[Field of the Invention]
[0001] The present invention relates to a method of supplying immunodynamicsrelated information.
[Background Arts]
[0002] Today, due to advance in technology, a variety of therapeutic means have been developed based on antitumor immunotherapies. However, a large part of the mechanism of cellular immunity is yet to be clarified, and its relevance in immunotherapy is being groped.
[0003] The inventor has previously proposed a method of measuring immunodynamics in blood analysis characterized in measuring antigen recognition function, etc., though the immunocompetent cells that were focused on at that time were not enough to comprehend the entire structure of immunodynamics (Patent Reference 1).
[Prior Art References]
[Patent References]
[Summary of the Invention]
[Problems to be Solved by the Invention]
[0005] An object of the present invention is to provide immunodynamicsrelated information that helps an easy comprehension of the immunodynamics of cellular immunity of a subject and can therefore be a guiding principle for therapy and prophylaxis for a disease and/or symptom of the subject.
[Means to Solve the Problems]
[0006] The inventor chose patients with prostate cancer as examples, carried out statistical analyses of cellcounts of immunocompetent cells in subjects' blood, and found out that the cellcounts of immunocompetent cells are associated with prognosis. The inventor further carried on the investigation and found that immunodynamicsrelated information which helps determination of therapy or prophylaxis for a disease and/or symptom can be obtained by analyzing the cellcounts of immunocompetent cells, and thus completed the invention.
[0007] Namely, the present invention relates to:
 <1> A method for supplying immunodynamicsrelated information for use in determining therapy or prophylaxis for a disease and/or symptom of a subject, comprising:
 (i) calculating a discriminant score by assigning the cellcount of each of multiple types of immunocompetent cells in blood collected from the subject into a discriminant function;
 (ii) determining a group into which the subject is to be sorted by the calculated discriminant score; and
 (iii) displaying immunodynamicsrelated information of the determined group;
wherein:
the discriminant function is obtained by performing a discriminant analysis for a data cluster containing data of the condition of an individual and the cellcount of each of multiple types of immunocompetent cells in blood collected from the individual in a number that allows for the discriminant analysis, wherein the condition of the individual is set an objective variable and the multiple types of immunocompetent cells are set as explanatory variables; and
the group into which the subject is to be sorted is one of the multiple groups that are divided from the data cluster used for obtaining the discriminant function using discriminant scores as indicator.
 <2> The method according to <1> above, wherein the condition of an individual is selected from a group consisting of health, a disease, a disorder, a symptom or prognosis.
 <3> The method according to <1> or <2> above, wherein the immunodynamicsrelated information is obtained by:
 (a) performing a multiple regression analysis for data of the cellcounts of n types of immunocompetent cells that constitute one group, wherein one type of immunocompetent cell among the n types of immunocompetent cells is set as an objective variable and n1 types of immunocompetent cells excluding the one type of immunocompetent cell that is set as the objective variable are set as explanatory variables, and wherein n is an integer of 4 or more;
 (b) ranking the n1 types of immunocompetent cells in descending order according to the magnitude of the absolute value of the standard partial regression coefficient obtained from the multiple regression analysis;
 (c) performing a regression analysis in which the one type of immunocompetent cell that is the objective variable in (a) above is set as an objective variable and the immunocompetent cell that is ranked as the first place in (b) above is set as explanatory variable, calculating the contribution ratio α_{1} which is considered as the influence degree of the firstplace ranked immunocompetent cell β_{1}; and
 (d) performing a multiple regression analysis in which the one type of immunocompetent cell that is the objective variable in (a) above is set as an objective variable and m types of immunocompetent cells from the first to the mth place ranked in (b) above are set as explanatory variables, calculating the contribution ratio α_{m}, and calculating the influence degree β_{m} of the immunocompetent cell ranked as mth place by the following formula:
for each of the immunocompetent cells ranked from the second to the mth place; wherein m is more than 3 and up to n1.
 <4> The method according to any one of <1> to <3> above, wherein the immunocompetent cells are three or more selected from a group consisting of: Th17+ lymphocyte, CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Th± lymphocyte, Th2 lymphocyte, Th+2 lymphocyte, Act.Th1 lymphocyte, Act.Th2 lymphocyte, Ti*DR lymphocyte, Ti± lymphocyte, Ti2 lymphocyte, Ti+2 lymphocyte, Tc*DR lymphocyte, Tc lymphocyte, Tc+ lymphocyte, Ts*DR lymphocyte, Tslymphocyte, Ts+ lymphocyte, NK cell, NKT cell, N3+ cell, monocyte, basophil, eosinophil and neutrophil.
 <5> The method according to <4> above, wherein the immunocompetent cells comprise Th17+ lymphocyte.
 <6> The method according to any one of <3> to <5> above, wherein the one type of immunocompetent cell that is set as the objective variable in (a) above is selected from a group consisting of: Tc*DR lymphocyte, CD20*DR lymphocyte, NK cell, NKT cell, basophil, eosinophil and neutrophil.
