the expression of CD69 molecule in lymph nodes is transiently increased when T cells recognize and activate antigens and can be widely used as an activation marker. The present inventors have found that the expression of the CD69 molecule of blood T cells also reflects the response to antigen in lymph nodes, and is useful as a liquid biopsy.
　このような知見に基づき、本発明を完成した。本発明は、以下を提供する。
［１］　乳酸菌の菌体外多糖を含む、ＣＤ４＋Ｔ細胞およびＣＤ８＋Ｔ細胞の少なくとも一方を活性化するための組成物。
［２］　ＣＤ４＋Ｔ細胞およびＣＤ８＋Ｔ細胞の少なくとも一方におけるＣＤ６９発現率を高めるための、１に記載の組成物。
［３］　ＣＤ６９発現率が、被験者に由来する末梢血Ｔ細胞の分析値に基づく値である、２に記載の組成物。 
［４］　ＣＤ８＋ＴＥＭからＣＤ８＋ＴＥＭＲＡへの成熟を促進するための、１～３のいずれか１項に記載の組成物。
［５］　乳酸菌が、ラクトバチルス属に分類されるものである、１～４のいずれか１項に記載の組成物。
［６］　乳酸菌が、ラクトバチルス・デルブルッキー・サブスピーシーズ・ブルガリクスに分類されるものである、１～５のいずれか１項に記載の組成物。
［７］　発酵乳である、１～６のいずれか１項に記載の組成物。
［８］　感染症またはがんの発症リスクを低減するための 非医療的方法であって、有効量の乳酸菌の菌体外多糖を含む組成物を対象に摂取させ、対象におけるＣＤ４＋Ｔ細胞、およびＣＤ８＋Ｔ細胞の少なくとも一方におけるＣＤ６９発現率を高める工程を含む、方法。
［９］　乳酸菌の菌体外多糖を用いる、ＣＤ４＋Ｔ細胞およびＣＤ８＋Ｔ細胞の少なくとも一方におけるＣＤ６９発現率を高める方法（ヒトに対する医療行為を除く。）。 Based on such knowledge, the present invention has been completed. The present invention provides the following.
[1] A composition for activating at least one of CD4 + T cells and CD8 + T cells, comprising a lactic acid bacteria exopolysaccharide.
[2] The composition according to 1, for increasing the CD69 expression rate in at least one of CD4 + T cells and CD8 + T cells.
[3] The composition according to 2, wherein the CD69 expression rate is a value based on an analysis value of peripheral blood T cells derived from the subject.
[4] The composition according to any one of items 1 to 3, for promoting maturation of CD8 + TEM to CD8 + TEMRA.
[5] The composition according to any one of items 1 to 4, wherein the lactic acid bacteria are classified into the genus Lactobacillus.
[6] The composition according to any one of [1] to [5], wherein the lactic acid bacterium is classified into Lactobacillus del Brooke subspecies Bulgaricus.
[7] The composition according to any one of 1 to 6, which is a fermented milk.
[8] A non-medical method for reducing the risk of developing an infectious disease or cancer, comprising ingesting a composition containing an effective amount of an exopolysaccharide of a lactic acid bacterium into a subject, the CD4 + T cell, and the CD8 + T Increasing the CD69 expression rate in at least one of the cells.
[9] A method for increasing the CD69 expression rate in at least one of CD4 + T cells and CD8 + T cells using an exopolysaccharide of a lactic acid bacterium (excluding medical practice for humans).
  また、本発明は、以下も提供する。
［１０］乳酸菌の菌体外多糖、またはこれを含む組成物を対象に（経口）投与する（摂取させる）工程を含む、ＣＤ４＋Ｔ細胞およびＣＤ８＋Ｔ細胞の少なくとも一方を活性化するための方法。
［１１］ＣＤ４＋Ｔ細胞およびＣＤ８＋Ｔ細胞の少なくとも一方を活性化するための組成物の製造における、乳酸菌の菌体外多糖の使用。
［１２］ＣＤ４＋Ｔ細胞およびＣＤ８＋Ｔ細胞の少なくとも一方の活性化方法において使用するための、乳酸菌の菌体外多糖、またはそれを含む組成物。
［１３］乳酸菌の菌体外多糖、またはそれを含む組成物を対象に（経口）投与する（摂取させる）工程を含む、対象におけるＣＤ４＋Ｔ細胞およびＣＤ８＋Ｔ細胞の少なくとも一方の活性化方法。
［１５］乳酸菌の菌体外多糖、またはそれを含む組成物を対象に（経口）投与する（摂取させる）工程を含む、対象におけるＣＤ６９発現率を向上させる、非治療的方法。
［１６］乳酸菌の菌体外多糖と医薬として許容可能な添加物を配合する工程を含む、ＣＤ４＋Ｔ細胞およびＣＤ８＋Ｔ細胞の少なくとも一方の活性化のための組成物の製造方法。 The present invention also provides the following.
[10] A method for activating at least one of CD4 + T cells and CD8 + T cells, comprising a step of (orally) administering (ingesting) an exopolysaccharide of a lactic acid bacterium or a composition containing the same to a subject.
[11] Use of an exopolysaccharide of a lactic acid bacterium in production of a composition for activating at least one of CD4 + T cells and CD8 + T cells.
[12] An exopolysaccharide of a lactic acid bacterium, or a composition containing the same, for use in a method for activating at least one of CD4 + T cells and CD8 + T cells.
[13] A method for activating at least one of CD4 + T cells and CD8 + T cells in a subject, comprising a step of (orally) administering (ingesting) the exopolysaccharide of a lactic acid bacterium or a composition containing the same to the subject.
[15] A non-therapeutic method for improving the CD69 expression rate in a subject, comprising the step of (orally) administering (ingesting) the exopolysaccharide of a lactic acid bacterium or a composition containing the same to the subject.
[16] A method for producing a composition for activating at least one of CD4 + T cells and CD8 + T cells, comprising a step of mixing an exopolysaccharide of a lactic acid bacterium with a pharmaceutically acceptable additive.
　本発明はまた、以下も提供する。
［１７］末梢血Ｔ細胞のＣＤ６９の、バイオマーカーとしての使用。
［１８］抗原特異的な活性化の指標としての、１７に記載の方法。
［１９］抗原特異的な免疫活性化の検査のための方法であって、末梢血Ｔ細胞におけるＣＤ６９を分析する工程を含む、方法。 The present invention also provides the following.
[17] Use of CD69 of peripheral blood T cells as a biomarker.
[18] The method according to 17, as an indicator of antigen-specific activation.
[19] A method for examining antigen-specific immune activation, comprising a step of analyzing CD69 in peripheral blood T cells.
　本発明によれば、ＣＤ４＋Ｔ細胞およびＣＤ８＋Ｔ細胞の少なくとも一方の活性化を効果的に促進できる。
　本発明の組成物の摂取・投与により、ＣＤ４＋Ｔ細胞およびＣＤ８＋Ｔ細胞の少なくとも一方の活性化を効果的に促進できるために、対象において腫瘍の増殖抑制や病原菌排除の促進が期待できる。
　本発明により、血液中T細胞のCD69の、リキッドバイオプシーとしての用途が提供される。 According to the present invention, activation of at least one of CD4 + T cells and CD8 + T cells can be effectively promoted.
Since the activation and / or administration of at least one of CD4 + T cells and CD8 + T cells can be effectively promoted by ingestion / administration of the composition of the present invention, suppression of tumor growth and promotion of elimination of pathogenic bacteria in a subject can be expected.
The present invention provides the use of CD69 of blood T cells as a liquid biopsy.