 <7> A method of evaluating NKcell activity using the influence degree according to any one of <3> to <6> by the following formula:
provided that, in multiple regression analysis in which NK cell is set as the objective variable, the standard partial regression coefficients of monocyte, Act.Th1 lymphocyte and Tc*DR lymphocyte are all positive.  <8> A method of evaluating NKcell ADCC (antibodydependent cellular cytotoxicity) activity using the influence degree according to any one of <3> to <6> by the following formula:
provided that, in multiple regression analysis in which NK cell is set as the objective variable, the standard partial regression coefficients of monocyte, Act.Th2 lymphocyte and CD20*DR lymphocyte are all positive.  <9> A method of evaluating NKTcell activity using the influence degree according to any one of <3> to <6> by the following formula:
provided that, in multiple regression analysis in which NKT cell is set as the objective variable, the standard partial regression coefficients of monocyte, Act.Th1 lymphocyte and Tc*DR lymphocyte are all positive.  <10> A method of evaluating NKTcell ADCC (antibodydependent cellular cytotoxicity) activity using the influence degree according to any one of <3> to <6> by the following formula:
provided that, in multiple regression analysis in which NKT cell is set as the objective variable, the standard partial regression coefficients of monocyte, Act.Th2 lymphocyte and CD20*DR lymphocyte are all positive.  <11> A method of evaluating killer Tcell activity using the influence degree according to any one of <3> to <6> by the following formula:
provided that, in multiple regression analysis in which Tc*DR lymphocyte is set as the objective variable, the standard partial regression coefficients of monocyte and Act.Th1 lymphocyte are both positive.  <12> A method of evaluating killer Tcell ADCC (antibodydependent cellular cytotoxicity) activity using the influence degree according to any one of <3> to <6> by the following formula:
provided that, in multiple regression analysis in which Tc*DR lymphocyte is set as the objective variable, the standard partial regression coefficients of monocyte, Act.Th2 lymphocyte and CD20*DR lymphocyte are all positive.  <13> A system for supplying immunodynamicsrelated information for use in determining therapy or prophylaxis for a disease and/or symptom of a subject, comprising:
 (i) a means to calculate a discriminant score by assigning the cellcount of each of multiple types of immunocompetent cells in blood collected from the subject into a discriminant function;
 (ii) a means to determine a group into which the subject is to be sorted by the calculated discriminant score; and
 (iii) a means to display immunodynamicsrelated information of the determined group; wherein:
the discriminant function is obtained by performing a discriminant analysis for a data cluster containing data of the condition of an individual and the cellcount of each of multiple types of immunocompetent cells in blood collected from the individual in a number that allows for the discriminant analysis, wherein the condition of the individual is set an objective variable and the multiple types of immunocompetent cells are set as explanatory variables; and
the group into which the subject is to be sorted is one of the multiple groups that are divided from the data cluster used for obtaining the discriminant function using discriminant scores as indicator.
 <14> A program to be run by a computer for supplying immunodynamicsrelated information for use in determining therapy or prophylaxis for a disease and/or symptom of a subject, comprising:
 (i) a step of calculating a discriminant score by assigning the cellcount of each of multiple types of immunocompetent cells in blood collected from the subject into a discriminant function;
 (ii) a step of determining a group into which the subject is to be sorted by the calculated discriminant score; and
 (iii) a step of displaying immunodynamicsrelated information of the determined group; wherein:
the discriminant function is obtained by performing a discriminant analysis for a data cluster containing data of the condition of an individual and the cellcount of each of multiple types of immunocompetent cells in blood collected from the individual in a number that allows for the discriminant analysis, wherein the condition of the individual is set an objective variable and the multiple types of immunocompetent cells are set as explanatory variables; and
the group into which the subject is to be sorted is one of the multiple groups that are divided from the data cluster used for obtaining the discriminant function using discriminant scores as indicator.
[Effects of the Invention]
[0008] According to the method of the present invention, immunodynamicsrelated information of a subject can be obtained from cellcounts of immunocompetent cells in blood of the subject. The immunodynamicsrelated information can be used as a guiding principle for determining therapy or prophylaxis for a disease and/or symptom depending on the immunodynamics of individual subject. For example, according to the present invention, immunodynamics of the subject's cellular immunity can easily be comprehended, and immunodynamicsrelated information can be provided which can be used as guiding principle for diagnosis of a disease and/or symptom of a subject, in particular pathology of diseases such as autoimmune diseases (asthma, atopic dermatoses, etc.), genetic diseases which cause immune disorder, diseases related to immunity against organ transplant, cancerous diseases, infectious diseases, diseases related to cellular immunity, and therapy or prophylaxis for these diseases. Moreover, it can be used for controlling the immunity when using an immunosuppressant, and for monitoring immunodynamics related to acute or chronic rejection caused upon an organ transplant.
[0009] In specific, by sorting and analyzing data from cases accumulated so far and data of different immunocompetent cells, immunodynamicsrelated information can be provided for each of the sorted groups. More appropriate information about immunodynamics can be obtained by sorting a subject into a specific group based on cellcount data of the immunocompetent cells obtained from the subject. This can further help determining an appropriate therapy or prophylaxis.
[0010] For example, when a multiple regression analysis is to be performed for the counts of 26 different immunocompetent cells in subject's blood, 26 times each of blood drawing and measurement are necessary in order to obtain data to be subjected to the analysis. By sorting the subject into the specific group based on data obtained in advance, an immunodynamics chart for helping determining an appropriate therapy or prophylaxis can be obtained in one blood drawing.