Ａ：ＥＰＳを経口投与したマウスのリンパ節Ｔ細胞刺激時のＩＦＮ－γ産生、Ｂ：ＥＰＳを経口投与したマウスの脾臓細胞のＣＤ６９発現率。A: IFN-γ production upon stimulation of lymph node T cells in mice orally administered with EPS, B: CD69 expression rate in spleen cells of mice orally administered with EPS. Ａ：発酵乳を摂取した対象の末梢血ＣＤ８＋Ｔ細胞におけるＴＥＭの存在比率、Ｂ：発酵乳を摂取した対象の末梢血ＣＤ８＋Ｔ細胞におけるＴＥＭＲＡの存在比率、Ｃ：発酵乳を摂取した対象の末梢血ＣＤ４＋ＴＥＭにおけるＣＤ６９発現率、Ｄ：発酵乳を摂取した対象の末梢血ＣＤ８＋ＴＥＭＲＡにおけるＣＤ６９発現率（点線：対照群、実線：発酵乳群）。A: Percentage of TEM in peripheral blood CD8 + T cells of a subject who has consumed fermented milk, B: Percentage of TEMRA in peripheral blood CD8 + T cells of a subject who has consumed fermented milk, C: Peripheral blood CD4 + TEM of a subject who has consumed fermented milk , D: CD69 expression rate in peripheral blood CD8 + TEMRA of a subject who ingested fermented milk (dotted line: control group, solid line: fermented milk group).
　以下、本発明を詳細に説明する。
　本発明は、乳酸菌の産生する菌体外多糖（ＥＰＳ）を有効成分とする組成物に関する。より詳細には、ＥＰＳを有効成分とし、ＣＤ４＋Ｔ細胞およびＣＤ８＋Ｔ細胞の少なくとも一方（以下、「ＣＤ４＋／ＣＤ８＋Ｔ細胞」という。）を活性化するための組成物に関する。 Hereinafter, the present invention will be described in detail.
The present invention relates to a composition containing an exopolysaccharide (EPS) produced by a lactic acid bacterium as an active ingredient. More specifically, the present invention relates to a composition for activating at least one of CD4 + T cells and CD8 + T cells (hereinafter, referred to as “CD4 + / CD8 + T cells”) using EPS as an active ingredient.
［有効成分］
  本発明の組成物は、有効成分として乳酸菌のＥＰＳを含む。 [Active ingredient]
The composition of the present invention contains lactic acid bacteria EPS as an active ingredient.
　乳酸菌とは、ブドウ糖を資化して対糖収率で５０％以上の乳酸を生産する微生物の総称で、生理学的性質としてグラム陽性菌の球菌または桿菌で、運動性なし、多くの場合胞子形成能なし（バシラス・コアギュランスのように胞子形成能のある乳酸菌もある。）、カタラーゼ陰性などの特徴を有しているものである。乳酸菌は古来、発酵乳等を介して世界各地で食されており、極めて安全性の高い微生物といえる。乳酸菌は、複数の属に分類される。本発明の組成物に含まれる乳酸菌のＥＰＳは、好ましくはラクトバチルス（Lactobacillus）属に分類されるラクトバチルス属乳酸菌により産生されたものである。 Lactic acid bacterium is a general term for microorganisms that utilize glucose to produce lactic acid at a yield of 50% or more in terms of sugar yield, and are physiologically gram-positive cocci or rods that have no motility and often have a sporulating ability. None (there is also a lactic acid bacterium capable of forming spores such as Bacillus coagulans) and catalase negative. Lactic acid bacteria have been eaten around the world through fermented milk and the like since ancient times, and can be said to be extremely safe microorganisms. Lactic acid bacteria are classified into several genera. The EPS of the lactic acid bacterium contained in the composition of the present invention is preferably produced by a lactic acid bacterium belonging to the genus Lactobacillus classified into the genus Lactobacillus.
　本発明の組成物に用いられるＥＰＳは、目的の効果を有する限り、特に限定されない。乳酸菌が産生するＥＰＳは、構造的に、ホモ多糖であるものとヘテロ多糖であるもの（例えば、ガラクトースとグルコースから構成されるもの）に分類され、リン酸化や硫酸化などの修飾を受けている場合もあるが、いずれも本発明の組成物の有効成分として用いることができる。好ましいＥＰＳの例の一つは、中性多糖体、および中性多糖体にリン酸基が付加した酸性多糖体の少なくとも一方を含むものである。このようなＥＰＳは、ラクトバチルス・デルブルッキー・サブスピーシーズ・ブルガリクス（Lactobacillus delbrueckii subsp. bulgaricus）や、ラクトコッカス・ラクティス・サブスピーシーズ・クレモリス（Lactococcus lactis subsp. cremoris）によって産生されることが知られている。本発明に用いられるＥＰＳは、一種でもよく、２種以上の組み合わせであってもよい。 ＥＰ The EPS used in the composition of the present invention is not particularly limited as long as it has the desired effect. EPS produced by lactic acid bacteria is structurally classified into homopolysaccharides and heteropolysaccharides (for example, those composed of galactose and glucose), and is modified by phosphorylation or sulfation. In some cases, any of them can be used as an active ingredient of the composition of the present invention. One example of a preferred EPS is one containing a neutral polysaccharide and / or an acidic polysaccharide having a phosphate group added to the neutral polysaccharide. Such EPS is known to be produced by Lactobacillus delbruchy subspecies bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus) or Lactococcus lactis subspecies cremoris (Lactococcus lactis subsp. Cremoris). ing. The EPS used in the present invention may be one kind or a combination of two or more kinds.
　本発明の組成物に用いられる特に好ましいＥＰＳを産生する乳酸菌の例は、ラクトバチルス属乳酸菌である。ラクトバチルス属乳酸菌としては、例えば、ブルガリクス種、カゼイ種、アシドフィルス種、プランタラム種などが挙げられる。これらのラクトバチルス属乳酸菌の中でも、本発明では、ブルガリクス種に分類される乳酸菌（ブルガリクス菌とも称する）であることが好ましい。さらに、ラクトバチルス属乳酸菌の中でも、ラクトバチルス・デルブルッキー・サブスピーシーズ・ブルガリクス（Lactobacillus delbrueckii subsp. bulgaricus）に分類されるものであることがより好ましい。特に好ましい態様においては、乳酸菌は、ラクトバチルス・デルブルッキー・サブスピーシーズ・ブルガリクスＯＬＬ１０７３　Ｒ－１菌（受託番号：ＦＥＲＭ  ＢＰ－１０７４１）（「ブルガリクス菌Ｒ－１株」と称することがある。）である。すなわち、本発明の組成物に用いられるＥＰＳの特に好ましい例の一つは、ブルガリクス菌Ｒ－１株が産生するＥＰＳである。 Examples of particularly preferred EPS-producing lactic acid bacteria used in the composition of the present invention are Lactobacillus lactic acid bacteria. Examples of lactic acid bacteria of the genus Lactobacillus include Bulgaricus spp., Casei spp., Acidophilus spp., Plantarum spp., And the like. Among these lactobacilli of the genus Lactobacillus, in the present invention, lactic acid bacteria classified as Bulgaricus species (also referred to as Bulgaricus bacteria) are preferable. Furthermore, among lactic acid bacteria belonging to the genus Lactobacillus, it is more preferable that the lactic acid bacterium is classified into Lactobacillus delbrueckii subsp. Bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus). In a particularly preferred embodiment, the lactic acid bacterium may be referred to as Lactobacillus delbruchy subspecies Bulgaricus OLL1073 @ R-1 (accession number: FERM @ BP-10741) ("Bulgarix strain R-1"). ). That is, one of the particularly preferred examples of the EPS used in the composition of the present invention is EPS produced by the B. bulgaricus strain R-1.
  ブルガリクス菌Ｒ－１株は、独立行政法人製品評価技術基盤機構特許生物寄託センター（ＩＰＯＤ，ＮＩＴＥ）（日本国千葉県木更津市かずさ鎌足２－５－８  １２０号室）にブタペスト条約に基づき、国際寄託されている（寄託者：株式会社 明治、寄託日：２００６年１１月２９日、受託番号：ＦＥＲＭ  ＢＰ－１０７４１）。 Based on the Budapest Treaty, the Bulgarix strain R-1 was approved by the National Institute of Technology and Evaluation Patent Organism Depositary Center (IPOD, NITE) (Room 2-5-8 @ 120 Kazusa Kamatari, Kisarazu-shi, Chiba, Japan) Deposited internationally (depositor: Meiji Co., Ltd., date of deposit: November 29, 2006, accession number: FERM @ BP-10741).