[Brief Description of Drawings]
[0011]
[Fig. 1] Fig. 1 represents survival rate curves of 59 cases each of GOOD, MODERATE and BAD groups (total 177 cases).
[Fig. 2] Fig. 2 represents a scatter plotting of discriminant scores of 177 cases.
[Fig. 3] Fig. 3 represents a scatter plotting of discriminant scores of 162 cases sorted in GOOD group.
[Fig. 4] Fig. 4 represents a scatter plotting of discriminant scores of 45 cases sorted in MODERATE group.
[Fig. 5] Fig. 5 represents scatter plotting of discriminant scores of 137 cases sorted into BAD group.
[Fig. 6] Fig. 6 represents an immunodynamics chart for Tcell immunity in GOOD/lower Xaxis group (n = 54).
[Fig. 7] Fig. 7 represents an immunodynamics chart for Tcell immunity in GOOD/middle Xaxis group (n = 54).
[Fig. 8] Fig. 8 represents an immunodynamics chart for NK cell in GOOD/higher Xaxis group (n = 54).
[Fig. 9] Fig. 9 represents an immunodynamics chart for NK cell in GOOD/middle Xaxis group (n = 26).
[Fig. 10] Fig. 10 represents an immunodynamics chart for Tcell immunity in GOOD group (n = 26).
[Fig. 11] Fig. 11 represents an immunodynamics chart for Bcell immunity in GOOD group (n = 26).
[Fig. 12] Fig. 12 represents an immunodynamics chart for basophil in GOOD group (n = 26).
[Fig. 13] Fig. 13 represents an immunodynamics chart for eosinophil in GOOD group (n = 26).
[Fig. 14] Fig. 14 represents an immunodynamics chart for neutrophil in GOOD group (n = 26).
[Fig. 15] Fig. 15 represents an immunodynamics chart for NK cell in MODERATE group (n = 45).
[Fig. 16] Fig. 16 represents an immunodynamics chart for Tcell immunity in MODERATE group (n = 35).
[Fig. 17] Fig. 17 represents an immunodynamics chart for Tcell immunity in BAD group (n = 40).
[Fig. 18] Fig. 18 represents an immunodynamics chart for eosinophil in BAD group (n = 26).
[Fig. 19] Fig. 19 represents an immunodynamics chart for basophil in BAD group (n = 26).
[Fig. 20] Fig. 20 represents an immunodynamics chart for neutrophil in BAD group (n = 26).
[Fig. 21] Fig. 21 represents an immunodynamics chart for Bcell immunity in BAD group (n = 26).
[Fig. 22] Fig. 22 represents an immunodynamics chart for NKT cell in BAD group (n = 26).
[Fig. 23] Fig. 23 represents an immunodynamics chart for NK cell in BAD group (n = 26).
[Fig. 24] Fig. 24 represents an immunodynamics chart for Tcell immunity in BAD group (n = 26).
[Fig. 25] Fig. 25 represents an immunodynamics chart for Tcell immunity in BAD/higher Xaxis group (n = 46).
[Fig. 26] Fig. 26 represents an immunodynamics chart for NKT cell in BAD/middle Xaxis group (n = 46).
[Fig. 27] Fig. 27 represents an immunodynamics chart for Tcell immunity in BAD/middle Xaxis group (n = 26).
[Fig. 28] Fig. 28 represents an immunodynamics chart for Bcell immunity in BAD/lower Xaxis group (n = 45).
[Fig. 29] Fig. 29 represents an immunodynamics chart for Tcell immunity in BAD/lower Xaxis group (n = 45).
[Fig. 30] Fig. 30 represents an immunodynamics chart for Tcell immunity in BAD/middle Yaxis group (n = 46).
[Fig. 31] Fig. 31 represents an immunodynamics chart for NKT cell in BAD/middle Yaxis group (n = 46).
[Fig. 32] Fig. 32 represents an immunodynamics chart for Bcell immunity in BAD/higher Yaxis group (n = 45).
[Fig. 33] Fig. 33 represents an immunodynamics chart for Bcell immunity in BAD/lower Yaxis group (n = 45).
[Fig. 34] Fig. 34 represents an immunodynamics chart for Tcell immunity (n=29).
[Fig. 35] Fig. 35 represents an immunodynamics chart for Bcell immunity (n=27).
[Fig. 36] Fig. 36 represents an immunodynamics chart for NKT cells(n=27).
[Fig. 37] Fig. 37 represents an immunodynamics chart for NK cell (n=28).
[Fig. 38] Fig. 38 represents an immunodynamics chart for basophil(n=28).
[Fig. 39] Fig. 39 represents an immunodynamics chart for eosinophil(n=27).
[Fig. 40] Fig. 40 represents an immunodynamics chart for neutrophil(n=27).
[Fig. 41] Fig. 41 represents transition of different indices in subjects with lingual cancer.