　本発明の組成物に含まれる乳酸菌のＥＰＳは、乳酸菌発酵物として含まれていてもよい。乳酸菌発酵物には、乳酸菌による発酵物自体のほか、その処理物が含まれる。乳酸菌発酵物自体には、例えば発酵乳（具体的には、ヨーグルト等）が含まれる。処理物には、例えば、粗精製物、発酵物をろ過、遠心分離、または膜分離で除菌して得られた培養濾液や培養上清液、培養濾液・培養上清液を濃縮した濃縮物、濃縮物の乾燥物が含まれる。 The EPS of lactic acid bacteria contained in the composition of the present invention may be contained as a fermented product of lactic acid bacteria. The fermented product of lactic acid bacteria includes a fermented product of lactic acid bacteria itself and a processed product thereof. The fermented lactic acid bacteria itself includes, for example, fermented milk (specifically, yogurt or the like). The processed product includes, for example, a crude filtrate, a culture filtrate or a culture supernatant obtained by removing bacteria by filtration, centrifugation, or membrane separation, and a concentrate obtained by concentrating the culture filtrate or culture supernatant. , And dried concentrates.
　乳酸菌のＥＰＳの調製方法は従来技術を利用することができ、より詳細な条件が必要な場合は、本明細書の実施例等を参照することができる。また、乳酸菌のＥＰＳを乳酸菌発酵物として調製する場合は、ＥＰＳを産生する乳酸菌をスターターとして原料乳に添加し、発酵させ、ＥＰＳを発酵物中に産生させることで、ＥＰＳを含む発酵乳が製造できる。発酵の際の条件、例えば、原料乳、発酵温度、発酵時間は、用いる乳酸菌がＥＰＳを産生することができれば特に制限されず、当業者であれば、適宜設定することができる。 従 来 Conventional methods can be used for preparing EPS of lactic acid bacteria. If more detailed conditions are required, the examples and the like in the present specification can be referred to. When EPS of lactic acid bacteria is prepared as a fermented product of lactic acid bacteria, lactic acid bacteria producing EPS are added to the raw milk as a starter, fermented, and EPS is produced in the fermented product, whereby fermented milk containing EPS is produced. it can. Conditions for fermentation, such as raw milk, fermentation temperature, and fermentation time, are not particularly limited as long as the lactic acid bacteria used can produce EPS, and those skilled in the art can appropriately set them.
［用途］
　本発明の組成物は、ＣＤ４＋Ｔ細胞およびＣＤ８＋Ｔ細胞の少なくとも一方（ＣＤ４＋／ＣＤ８＋Ｔ細胞）を活性化するために用いることができる。本発明に関し、ＣＤ４＋／ＣＤ８＋Ｔ細胞の活性化を促進するというときは、特に記載した場合を除き、抗原特異的なＣＤ４＋／ＣＤ８＋Ｔ細胞の活性化を促進することをいう。ＣＤ４＋／ＣＤ８＋Ｔ細胞の活性化を促進することには、ＣＤ４＋／ＣＤ８＋Ｔ細胞においてＣＤ６９発現率を高めること、ＣＤ８＋ＴＥＭからＣＤ８＋ＴＥＭＲＡへの成熟の進行を促進することが含まれる。 [Use]
The composition of the present invention can be used to activate at least one of CD4 + T cells and CD8 + T cells (CD4 + / CD8 + T cells). In the present invention, when promoting the activation of CD4 + / CD8 + T cells, it refers to promoting the activation of antigen-specific CD4 + / CD8 + T cells, unless otherwise specified. Enhancing the activation of CD4 + / CD8 + T cells includes increasing CD69 expression in CD4 + / CD8 + T cells and promoting the progression of maturation from CD8 + TEM to CD8 + TEMRA.
　本発明の組成物は、ＣＤ４＋／ＣＤ８＋Ｔ細胞においてＣＤ６９発現率を高めることができる。ＣＤ６９は、Ｔ細胞やＢ細胞を抗原で刺激すると数時間以内に発現が上昇するために、初期の活性化マーカー分子として、リンパ球の活性化の指標となる。ＣＤ６９発現率とは、全当該細胞に占めるＣＤ６９を発現している細胞の割合をいう。蛍光標識した抗ＣＤ６９抗体を用いてフローサイトメーターによる検出を行い、全当該細胞における標識細胞の割合として測定できる。ＣＤ６９発現率、および後述するＴＥＭおよびＴＥＭＲＡの存在比率の評価において、ＣＤ３陽性かつＣＤ４陽性の単核細胞をＣＤ４＋Ｔ細胞とすることができ、ＣＤ３陽性かつＣＤ８陽性の単核細胞をＣＤ８＋Ｔ細胞とすることができる。 組成 The composition of the present invention can increase the CD69 expression rate in CD4 + / CD8 + T cells. Since CD69 increases its expression within several hours when T cells or B cells are stimulated with an antigen, it serves as an early activation marker molecule and serves as an indicator of lymphocyte activation. The expression ratio of CD69 refers to the ratio of cells expressing CD69 to all the cells. Detection by a flow cytometer is performed using a fluorescently labeled anti-CD69 antibody, which can be measured as the ratio of labeled cells in all the cells. In the evaluation of the CD69 expression rate and the abundance ratio of TEM and TEMRA described below, CD3 + and CD4 + mononuclear cells can be CD4 + T cells, and CD3 + and CD8 + mononuclear cells are CD8 + T cells. Can be.
　本発明の組成物によるＣＤ６９発現率の向上は、脾臓においても確認でき、また末梢血においても確認できる。そのため、ＣＤ６９発現率は、被験者に由来する末梢血Ｔ細胞の分析値に基づく値とすることができる。分析とは、分析対象分子の、存在の有無、および存在する場合はその程度（量）のいずれかを測定することをいう。本発明者らの検討によると、本発明に係るＥＰＳの摂取は、マウスのリンパ節内Ｔ細胞のＩＦＮ－γ産生を高めるとともに、脾臓においてＣＤ６９の発現率を向上させる効果を有した。ＥＰＳの摂取はまた、ヒトにおいては末梢血中のＴ細胞のＣＤ６９の発現率を向上する効果を有した。そのためヒトにおける末梢血中のＴ細胞のＣＤ６９の発現率向上の際には、リンパ節内Ｔ細胞のＩＦＮ－γ産生の向上と脾臓でのＣＤ６９の発現率の向上が起こっているはずであり、末梢血中のＴ細胞のＣＤ６９の発現率向上は抗原への反応性の向上を反映していると考えられる。すなわち、血中Ｔ細胞のＣＤ６９発現率を測定することは、内視鏡や針を使って組織を採取する従来の生検（バイオプシー）に代えて、より採取容易な体液（例えば、血液）を用いて診断や治療効果予測のための技術（リキッドバイオプシー）としても有用である。 (4) The improvement of the CD69 expression rate by the composition of the present invention can be confirmed in the spleen and also in the peripheral blood. Therefore, the CD69 expression rate can be a value based on the analysis value of peripheral blood T cells derived from the subject. The analysis refers to measuring any of the presence or absence and, if present, the degree (amount) of the molecule to be analyzed. According to the studies by the present inventors, ingestion of the EPS according to the present invention had the effect of increasing IFN-γ production of T cells in lymph nodes of mice and improving the expression rate of CD69 in the spleen. Ingestion of EPS also had the effect of increasing the expression rate of CD69 on T cells in peripheral blood in humans. Therefore, when improving the expression rate of CD69 of T cells in peripheral blood in humans, the IFN-γ production of T cells in lymph nodes and the expression rate of CD69 in spleen should be improved. It is considered that the improvement in the CD69 expression rate of T cells in peripheral blood reflects an increase in the reactivity to antigen. That is, measuring the CD69 expression rate of blood T cells can be achieved by replacing a conventional biopsy (biopsy) of collecting tissue using an endoscope or a needle with a body fluid (eg, blood) that is easier to collect. It is also useful as a technique (liquid biopsy) for diagnosis and treatment effect prediction.
　本発明の組成物は、ＣＤ８＋Ｔ細胞のＴＥＭからＴＥＭＲＡへの成熟を促進することができる。すなわち、ＣＤ８＋ＴＥＭＲＡの存在比率を高めることができる。ヒトのＣＤ８＋Ｔ細胞は表面マーカーおよび機能（サイトカイン産生や細胞傷害）によって、さらに分類することができる。詳細には、ナイーブＴ細胞（ＴＮ）（ＣＣＲ７＋ＣＤ４５ＲＡ＋）、セントラルメモリーＴ細胞（ＴＣＭ）（ＣＣＲ７＋ＣＤ４５ＲＡ－）、エフェクターメモリーＴ細胞（ＴＥＭ）（ＣＣＲ７－ＣＤ４５ＲＡ－）、ＣＤ４５ＲＡ＋エフェクターメモリーＴ細胞（ＴＥＭＲＡ）（ＣＣＲ７－ＣＤ４５ＲＡ＋）である。ＴＥＭＲＡは、メモリーＴ細胞のサブセットの中で，分化・成熟の進んだものであるため、より活性化された状態であるといえる。なおＣＤ４５ＲＡとＣＤ４５ＲＯはアイソフォームであり、ＣＤ４５ＲＡは、場合によりＣＤ４５ＲＯと読み替えることができる。末梢血Ｔ細胞リンパ球は、２つの異なるポピュレーション、ＣＤ４５ＲＡ－ＣＤ４５ＲＯ＋とＣＤ４５ＲＡ＋ＣＤ４５ＲＯ－に分けることができる。ＣＤ４５ＲＡはまた、髄質胸腺細胞及びナイーブ／休止期の末梢ＣＤ４＋リンパ球に発現する。ＣＤ４５ＲＯは皮質胸腺細胞、ＣＤ４＋メモリーＴ細胞サブセット、及び活性化Ｔ細胞に発現する。ＣＤ４５ＲＯは、単球と顆粒球にも弱く発現する。 組成 The composition of the present invention can promote the maturation of CD8 + T cells from TEM to TEMRA. That is, the existence ratio of CD8 + TEMRA can be increased. Human CD8 + T cells can be further classified by surface marker and function (cytokine production or cytotoxicity). Specifically, naive T cells (TN) (CCR7 + CD45RA +), central memory T cells (TCM) (CCR7 + CD45RA-), effector memory T cells (TEM) (CCR7-CD45RA-), CD45RA + effector memory T cells (TEMRA) (CCR7) -CD45RA +). TEMRA is a more activated state because of its advanced differentiation and maturation in a subset of memory T cells. Note that CD45RA and CD45RO are isoforms, and CD45RA can be replaced with CD45RO in some cases. Peripheral blood T cell lymphocytes can be divided into two different populations, CD45RA-CD45RO + and CD45RA + CD45RO-. CD45RA is also expressed on medullary thymocytes and naive / resting peripheral CD4 + lymphocytes. CD45RO is expressed on cortical thymocytes, CD4 + memory T cell subsets, and activated T cells. CD45RO is also weakly expressed on monocytes and granulocytes.
　ＣＤ８＋ＴＥＭとは、ＣＤ４５ＲＯ陽性ＣＣＲ７陰性であるＣＤ８＋Ｔ細胞をいう。ＣＤ８＋ＴＥＭＲＡとは、ＣＤ４５ＲＯ陰性ＣＣＲ７陰性であるＣＤ８＋Ｔ細胞をいう。
なお、ＣＤ８＋ＴＥＭ存在比率、およびＣＤ８＋ＴＥＭＲＡ存在比率は、全ＣＤ８＋Ｔ細胞（ＣＤ３陽性ＣＤ８陽性）におけるＴＥＭ細胞（ＣＤ４５ＲＯ陽性ＣＣＲ７陰性）およびＴＥＭＲＡ細胞（ＣＤ４５ＲＯ陰性ＣＣＲ７陰性）比率として定義することができる。ＣＤ４＋Ｔ細胞についても同様である。 CD8 + TEM refers to CD8 + T cells that are CD45RO positive CCR7 negative. CD8 + TEMRA refers to CD8 + T cells that are CD45RO negative CCR7 negative.
The CD8 + TEM presence ratio and CD8 + TEMRA presence ratio can be defined as the ratio of TEM cells (CD45RO-positive CCR7-negative) and TEMRA cells (CD45RO-negative CCR7-negative) in all CD8 + T cells (CD3 + CD8 +). The same applies to CD4 + T cells.
　ＣＤ４＋／ＣＤ８＋Ｔ細胞の活性化による効果には、例えば、病原体による感染症に対して、感染のリスクを低減する、発症のリスクを低減する、感染した対象において、病原体の増殖を抑制する、病原体や病原体に感染した細胞を殺傷する、がんの発症のリスクを低減する、がん細胞の増殖を抑制する、がん細胞を殺傷する、がんを治癒させる、がんが転移・再発するのを防ぐことが含まれる。病原体の例は、各種の、ウイルス、細菌、真菌、マイコプラズマ、寄生虫等である。がんの例は、悪性黒色腫、非小細胞肺がん、腎細胞がん、ホジキンリンパ腫、頭頸部がん、胃がん、尿路上皮がん、メルケル細胞がん等である。本発明の組成物は、これらのいずれに対するＣＤ４＋／ＣＤ８＋Ｔ細胞の活性化に関しても、用いることができる。 The effects of CD4 + / CD8 + T cell activation include, for example, reducing the risk of infection, reducing the risk of onset, suppressing the growth of pathogens in infected subjects, reducing the risk of infection by pathogens, Kill cells infected by pathogens, reduce the risk of developing cancer, suppress cancer cell growth, kill cancer cells, cure cancer, prevent cancer from metastasis and recurrence Prevention is included. Examples of pathogens are various viruses, bacteria, fungi, mycoplasmas, parasites and the like. Examples of cancer are malignant melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, stomach cancer, urothelial cancer, Merkel cell carcinoma and the like. The compositions of the present invention can be used for activation of CD4 + / CD8 + T cells against any of these.
　本発明の組成物は、ＣＤ４＋／ＣＤ８＋Ｔ細胞の活性化が、感染症やがんの処置のための他の療法と一緒に行われる場合であっても、用いることができる。そのような他の療法には、薬物による療法、手術、放射線治療、他の免疫療法（例えば、サイトカイン療法、免疫賦活剤の投与、免疫細胞移植、等）、健康食品やサプリメントの摂取、運動療法等がある。 組成 The compositions of the present invention can be used even when CD4 + / CD8 + T cell activation is performed in conjunction with other therapies for the treatment of infectious diseases and cancer. Such other therapies include drug therapy, surgery, radiation therapy, other immunotherapy (eg, cytokine therapy, administration of immunostimulants, immune cell transplantation, etc.), ingestion of health foods and supplements, exercise therapy Etc.
　なお、本発明の組成物に係る用途からは、次のものを除いてもよい：
・ＥＰＳを有効成分とする、抗原で免疫された脾臓細胞を抗原およびＥＰＳを含む培地で培養した際のインターフェロン－γ（ＩＦＮ－γ）の産生促進に基づく、細胞性免疫活性化組成物；
・ＣＤ４αβＴ細胞、ＣＤ８αβＴ細胞、γδＴ細胞、Ｔｈ１７細胞、およびＩＬＣ３細胞からなる群より選択される少なくとも一つの細胞による、ＩＦＮ－γ産生を促進させるための組成物。 From the uses according to the composition of the present invention, the following may be excluded:
A cellular immunity activating composition comprising EPS as an active ingredient, which is based on promoting the production of interferon-γ (IFN-γ) when spleen cells immunized with an antigen are cultured in a medium containing the antigen and EPS;
-A composition for promoting IFN-γ production by at least one cell selected from the group consisting of CD4αβ T cells, CD8αβ T cells, γδ T cells, Th17 cells, and ILC3 cells.