[Fig. 42] Fig. 42 represents transition of different indices in subjects with prostate cancer. Cultured Living Cell Count indicates the number of lymphocytes that were collected from 40 ml of venous blood drawn from the subject, cultured, and still viable on the day of administering blood transfusion. Peripheral Blood NKActivity is the activity of NK cells collected from peripheral blood of the subject, measured by flowcytometry using calceinAM fluorescent staining dye (effectortarget ratio; 12:1). Cultured NKActivity (the activity of cultured NK cells) is the activity of NK cells collected from 40 ml of venous blood drawn from the subject and cultured, measured by flowcytometry using calceinAM fluorescent staining dye (effectortarget ratio; 12:1). Killer Activity is the activity of killer Tcells collected from 40 ml of venous blood drawn from the subject and cultured, measured by flowcytometry using calceinAM fluorescent staining dye (effector target ratio; 12:1). Cultured NK Cell Count (the number of cultured NK cells) indicates the number of NK cells that were collected from 40 ml of venous blood drawn from the subject and cultured. Cell counts are given in a unit of *10^{7} cells. CD3+CD8+αβT cell indicates the number of CD3+CD8+αβT cells that were collected from 40 ml of venous blood drawn from the subject and cultured. This is a cell fraction which contains killer Tcells and given in a unit of *10^{9} cells. CD3+CD4+αβT cell indicates the number of CD3+CD4+αβT cells that were collected from 40 ml of venous blood drawn from the subject and cultured. This is a cell fraction which contains helper Tcells and given in a unit of *10^{7} cells.
[Fig. 43] Fig. 43 represents transition of killer Tcell activity index and killer Tcell ADCC activity index in a subject with prostate cancer.
[Fig. 44] Fig. 44 represents transition of NKcell activity index and NKcell ADCC activity index in a subject with prostate cancer.
[Fig. 45] Fig. 45 represents transition of NKTcell activity index and NKTcell ADCC activity index in a subject with prostate cancer.
[Fig. 46] Fig. 46 represents a scatter plot of discriminant scores of 267 cases obtained by a discriminant function generated by targeting the 267 cases.
[Description of Embodiments]
[0012] The present invention relates to a method for supplying immunodynamicsrelated information for use in determining therapy or prophylaxis for a disease and/or symptom of a subject.
[0013] In the present invention, a subject may be any living organism, and is preferably a mammal including, for example, a primate such as human and chimpanzee, a rodent such as mouse, rat, guinea pig and hamster, and cattle, camel, goat, sheep, horse, rabbit, dog and cat, and is more preferably, human.
[0014] In the present invention, a disease and/or symptom is not particularly limited, but, for example, a disease and/or symptom associated with immunity. More specifically, it includes autoimmune diseases (such as asthma, atopic dermatoses, chronic inflammatory demyelinating polyneuropathy/multifocal motor neuropathy), genetic diseases which cause immune disorder, diseases related to immunity against organ transplant, cancerous diseases, infectious diseases, viral diseases related to cellular immunity (such as serum hepatitis), and amyotrophic lateral sclerosis, etc.
[0015] An autoimmune disease includes, such as, e.g., asthma, pollinosis, atopic dermatitis, sarcoidosis, Wegener's granulomatous angiitis, collagen disease overlap syndrome, infertility, pernicious anemia, GuillainBarre syndrome, myasthenia gravis, chronic gastritis, chronic atrophic gastritis, autoimmune hepatitis, primary biliary cholangitis, ulcerative colitis, Crohn's disease, autoimmune pancreatitis, Takayasu's arteritis, Goodpasture's syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, autoimmune hemolytic anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease (Basedow's disease), Hashimoto's disease (chronic thyroiditis), primary hypothyroidism, idiopathic Addison's disease, type 1 diabetes, insulinresistant diabetes, chronic discoid lupus erythematosus, localized scleroderma, pemphigus, pustular psoriasis, plaque psoriasis, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, alopecia areata, vitiligo, leukoderma acquisitum centrifugum Sutton/Sutton nevus, Harada disease, autoimmune optic neuropathy, autoimmune inner ear disorder, idiopathic azoospermia, rheumatoid arthritis, systemic lupus erythematosus, antiphospholipid antibody syndrome, polymyositis, dermatomyositis, scleroderma, Sjogren syndrome, IgG4related disease, vasculitic syndrome, mixed connective tissue disease, familial Mediterranean fever, PAPA syndrome (pyogenic arthritis, pyoderma gangrenosum, and acne), ankylosing spondylitis, giant cell arteritis, cryopyrinassociated periodic syndrome, CrowFukase syndrome, polyarteritis nodosa, thrombotic thrombocytopenic purpura, primary immunodeficiency syndrome, microscopic polyangiitis, hyperlgD syndrome, antiglomerular basement membrane nephritis, eosinophilic gastrointestinal disorder, eosinophilic granulomatosis with polyangiitis, eosinophilic sinusitis, relapsing polychondritis, autoimmune hemorrhaphilia XIII, purpura nephritis, adultonset Still's disease, systemic juvenile idiopathic arthritis, systemic scleroderma, granulomatosis with polyangiitis, TNF receptorassociated periodic syndrome, NakajoNishimura syndrome, inclusion body myositis, Blau syndrome, Behcet's disease, and chronic inflammatory demyelinating polyneuropathy/multifocal motor neuropathy.