［組成物］
（食品組成物等）
　本発明の組成物は、食品組成物または医薬組成物とすることができる。食品および医薬品は、特に記載した場合を除き、ヒトのためのもののみならず、ヒト以外の動物のためのものを含む。食品は、特に記載した場合を除き、一般食品、機能性食品、栄養組成物を含み、また治療食（治療の目的を果たすもの。医師が食事箋を出し、それに従い栄養士等が作成した献立に基づいて調理されたもの。）、食事療法食、成分調整食、介護食、治療支援用食品を含む。食品は、特に記載した場合を除き、固形物のみならず、液状のもの、例えば飲料、ドリンク剤、流動食、およびスープを含む。機能性食品とは、生体に所定の機能性を付与できる食品をいい、例えば、特定保健用食品（条件付きトクホ［特定保健用食品］を含む）、機能性表示食品、栄養機能食品を含む保健機能食品、特別用途食品、栄養補助食品、健康補助食品、サプリメント（例えば、錠剤、被覆錠、糖衣錠、カプセル、液剤等の各種の剤型のもの）、美容食品（例えば、ダイエット食品）等の、健康食品の全般を包含している。また、本発明において「機能性食品」とは、コーデックス（FAO/WHO合同食品規格委員会）の食品規格に基づく健康強調表示（Health claim）が適用される健康食品を包含している。 [Composition]
(Food composition, etc.)
The composition of the present invention can be a food composition or a pharmaceutical composition. Foods and pharmaceuticals include not only those for humans but also those for non-human animals, unless otherwise indicated. Unless otherwise specified, foods include general foods, functional foods, and nutritional compositions. Cooked on the basis of food), dietary meals, ingredient-adjusted meals, nursing care meals, and treatment support foods. The food includes not only solids but also liquids such as beverages, drinks, liquid foods, and soups, unless otherwise specified. Functional foods refer to foods that can impart predetermined functionality to living organisms, and include, for example, health foods including specified health foods (including conditional Tokuho [specified health foods]), functionally labeled foods, and nutritionally functional foods. Functional foods, special use foods, dietary supplements, health supplements, supplements (for example, tablets, coated tablets, dragees, capsules, various types of liquids such as liquids), beauty foods (for example, diet foods), etc. Includes a whole range of health foods. In the present invention, the “functional food” includes a health food to which a health claim based on a food standard of the Codex (FAO / WHO Joint Food Standards Committee) is applied.
（対象） 
　本発明の組成物は、ＣＤ４＋／ＣＤ８＋Ｔ細胞の活性化が好ましい対象に、摂取させる・投与するのに適している。このような対象には、乳幼児、子ども、成人（１５歳以上）、中高年者、高齢者（６５歳以上）、病中病後の者、妊婦、産婦、男性、女性が含まれる。また本発明の組成物は、有効成分が食経験の長いＥＰＳであるため、長期間の摂取に適しているため、日常生活における病気になりにくい体づくりが重要な高齢者に、特に適している。 (Target)
The composition of the present invention is suitable for ingestion / administration of a subject in which activation of CD4 + / CD8 + T cells is preferred. Such subjects include infants, children, adults (aged 15 and over), middle-aged and elderly, elderly (aged 65 and over), persons ill and ill, pregnant women, maternal women, men, and women. In addition, the composition of the present invention is particularly suitable for elderly people who are important to build a body that is less susceptible to illness in daily life because the active ingredient is EPS with a long eating experience and is suitable for long-term ingestion. .
（投与経路）
　本発明の組成物は、それを非経口的に、例えば経管的（胃瘻、腸瘻）に投与してもよいし、経鼻的に投与してもよいし、経口的に投与してもよいが、経口的に投与することが好ましい。 (Administration route)
The composition of the present invention may be administered parenterally, for example, transluminally (gastrostomy, enterostomy), nasally, or orally. Oral administration is preferred.
（有効成分の含有量・用量）
　本発明の組成物における、乳酸菌のＥＰＳの含有量は、目的の効果が発揮される量であればよい。組成物は、その被験体の年齢、体重、症状等の種々の要因を考慮して、その投与量または摂取量を適宜設定することができるが、一日量あたりの乳酸菌のＥＰＳの量は、例えば0.1 mg以上とすることができ、0.6 mg以上とすることが好ましく、1 mg以上とすることがより好ましく、3 mg以上とすることが特に好ましい。一日量あたりのＥＰＳの量の上限値は、下限値がいずれの場合であっても、500 mg以下とすることができ、300 mg以下とすることが好ましく、250 mg以下とすることが特に好ましい。 (Content and dosage of active ingredient)
The EPS content of the lactic acid bacterium in the composition of the present invention may be any amount as long as the desired effect is exhibited. The composition, the age of the subject, weight, taking into account various factors such as symptoms, the dose or intake can be appropriately set, the amount of EPS of lactic acid bacteria per daily dose, For example, it can be 0.1 mg or more, preferably 0.6 mg or more, more preferably 1 mg or more, and particularly preferably 3 mg or more. The upper limit of the amount of EPS per day, regardless of the lower limit, can be 500 mg or less, preferably 300 mg or less, and particularly preferably 250 mg or less. preferable.
　１投与または１食あたり、すなわち一回量あたりの乳酸菌のＥＰＳの量は、例えば0.03 mg以上とすることができ、0.2 mg以上とすることが好ましく、1 mg以上とすることがより好ましい。一回量あたりのＥＰＳの量の上限値は、下限値がいずれの場合であっても、200 mg以下とすることができ、100 mg以下とすることが好ましく、70 mg以下とすることがより好ましく、30 mg以下とすることが特に好ましい。 The amount of EPS of lactic acid bacteria per administration or per serving, that is, per dose, can be, for example, 0.03 mg or more, preferably 0.2 mg or more, and more preferably 1 mg or more. The upper limit of the amount of EPS per dose can be 200 mg or less, preferably 100 mg or less, and more preferably 70 mg or less, regardless of the lower limit. It is particularly preferable that the content be 30 mg or less.
　本発明の組成物における、乳酸菌のＥＰＳを発酵乳のような組成物として用いる場合、組成物としての一日量は、例えば30 g以上とすることができ、50 g以上とすることが好ましく、60 g以上とすることがより好ましく、100 g以上とすることが特に好ましい。発酵乳としての一日量の上限値は、下限値がいずれの場合であっても、例えば1500 g以下とすることができ、1200 g以下とすることが好ましく、900 g以下とすることがより好ましく、600 g以下とすることがより好ましい。 In the composition of the present invention, when EPS of lactic acid bacteria is used as a composition such as fermented milk, the daily dose as the composition can be, for example, 30 mg or more, and preferably 50 mg or more, It is more preferably at least 60 μg, particularly preferably at least 100 μg. The upper limit of the daily amount as fermented milk, in any case, the lower limit can be, for example, 1500 g or less, preferably 1200 g or less, more preferably 900 g or less More preferably, it is more preferably 600 mg or less.
　組成物としての一回量は、例えば10 g以上とすることができ、20 g以上とすることが好ましく、30 g以上とすることがより好ましい。組成物としての一回量の上限値は、下限値がいずれの場合であっても、例えば500 g以下とすることができ、400 g以下とすることが好ましく、200 g以下とすることがより好ましく、125 g以下とすることが特に好ましい。 の 一 A single dose of the composition can be, for example, 10 μg or more, preferably 20 μg or more, and more preferably 30 μg or more. The upper limit of the single dose of the composition, regardless of the lower limit, can be, for example, 500 g or less, preferably 400 g or less, more preferably 200 g or less. It is particularly preferable that the content be 125 mg or less.
　組成物は、一日１回の投与・摂取としてもよいし、一日複数回、例えば食事毎の３回の投与としてもよい。組成物は、食経験豊富な乳酸菌のＥＰＳを有効成分としている。そのため、本発明の組成物は、繰り返し、または長期間にわたって摂取してもよく、例えば３日以上、好ましくは１週間以上、より好ましくは４週間以上、特に好ましくは１カ月以上、続けて投与・摂取することができる。 The composition may be administered and taken once a day, or may be administered several times a day, for example, three times for each meal. The composition contains, as an active ingredient, EPS of a lactic acid bacterium with a good dietary experience. Therefore, the composition of the present invention may be taken repeatedly or over a long period, for example, continuously administered for 3 days or more, preferably 1 week or more, more preferably 4 weeks or more, particularly preferably 1 month or more. Can be taken.