[0016] Cancerous diseases are, for example, cancers and sarcomas including, such as, e.g., brain tumor (such as malignant glioma and glioblastoma), lung cancer (adenocarcinoma, squamous cell carcinoma, large cell carcinoma, small cell carcinoma), mediastinal tumor, head and neck cancer such as nasopharyngeal cancer, laryngeal cancer, lingual cancer, oral (mucosal) cancer and gingival cancer; esophageal cancer, gastric cancer, colorectal cancer, rectal cancer, anal cancer, liver cancer (including those originated from hepatic cirrhosis due to hepatitis B or C or other alcoholic or lipogenous hepatic cirrhosis), cancer of gastrointestinal system such as gallbladder/ bile duct cancer, pancreatic cancer; gynecologic cancer such as breast cancer, cervical cancer, uterine body cancer, ovarian cancer and endometrial cancer; urogenital cancer such as renal cell carcinoma, ureteropelvic cancer, bladder cancer, prostate cancer, testicular tumor, urethral cancer and penile cancer; osteosarcoma, soft tissue leiomyosarcoma, rhabdomyosarcoma, malignant melanoma, skin cancer; leukemia such as adult Tcell leukemia, EpsteinBarr virus infection (infectious mononucleosis, Burkitt's lymphoma, some nasopharyngeal cancers), Hodgkin's lymphoma and hairy cell leukemia, and hematologic diseases such as malignant lymphoma.
[0017] Infectious diseases include, such as, e.g., viral infection, bacterial infection, fungal infection, protozoan parasite infection and helminthic parasite infection.
[0018] Viral infections include, such as, e.g., common cold, norovirus infection, rotavirus infection, influenza virus infection, viral hepatitis, viral meningitis, acquired immunodeficiency syndrome (AIDS), adult Tcell leukemia, Ebola hemorrhagic fever, yellow fever, common cold syndrome, rabies, cytomegalovirus infection, severe acute respiratory syndrome (SARS), progressive multifocal leukoencephalopathy, chickenpox/herpes zoster, herpes simplex, handfootandmouth disease, dengue fever, erythema infectiosum, infectious mononucleosis, smallpox, rubella, acute anterior poliomyelitis (polio), measles, pharyngoconjunctival fever (pool fever), Marburg hemorrhagic fever, hantavirus renal hemorrhagic fever, Lassa fever, South American hemorrhagic fever, Middle East respiratory syndrome (MERS), mumps, West Nile fever, herpangina, chikungunya fever.
[0019] Bacterial infection include a variety of infections by, such as, e.g., Streptococcus (e.g., Group A β hemolytic streptococcus,
Streptococcus pneumoniae),
Staphylococcus aureus (MSSA, MRSA),
Staphylococcus epidermidis, Enterococcus, Listeria,
Neisseria meningitidis, Neisseria gonorrhoeae, pathogenic
Escherichia coli (e.g., O157:H7), Klebsiella (
Klebsiella pneumoniae), Proteus,
Bordetella pertussis, Pseudomonas aeruginosa, Serratia, Citrobacter, Acinetobacter, Enterobacter, Mycoplasma, Clostridium, and tuberculosis, cholera, plague, diphtheria, dysentery, scarlet fever, anthrax, syphilis, tetanus, Hansen's disease, Legionella pneumonia, leptospirosis, Lyme disease, tularemia and Qfever.
[0020] In the present invention, therapy can be any therapy known for the disease or symptom, and may be, without being particularly limited, for example, a therapy that utilizes immune function such as immune checkpoint inhibitor therapy, cytokine therapy, cell adoptive immunity therapy (e.g., αβT cell, γδT cell, NK cell and NKT cell), regenerative immunotherapy with iPS cells and genetically modified T cell therapy (CART: Chimeric Antigen Receptor Tcell Therapy).
[0021] In the present invention, prophylaxis can be any prophylaxis known for the disease or symptom, and may be, without being particularly limited, for example, a prophylaxis that enhances immune function by an intake or administration of an useful substance. The useful substances include, for example, fungus such as Agaricus, Reishi, Polyporaceae (Hoelen), Cordyceps, Shiitake mushroom, Shiitake mushroom extract, AHCC (Active Hexose Correlated Compound)® or a functional food, extract or supplement made of fungus; Chinese herbal medicines such as juzentaihoto, hochuekkito and saireito; lipidlowering drug; supplements such as vitamins such as vitamin D3.
[0022] In the present invention, immunodynamicsrelated information is information for determining whether different immunocompetent cells are coordinately functioning or whether their differentiation and proliferation is stagnated/inhibited, and it means, for example, information about analyses of the degree or condition of the correlation between different immunocompetent cells.
[0023] More specifically, information is about analyses of the degree of coordination to, e.g., Tc*DR lymphocyte, CD20*DR lymphocyte, NK cell, NKT cell, basophil, eosinophils or neutrophils by other immunocompetent cells. Moreover, immunodynamicsrelated information can be expressed as an immunodynamics chart. By being expressed as an immunodynamics chart, the condition of immunodynamics will more easily be determined or understood. Such information is extremely useful in determining the immunocompetent cell to be targeted or the correlation between immunocompetent cells for therapy or prophylaxis of a disease or symptom.
[0024] In the present invention, the degree of coordination is expressed, for example, by a influence degree (the area of a circle in an immunodynamics chart) or by either positive or negative value of the partial regression coefficient (a type of arrow in the immunodynamics chart).
[0025] The method for supplying immunodynamicsrelated information of the present invention, in one embodiment, comprises:
 (i) calculating a discriminant score by assigning the cellcount of each of multiple types of immunocompetent cells in blood collected from the subject into a discriminant function;
 (ii) determining a group into which the subject is to be sorted by the calculated discriminant score; and
 (iii) displaying immunodynamicsrelated information of the determined group.
[0026] Here, the discriminant function can be obtained by performing a discriminant analysis for a data cluster containing data of the condition of an individual and the cellcount of each of multiple types of immunocompetent cells in blood collected from the individual in a number that allows for the discriminant analysis, wherein the condition of the individual is set as an objective variable, and the multiple types of immunocompetent cells are set as explanatory variables.
[0027] In the present invention, the condition of an individual is such as, e.g., health, a disease, a disorder, a symptom or prognosis of an individual. The condition of an individual can be typified according to the necessity. It is typified, for example, by degree of health, degree of symptoms, type or degree of the disease, type or degree of the disorder, and degree of the prognosis. Typification can also be, for example, classification by the values of biomarkers, by disease stages, by the rate of complete remission, by survival rate such as threeyear or fiveyear survival rate.
[0028] In the present invention, the number of individuals is any number. For example, a plurality of data can be obtained from one individual.
[0029] Moreover, the group into which the subject is to be sorted is one of the multiple groups that are divided from the data cluster used for obtaining the discriminant function using discriminant scores as indicator.
[0030] In the present invention, a discriminant analysis encompasses a discriminant analysis which involves two groups of objective variables, as well as a multiple discriminant analysis or canonical discriminant analysis which involves three or more groups of objective variables.
[0031] In the present invention, a data cluster means a cluster of data which comprises at least data of the condition of an individual and data of the cellcounts of multiple immunocompetent cells in blood collected from the individual, and the data cluster comprises such data in a number that allows for the discriminant analysis. Here, the number that allows for the discriminant analysis is a number which exceeds the number of explanatory variables by at least one in the discriminant analysis. The data cluster can be divided into multiple groups using the discriminant score as an indicator.
[0032] In the present invention, an immunocompetent cell means any cell which is in charge of immune response, and includes, such as, without being particularly limited, e.g., a leukocyte, monocyte, basophil, eosinophil, neutrophil, CD3positive lymphocyte (CD3), CD4positive lymphocyte (CD4), CD8positive lymphocyte (CD8), CD20*DR lymphocyte (CD20
^{∗}DR), Ti lymphocyte (inducer T cell; Ti), Ti2 lymphocyte (Ti2), Ti± lymphocyte (Ti±), Ti+2 lymphocyte (Ti+2), Th lymphocyte (helper T cell; Th), Th1 lymphocyte (helper Th1 lymphocyte; helperTh1 cell; Th1), Th2 lymphocyte (helperTh2 lymphocyte; helperTh2 cell; Th2), Th2 lymphocyte (Th2), Th± lymphocyte (Th±), Th+2 lymphocyte (Th+2), Th17+ lymphocyte (Th17+), Ts lymphocyte (suppressor T cell; Ts), Ts lymphocyte (Ts), Ts+ lymphocyte (Ts+), Tc lymphocyte (cytotoxic T cell; Tc), Tc lymphocyte (Tc), Tc+ lymphocyte (Tc+), Act.s/cT lymphocyte (activated suppressor/cytotoxic T cell; Act.s/cT), Ts*DR lymphocyte (activated suppressor T cell; Ts*DR), Tc*DR lymphocyte (activated cytotoxic T cell; Tc*DR), Act.i/h T lymphocytes (activated inducer/helper T cell; activated inducer/helper T cell; Act.i/hT), Th*DR (activated helper T cell; activated helper T cell; Th*DR), Ti*DR lymphocyte (activated inducerT cell; Ti*DR), Act.Th1 lymphocyte (activated helper Th1 cell; Act.Th1), Act.Th2 lymphocyte (activated helper Th2 cell; Act.Th2), NK cell (natural killer cell; NK), NKT cell (natural killer T cell; NKT) and N3+ cell (N3+). For any one of the immunocompetent cells described above, the abbreviation described in parentheses may be used.
[0033] In the present invention, the cellcount of an immunocompetent cell can be counted or calculated according to a conventional method.
[0034] For instance, the number of leukocytes, lymphocytes, monocyte, basophil, eosinophils or neutrophils can be counted to 1 digit of real number by subjecting the blood drawn from the subject to a general automatic hemocyte counting apparatus. The unit is, for example, in cells/cubic millimeter (mm
^{3} = µL).
[0035] Lymphocytes are single or multistained for the abundance ratio of various cellsurface markers such as, e.g., CD3, CD4, CD8, CD11b, CD16, CD20, CD25, CD45RA, CD56, CD122, CD161, FoxP3, HLADR, IFNγ and IL4 using antibodies, and measured by flowcytometry.
[0036] The lymphocytes are defined as described below by the combination of various cellsurface markers such as, e.g., CD3, CD4, CD8, CD11b, CD16, CD20, CD25, CD45RA, CD56, CD122, CD161, FoxP3, HLADR, IFNγ and IL4.