（他の成分、添加剤）
　本発明の組成物は、食品または医薬品として許容可能な他の有効成分や栄養成分を含んでいてもよい。そのような成分の例は、アミノ酸類（例えば、リジン、アルギニン、グリシン、アラニン、グルタミン酸、ロイシン、イソロイシン、バリン）、糖質（グルコース、ショ糖、果糖、麦芽糖、トレハロース、エリスリトール、マルチトール、パラチノース、キシリトール、デキストリン）、電解質（例えば、ナトリウム、カリウム、カルシウム、マグネシウム）、ビタミン（例えば、ビタミンＡ、ビタミンＢ１、ビタミンＢ２、ビタミンＢ６、ビタミンＢ１２、ビタミンＣ、ビタミンＤ、ビタミンＥ、ビタミンＫ、ビオチン、葉酸、パントテン酸およびニコチン酸類）、ミネラル（例えば、銅、亜鉛、鉄、コバルト、マンガン）、抗生物質、食物繊維、タンパク質、脂質等である。 (Other components and additives)
The compositions of the present invention may include other active or nutritional ingredients that are food or pharmaceutically acceptable. Examples of such components include amino acids (eg, lysine, arginine, glycine, alanine, glutamic acid, leucine, isoleucine, valine), carbohydrates (glucose, sucrose, fructose, maltose, trehalose, erythritol, maltitol, palatinose). , Xylitol, dextrin), electrolytes (eg, sodium, potassium, calcium, magnesium), vitamins (eg, vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, Biotin, folic acid, pantothenic acid and nicotinic acids), minerals (eg, copper, zinc, iron, cobalt, manganese), antibiotics, dietary fiber, proteins, lipids and the like.
　また組成物は、食品または医薬として許容される添加物をさらに含んでいてもよい。そのような添加物の例は、不活性担体（固体や液体担体）、賦形剤、界面活性剤、結合剤、崩壊剤、滑沢剤、溶解補助剤、懸濁化剤、コーティング剤、着色剤、保存剤、緩衝剤、pH調整剤、乳化剤、安定剤、甘味料、酸化防止剤、香料、酸味料、天然物である。より具体的には、水、他の水性溶媒、製薬上で許容される有機溶媒、コラーゲン、ポリビニルアルコール、ポリビニルピロリドン、カルボキシビニルポリマー、アルギン酸ナトリウム、水溶性デキストラン、水溶性デキストリン、カルボキシメチルスターチナトリウム、ペクチン、キサンタンガム、アラビアゴム、カゼイン、ゼラチン、寒天、グリセリン、プロピレングリコール、ポリエチレングリコール、ワセリン、パラフィン、ステアリルアルコール、ステアリン酸、ヒト血清アルブミン、マンニトール、ソルビトール、ラクトース、スクラロース、ステビア、アスパルテーム、アセスルファムカリウム、クエン酸、乳酸、りんご酸、酒石酸、リン酸、酢酸、果汁、野菜汁等である。 The composition may further include a food or a pharmaceutically acceptable additive. Examples of such additives are inert carriers (solid or liquid carriers), excipients, surfactants, binders, disintegrants, lubricants, solubilizers, suspending agents, coatings, coloring. Agents, preservatives, buffers, pH adjusters, emulsifiers, stabilizers, sweeteners, antioxidants, flavors, acidulants, and natural products. More specifically, water, other aqueous solvents, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, water-soluble dextrin, sodium carboxymethyl starch, Pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, sucralose, stevia, aspartame, acesulfame potassium, Citric acid, lactic acid, malic acid, tartaric acid, phosphoric acid, acetic acid, fruit juice, vegetable juice and the like.
（剤型・形態）
　本発明の医薬組成物は、経口投与に適した、錠剤、顆粒剤、散剤、丸剤、カプセル剤等の固形製剤、液剤、懸濁剤、シロップ剤等の液体製剤、ジェル剤、エアロゾル剤等の任意の剤型にすることができる。 (Dosage form / form)
The pharmaceutical composition of the present invention is suitable for oral administration, such as tablets, granules, powders, pills, capsules and other solid preparations, liquid preparations such as liquid preparations, suspensions and syrups, gel preparations, aerosol preparations and the like. In any dosage form.
　本発明の食品組成物は、固体、液体、混合物、懸濁液、粉末、顆粒、ペースト、ゼリー、ゲル、カプセル等の任意の形態に調製されたものであってよい。また、本発明に係る食品組成物は、乳製品、サプリメント、菓子、飲料、ドリンク剤、調味料、加工食品、惣菜、スープ等の任意の形態にすることができる。より具体的には、本発明の組成物は、乳飲料、清涼飲料、発酵乳、ヨーグルト、アイスクリーム、タブレット、チーズ、パン、ビスケット、クラッカー、ピッツァクラスト、調製粉乳、流動食、病者用食品、栄養食品、冷凍食品、加工食品等の形態とすることができ、また飲料や食品に混合して摂取するための、顆粒、粉末、ペースト、濃厚液等の形態とすることができる。 The food composition of the present invention may be prepared in any form such as solid, liquid, mixture, suspension, powder, granule, paste, jelly, gel, capsule and the like. Further, the food composition according to the present invention can be in any form such as dairy products, supplements, confectionery, beverages, drinks, seasonings, processed foods, prepared dishes, soups, and the like. More specifically, the composition of the present invention comprises milk drink, soft drink, fermented milk, yogurt, ice cream, tablet, cheese, bread, biscuit, cracker, pizza crust, powdered milk, liquid food, food for the sick , Nutritional foods, frozen foods, processed foods, and the like, and granules, powders, pastes, concentrated liquids, and the like for mixing and ingesting into beverages and foods.
（その他）
　本発明の組成物の製造において、乳酸菌のＥＰＳの配合の段階は、適宜選択することができる。乳酸菌のＥＰＳの特性を著しく損なわない限り配合の段階は特に制限されない。例えば、製造の初期の段階に、原材料に混合して配合することができる。あるいは、本発明の組成物を発酵乳として実施する場合は、ＥＰＳを産生する乳酸菌をスターターとして原料乳に添加し、発酵させ、ＥＰＳを産生させることで、ＥＰＳを含む発酵乳が製造できる。 (Other)
In the production of the composition of the present invention, the stage of compounding the lactic acid bacteria with EPS can be appropriately selected. The blending step is not particularly limited as long as the EPS characteristics of the lactic acid bacteria are not significantly impaired. For example, it can be mixed and blended with raw materials at an early stage of production. Alternatively, when the composition of the present invention is used as fermented milk, a lactic acid bacterium that produces EPS is added to the raw milk as a starter, fermented, and EPS is produced, whereby fermented milk containing EPS can be produced.
　本発明の組成物には、ＣＤ４＋／ＣＤ８＋Ｔ細胞の活性化のために用いることができる旨を表示することができ、また特定の対象に対して摂取を薦める旨を表示することができる。表示は、直接的にまたは間接的にすることができ、直接的な表示の例は、製品自体、パッケージ、容器、ラベル、タグ等の有体物への記載であり、間接的な表示の例は、ウェブサイト、店頭、パンフレット、展示会、メディアセミナー等のセミナー、書籍、新聞、雑誌、テレビ、ラジオ、郵送物、電子メール、音声等の、場所または手段による、広告・宣伝活動を含む。 組成 The composition of the present invention can indicate that it can be used for activating CD4 + / CD8 + T cells, and can indicate that it is recommended to be taken to a specific subject. Labeling can be direct or indirect, examples of direct labeling are descriptions on tangible objects such as products themselves, packages, containers, labels, tags, etc. Examples of indirect labeling are: Includes advertising and promotion activities by location or means, such as websites, storefronts, brochures, exhibitions, seminars such as media seminars, books, newspapers, magazines, televisions, radios, mailings, emails, voices, etc.