[0037] A CD3positive lymphocyte means a lymphocyte which is CD3positive. This is similar for representation such as a CD4positive or CD8positive lymphocyte.
[0038] The number of CD3positive lymphocytes can be calculated, for example, by multiplying either the percentage of CD3positives in the result of a doublestaining flowcytometry analysis for CD3 and CD161 (i.e., the sum of the percentage of [CD3positive and CD161negative] plus the percentage of [CD3positive and CD161positive]) or the percentage of CD3positives in the result of a quadruplestaining flowcytometry analysis for CD11b, CD122, CD3 and CD8 by the number of lymphocytes. For instance, the values of these may be compared to adopt smaller value.
[0039] The number of CD4positive lymphocytes can be calculated, for example, by using either of the percentage of CD4positives in the result of a doublestaining flowcytometry analysis for HLADR and CD4 (i.e., the sum of the percentage of [CD4positive and HLADRnegative] and the percentage of [CD4positive and HLADRpositive]) or the percentage of CD4positives in the result of a quadruplestaining flowcytometry analysis for CD45RA, FoxP3, CD4 and CD25, and multiplying this by the number of lymphocytes. For instance, the values of these may be compared to adopt smaller value.
[0040] The number of CD8positive lymphocytes can be calculated, for example, by multiplying the percentage of CD8positives in the result of a doublestaining flowcytometry analysis for HLADR and CD8 (i.e., the sum of the percentage of [CD8positive and HLADRnegative] and the percentage of [CD8positive and HLADRpositive]) by the number of lymphocytes.
[0041] CD20*DR lymphocyte means a lymphocyte that is CD20positive and HLADRpositive.
[0042] The number of CD20*DR lymphocytes can be calculated, for example, by multiplying the percentage of [CD20positive and HLADRpositive] in the result of a doublestaining flowcytometry analysis for HLADR and CD20 by the number of lymphocytes.
[0043] Ti lymphocyte (inducer T cell) means a lymphocyte that is CD4positive and CD45RApositive.
[0044] The number of Ti lymphocytes can be calculated, for example, by multiplying the percentage of [CD4positive and CD45RApositive] in the result of a quadruplestaining flowcytometry analysis for CD45RA, FoxP3, CD4 and CD25 by the number of lymphocytes.
[0045] Ti2 lymphocyte means a lymphocyte that is CD4positive, CD45RApositive, CD25negative, and FoxP3negative.
[0046] The number of Ti2 lymphocytes can be calculated, for example, by multiplying the percentage of [CD4positive and CD45RApositive] and the percentage of [CD4positive, CD45RApositive, CD25negative and FoxP3negative] in the result of a quadruplestaining flowcytometry analysis for CD45RA, FoxP3, CD4 and CD25, and multiplying this by the number of lymphocytes.
[0047] Ti± lymphocyte means a lymphocyte that is CD4positive, CD45RApositive, CD25positive, and FoxP3negative.
[0048] The number of Ti± lymphocytes can be calculated, for example, by multiplying the percentage of [CD4positive and CD45RApositive] and the percentage of [CD4positive, CD45RApositive, CD25positive and FoxP3negative] in the result of a quadruplestaining flowcytometry analysis for CD45RA, FoxP3, CD4 and CD25, and multiplying this by the number of lymphocytes.
[0049] Ti+2 lymphocyte means a lymphocyte that is CD4positive, CD45RApositive, CD25positive, and FoxP3positive.
[0050] The number of Ti+2 lymphocytes can be calculated, for example, by multiplying the percentage of [CD4positive and CD45RApositive] and the percentage of [CD4positive, CD45RApositive, CD25positive and FoxP3positive] in the result of a quadruplestaining flowcytometry analysis for CD45RA, FoxP3, CD4 and CD25, and multiplying this by the number of lymphocytes.
[0051] Th lymphocyte (helper T cell) means a lymphocyte that is CD4positive, and CD45RAnegative.
[0052] The number of Th lymphocytes can be calculated, for example, by multiplying the percentage of [CD4positive and CD45RAnegative] in the result of a quadruplestaining flowcytometry analysis for CD45RA, FoxP3, CD4 and CD25 by the number of lymphocytes.
[0053] Th1 lymphocyte (helper Th1 lymphocyte; helper Th1 cell) means a lymphocyte that is CD4positive, CD45RAnegative, IFNγpositive, and IL4negative.
[0054] The number of Th1 lymphocytes can be calculated by multiplying the number of Th lymphocytes by, e.g., the percentage of [CD4positive, IFNγpositive and IL4negative] in the results of triple staining analysis by flowcytometry for CD4, IFNγ and IL4.
[0055] Th2 lymphocyte (helper Th2 lymphocyte; helper Th2 cell) means a lymphocyte that is CD4positive, CD45RAnegative, IFNγnegative, and IL4positive.
[0056] The number of Th2 lymphocytes can be calculated, for example, by multiplying the number of Th lymphocytes by the percentage of [CD4positive, IFNγnegative and IL4positive] in the results of triple staining analysis by flowcytometry for CD4, IFNγ and IL4.
[0057] Th2 lymphocyte means a lymphocyte that is CD4positive, CD45RAnegative, CD25negative, and FoxP3negative.