  以下、実施例を用いて、本発明をさらに具体的に説明する。但し、本発明の技術的範囲は、これら実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
＜EPS（菌体外多糖体）の調製＞
　実施例1においては、10質量%脱脂粉乳培地でLactobacillus delbrueckii subsp. bulgaricus OLL1073R-1を培養して得た培養物中のEPSを精製した。すなわち、37℃で18時間培養した培養物に、終濃度10質量%になるようトリクロロ酢酸を加えて変性タンパク質を除去し、冷エタノールを加えて4℃で2時間静置してEPSを含む沈殿物を得た。これを、透析膜（分画分子量6,000 - 8,000）を用いてMilliQ水に対して透析し、核酸とタンパク質を酵素分解した後、再度エタノール沈殿を行って沈殿物を得た。これをMilliQ水に溶解し、再度透析を行った後に凍結乾燥を行ってEPSを精製した。 <Preparation of EPS (Extracellular Polysaccharide)>
In Example 1, EPS in a culture obtained by culturing Lactobacillus delbrueckii subsp. Bulgaricus OLL1073R-1 in a 10% by mass skim milk medium was purified. That is, the denatured proteins were removed by adding trichloroacetic acid to a final concentration of 10% by mass to the culture cultured at 37 ° C. for 18 hours, and cold ethanol was added thereto, and the mixture was allowed to stand at 4 ° C. for 2 hours to precipitate EPS. I got something. This was dialyzed against MilliQ water using a dialysis membrane (fraction molecular weight: 6,000 to 8,000) to decompose nucleic acids and proteins, and then subjected to ethanol precipitation again to obtain a precipitate. This was dissolved in MilliQ water, dialyzed again, and then freeze-dried to purify EPS.
＜発酵乳の調製＞
　実施例2においては、生乳、脱脂粉乳、クリーム、砂糖、ステビアを含む混合物に、Lactobacillus delbrueckii subsp. bulgaricusOLL1073R-1とサーモフィラス菌をスターターとして添加して発酵させ、発酵乳を製造した。また、臨床試験のプラセボとして、pHや酸度を合わせて発酵乳と識別不可能に調整した酸性乳飲料を用いた。 <Preparation of fermented milk>
In Example 2, Lactobacillus delbrueckii subsp. BulgaricusOLL1073R-1 and a thermophilus bacterium were added as a starter to a mixture containing raw milk, skim milk powder, cream, sugar, and stevia, and fermented to produce fermented milk. In addition, as a placebo in a clinical test, an acidic milk beverage adjusted to be indistinguishable from fermented milk by adjusting pH and acidity was used.
＜実施例1＞
　C57BL/6Nマウス、メス、6週齢(日本チャールスリバー)の計12匹を、対照群(n=6)とEPS群(n=6)に群分けした。対照群には蒸留水を、EPS群には上記EPS水溶液(250μg/ml)をそれぞれ400μl/匹強制経口投与した。経口投与は解剖前日まで毎日1回実施した。経口投与を開始した3週間後に、次に示す免疫組成物（T細胞に抗原特異的な免疫反応を起こさせる）をマウス全匹の右フットパッドに皮下投与した。組成は、TiterMax Gold (TiterMax社) 25μl、H-2Db human gp100 peptide (配列KVPRNQDWL、MBL社) 5μl、I-AbHBc helper peptide (配列TPPAYRPPNAPIL、MBL社) 5μl、PBS 15μlの計50μl/匹をよくエマルジョン化したのち用いた。免疫組成物の投与1週間後にマウス全匹を安楽殺し、腫脹している右膝窩リンパ節および脾臓を採取した。それぞれの器官を処理して得た細胞懸濁液を試験に用いて両群を比較した。なお、H-2Db human gp100 peptideは、細胞傷害性T細胞（CTL）に抗原特異的な刺激を入れることができるため、サイトカインの産生、細胞表面抗原の発現、細胞傷害性活性を確認する際の刺激、抗原特異的CTLの誘導などに使用できる試薬である。また、I-AbHBc  helper peptideは、CD4陽性T細胞に抗原特異的な刺激を入れることができるため、サイトカインの産生、抗原特異的CTLの誘導補助などに使用できる試薬である。 <Example 1>
A total of 12 C57BL / 6N mice, female, and 6 weeks old (Charles River Japan) were divided into a control group (n = 6) and an EPS group (n = 6). Distilled water was administered to the control group, and the EPS aqueous solution (250 μg / ml) was forcibly administered 400 μl / animal to the EPS group. Oral administration was performed once daily until the day before the dissection. Three weeks after the start of oral administration, the following immune composition (which causes an antigen-specific immune response to T cells) was subcutaneously administered to the right footpad of all mice. The composition is 25 μl of TiterMax Gold (TiterMax), 5 μl of H-2D b human gp100 peptide (sequence KVPRNQDWL, MBL), 5 μl of IA b HBc helper peptide (sequence TPPAYRPPNAPIL, MBL), and 15 μl of PBS, a total of 50 μl / animal. It was used after emulsification. One week after the administration of the immune composition, all mice were euthanized, and the swollen right popliteal lymph node and spleen were collected. A cell suspension obtained by treating each organ was used in the test to compare the two groups. Since the H-2D b human gp100 peptide can stimulate antigen-specific stimuli to cytotoxic T cells (CTLs), it can be used to confirm cytokine production, cell surface antigen expression, and cytotoxic activity. It is a reagent that can be used for stimulating DNA, inducing antigen-specific CTL, and the like. The IA b HBc helper peptide is a reagent that can be used for production of cytokines, assistance for induction of antigen-specific CTL, and the like, because it can stimulate CD4-positive T cells with antigen-specific stimulation.
　生細胞数1×106cells/mlに調整したリンパ節細胞を1μg/mlの抗マウスCD3抗体(日本BD社)でT細胞を刺激培養(37℃, CO2濃度:5%, 48時間)し、培養上清のIFN-γ濃度をELISA法で測定(OptEIAkit、日本BD社)した。その結果、対照群と比較してEPS群のリンパ節細胞は有意に(p<0.001)高いIFN-γ産生を示した(図1A)。この結果は、EPSの摂取によって、抗原反応を模したリンパ節においてT細胞が活性化し、サイトカイン産生能が高まったことを意味する。 Stimulated culture of T cells with 1 μg / ml anti-mouse CD3 antibody (BD Japan) at 37 ° C, CO 2 concentration: 5%, 48 hours, with lymph node cells adjusted to a live cell count of 1 × 10 6 cells / ml Then, the IFN-γ concentration of the culture supernatant was measured by ELISA (OptEIAkit, Japan BD). As a result, the lymph node cells in the EPS group showed significantly (p <0.001) higher IFN-γ production than the control group (FIG. 1A). This result indicates that the intake of EPS activated T cells in the lymph node mimicking the antigen reaction and increased the ability to produce cytokines.
　生細胞数1×106cells/匹に調整した脾臓細胞を蛍光標識した抗マウスCD3抗体、抗マウスCD4抗体、抗マウスCD8抗体、抗マウスCD69抗体(いずれも日本BD社)を含む抗体MIXで染色し、フローサイトメーター(FACS Verse、日本BD社)を用いてCD4+T細胞およびCD8+T細胞のCD69発現率を測定した。その結果、対照群と比較してEPS群の脾細胞は、CD4+T細胞(p=0.045)およびCD8+T細胞(p=0.002)のいずれも、CD69発現率が有意に高かった(図1B)。 Antibody MIX containing anti-mouse CD3 antibody, anti-mouse CD4 antibody, anti-mouse CD8 antibody, anti-mouse CD69 antibody (all of which are BD Japan) fluorescently labeled spleen cells adjusted to 1 × 10 6 cells / animal The cells were stained, and CD69 expression rates of CD4 + T cells and CD8 + T cells were measured using a flow cytometer (FACS Verse, BD Japan). As a result, as compared to the control group, splenocytes of the EPS group, both CD4 + T cells (p = 0.045) and CD8 + T cells (p = 0.002), CD69 expression rate was significantly higher (Figure 1B ).
　このことから、上記EPSの摂取によって、マウスの脾臓においてCD69発現T細胞が増加し、リンパ節におけるT細胞の活性化が起きたことが分かった。 This indicates that the ingestion of the EPS increased the number of CD69-expressing T cells in the spleen of mice and activated T cells in lymph nodes.
＜実施例2＞
　65歳以上(65 - 81歳)の健常高齢者男女24名を、対照群(n=12)と発酵乳群(n=12)に群分けし、プラセボ対照二重盲検群間比較試験を実施した。対照群にはプラセボを、発酵乳群には上記発酵乳をそれぞれ112ml(119g)毎日摂取させ、摂取前後の末梢血をBDバキュテイナ CPT 単核球分離用採血管(日本BD社)で処理し、得たPBMC（末梢血単核細胞）を試験に用いて両群を比較した。 <Example 2>
Twenty-four healthy men and women aged 65 or older (65-81 years) were divided into a control group (n = 12) and a fermented milk group (n = 12). Carried out. The control group was given a placebo, the fermented milk group was ingested with 112 ml (119 g) of the above fermented milk each day, and the peripheral blood before and after ingestion was treated with a BD vacutainer CPT blood collection tube for mononuclear cell separation (BD Japan), The obtained PBMC (peripheral blood mononuclear cells) were used for the test to compare the two groups.
　細胞数1×106cells/検体に調整したPBMCを抗ヒトCD3抗体、抗ヒトCD8抗体、抗ヒトCD45RO抗体、抗ヒトCCR7抗体、抗ヒトCD69抗体(いずれも日本BD社)を含む抗体MIXで染色し、フローサイトメーター(FACS Verse、日本BD社)を用いてCD4+TEMおよびCD4+TEMRA存在比率、CD8+TEMおよびCD8+TEMRA存在比率、CD4+TEMおよびCD8+TEMRAのCD69発現率を測定した。その結果、対照群と比較して発酵乳群の末梢血CD8+T細胞はTEMからTEMRAへの成熟の進行が認められた(図2A, 図2B)。また、CD4+TEMのCD69発現率は有意に(p=0.030)増加し、CD8+TEMRAのCD69発現率も増加傾向(p=0.088)が認められた(図2C, 図2D)。なお、CD4+TEM存在比率、およびCD4+TEMRA存在比率は、全CD4+T細胞（CD3陽性CD4陽性）におけるTEM細胞(CD45RO陽性CCR7陰性)およびTEMRA細胞（CD45RO陰性CCR7陰性）比率と定義した。CD8+T細胞についても同様とした。また、CD4+TEMのCD69発現率は、全CD4+TEM細胞（CD3陽性CD4陽性、かつCD45RO陽性CCR7陰性）におけるCD69陽性細胞比率と定義した。CD8+TEMRAのCD69発現率についても同様とした。 PBMC adjusted to a cell count of 1 × 10 6 cells / sample was subjected to antibody MIX containing anti-human CD3 antibody, anti-human CD8 antibody, anti-human CD45RO antibody, anti-human CCR7 antibody, and anti-human CD69 antibody (all BD Japan). Stain and measure CD4 + TEM and CD4 + TEMRA abundance ratio, CD8 + TEM and CD8 + TEMRA abundance ratio, and CD69 expression ratio of CD4 + TEM and CD8 + TEMRA using flow cytometer (FACS Verse, Japan BD) did. As a result, the progression of maturation from TEM to TEMRA was observed in the peripheral blood CD8 + T cells of the fermented milk group as compared with the control group (FIGS. 2A and 2B). The CD69 expression rate of CD4 + TEM increased significantly (p = 0.030), and the CD69 expression rate of CD8 + TEMRA also tended to increase (p = 0.088) (FIGS. 2C and 2D). The CD4 + TEM presence ratio and CD4 + TEMRA presence ratio were defined as the ratio of TEM cells (CD45RO-positive CCR7-negative) and TEMRA cells (CD45RO-negative CCR7-negative) in all CD4 + T cells (CD3 + CD4 +). The same applies to CD8 + T cells. The CD69 expression rate of CD4 + TEM was defined as the ratio of CD69-positive cells in all CD4 + TEM cells (CD3-positive CD4-positive and CD45RO-positive CCR7-negative). The same was applied to the CD69 expression rate of CD8 + TEMRA.
　この結果は、上記発酵乳の摂取によって、抗原に対してT細胞が高い反応性を示してより強く活性化しただけでなく、CD8+T細胞に対しては自身の分化・成熟を促進する影響もあったことを意味する。 This result indicates that the intake of fermented milk not only activated T cells with high reactivity to antigens and activated them, but also promoted differentiation and maturation of CD8 + T cells. Means that there was also.
　さらに、本試験で用いた発酵乳に含まれるEPSは、リンパ節においてIFN-γの産生を促進させる効果を有することが分かっていることや（実施例1）、抗原に対して特異的に誘導されるTEM細胞がより成熟型のTEMRA細胞に分化したことから（実施例2）、本試験における血液中T細胞のCD69の発現は、抗原への反応性を反映していると考えられる。通常、ヒトにおいてリンパ節内のT細胞のCD69発現を調べるのは難しいが、実施例1、2の結果から、血中T細胞のCD69の発現を調べるだけで（リキッドバイオプシー）、抗原に対するT細胞の反応を調べることができると分かった。 Furthermore, it was found that EPS contained in the fermented milk used in this test had the effect of promoting IFN-γ production in lymph nodes (Example 1), and that it was specifically induced to antigens. Since the obtained TEM cells differentiated into more mature TEMRA cells (Example 2), the expression of CD69 on blood T cells in this test is considered to reflect the reactivity to antigen. Normally, it is difficult to examine CD69 expression of T cells in lymph nodes in humans. However, from the results of Examples 1 and 2, it is only necessary to examine the expression of CD69 on blood T cells (liquid biopsy) to obtain T cells against antigen. It was found that the reaction could be examined.

Claims (9)

  1. 　乳酸菌の菌体外多糖を含む、ＣＤ４＋Ｔ細胞およびＣＤ８＋Ｔ細胞の少なくとも一方を活性化するための組成物。 (4) A composition for activating at least one of CD4 + T cells and CD8 + T cells, which comprises an exopolysaccharide of a lactic acid bacterium.
  2. 　ＣＤ４＋Ｔ細胞およびＣＤ８＋Ｔ細胞の少なくとも一方におけるＣＤ６９発現率を高めるための、請求項１に記載の組成物。 The composition according to claim 1, for increasing the CD69 expression rate in at least one of CD4 + T cells and CD8 + T cells.
  3. 　ＣＤ６９発現率が、被験者に由来する末梢血Ｔ細胞の分析値に基づく値である、請求項２に記載の組成物。 The composition according to claim 2, wherein the CD69 expression rate is a value based on an analysis value of peripheral blood T cells derived from the subject.
  4. 　ＣＤ８＋ＴＥＭからＣＤ８＋ＴＥＭＲＡへの成熟を促進するための、請求項１～３のいずれか１項に記載の組成物。 4. The composition according to any one of claims 1 to 3, for promoting maturation of CD8 + TEM to CD8 + TEMRA.
  5. 　乳酸菌が、ラクトバチルス属に分類されるものである、請求項１～４のいずれか１項に記載の組成物。 The composition according to any one of claims 1 to 4, wherein the lactic acid bacteria are classified into the genus Lactobacillus.
  6. 　乳酸菌が、ラクトバチルス・デルブルッキー・サブスピーシーズ・ブルガリクスに分類されるものである、請求項１～５のいずれか１項に記載の組成物。 (6) The composition according to any one of (1) to (5), wherein the lactic acid bacterium is classified into Lactobacillus del Brooke subspecies Bulgaricus.
  7. 　発酵乳である、請求項１～６のいずれか１項に記載の組成物。 The composition according to any one of claims 1 to 6, which is a fermented milk.
  8. 　感染症またはがんの発症リスクを低減するための非医療的方法であって、有効量の乳酸菌の菌体外多糖を含む組成物を対象に摂取させ、対象におけるＣＤ４＋Ｔ細胞、およびＣＤ８＋Ｔ細胞の少なくとも一方におけるＣＤ６９発現率を高める工程を含む、方法。 A non-medical method for reducing the risk of developing an infectious disease or cancer, comprising causing a subject to ingest a composition comprising an effective amount of an exopolysaccharide of a lactic acid bacterium, wherein at least one of CD4 + T cells and CD8 + T cells in the subject is consumed. A method comprising the step of increasing the CD69 expression rate on one side.
  9. 　乳酸菌の菌体外多糖を用いる、ＣＤ４＋Ｔ細胞およびＣＤ８＋Ｔ細胞の少なくとも一方におけるＣＤ６９発現率を高める方法（ヒトに対する医療行為を除く。）。 (4) A method for increasing the CD69 expression rate in at least one of CD4 + T cells and CD8 + T cells using an exopolysaccharide of a lactic acid bacterium (excluding medical practice for humans).
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