[0058] The number of Th2 lymphocytes can be calculated, for example, by multiplying the percentage of [CD4positive and CD45RAnegative] and the percentage of [CD4positive, CD45RAnegative, CD25negative and FoxP3negative] in the result of a quadruplestaining flowcytometry analysis for CD45RA, FoxP3, CD4 and CD25, and multiplying this by the number of lymphocytes.
[0059] Th± lymphocyte means a lymphocyte that is CD4positive, CD45RAnegative, CD25positive, and FoxP3negative.
[0060] The number of Th± lymphocytes can be calculated, for example, by multiplying the percentage of [CD4positive and CD45RAnegative] and the percentage of [CD4positive, CD45RAnegative, CD25positive and FoxP3negative] in the result of a quadruple staining flowcytometry analysis for CD45RA, FoxP3, CD4 and CD25, and multiplying this by the number of lymphocytes.
[0061] Th+2 lymphocyte means a lymphocyte that is CD4positive, CD45RAnegative, CD25positive, and FoxP3positive.
[0062] The number of Th+2 lymphocytes can be calculated, for example, by multiplying the percentage of [CD4positive and CD45RAnegative] and the percentage of [CD4positive, CD45RAnegative, CD25positive and FoxP3positive] in the result of a quadruplestaining flowcytometry analysis for CD45RA, FoxP3, CD4 and CD25, and multiplying this by the number of lymphocytes.
[0063] Th17+ lymphocyte means a lymphocyte that is CD4positive, IFNγpositive and IL17positive.
[0064] The number of Th17+ lymphocytes can be calculated, for example, by multiplying the percentage of [IFNγpositive and IL17positive] in the results of triple staining analysis by flowcytometry for IFNγ, CD4 and IL17 by the number of CD4 lymphocytes.
[0065] Act.i/h T lymphocytes (Activated inducer/helper T Cell) means a lymphocyte that is CD4positive and HLADRpositive.
[0066] The number of Act.i/h T lymphocytes can be calculated, for example, by multiplying the percentage of [CD4positive and HLADRpositive] in the result of a doublestaining flowcytometry analysis for HLADR and CD4 by the number of lymphocytes.
[0067] The number of Ti*DR lymphocytes (Activated inducer T cell) can be calculated, for example, by multiplying Act.i/h T lymphocytes and the number of Ti lymphocytes/(the number of Th lymphocytes + the number of Ti lymphocytes).
[0068] The number of Th*DR lymphocytes (Activated helper T cells) can be calculated, for example, by multiplying the number of Act.i/h T lymphocytes by [the number of Th lymphocytes ÷ (the number of Th lymphocytes + the number of Ti lymphocytes)].
[0069] The number of Act.Th1 lymphocytes can be calculated, for example, by multiplying the number of Th*DR lymphocytes by [the number of Th1 lymphocytes ÷ (the number of Th1 lymphocytes + the number of Th2 lymphocytes)].
[0070] The number of Act.Th2 lymphocytes can be calculated, for example, by multiplying the number of Th*DR lymphocytes by [the number of Th2 lymphocytes ÷ (the number of Th1 lymphocytes + the number of Th2 lymphocytes)].
[0071] Ts lymphocyte (Suppressor T cell) means a lymphocyte that is CD8positive and CD11bpositive.
[0072] The number of Ts lymphocytes can be calculated, for example, by multiplying the percentage of [CD8positive and CD11bpositive] in the result of a quadruplestaining flowcytometry analysis for CD11b, CD122, CD3 and CD8 by the number of lymphocytes.
[0073] Ts lymphocyte means a lymphocyte that is CD8positive, CD11bpositive and CD122negative.
[0074] The number of Ts lymphocytes can be calculated, for example, by multiplying the percentage of [CD8positive and CD11bpositive] and the percentage of [CD11bpositive and CD122negative] in the result of a quadruplestaining flowcytometry analysis for CD11b, CD122, CD3 and CD8, and multiplying this by the number of lymphocytes.
[0075] Ts+ lymphocyte means a lymphocyte that is CD8positive, CD11bpositive and CD122positive.
[0076] The number of Ts+ lymphocytes can be calculated, for example, by multiplying the percentage of [CD8positive and CD11bpositive] and the percentage of [CD11bpositive and CD122positive] in the result of a quadruplestaining flowcytometry analysis for CD11b, CD122, CD3 and CD8, and multiplying this by the number of lymphocytes.
[0077] Tc lymphocyte (Cytotoxic T cell) means a lymphocyte that is CD8positive and CD11bnegative.
[0078] The number of Tc lymphocytes can be calculated, for example, by multiplying the percentage of [CD8positive and CD11bnegative] in the result of a quadruplestaining flowcytometry analysis for CD11b, CD122, CD3 and CD8 by the number of lymphocytes.
[0079] Tc lymphocyte means a lymphocyte that is CD8positive, CD11bnegative, and CD122negative.
[0080] The number of Tc lymphocytes can be calculated, for example, by multiplying the percentage of [CD8positive and CD11bnegative] and the percentage of [CD11bnegative and CD122negative] in the result of a quadruplestaining flowcytometry analysis for CD11b, CD122, CD3 and CD8, and multiplying this by the number of lymphocytes.
[0081] Tc+ lymphocyte means a lymphocyte that is CD8positive, CD11bnegative, and CD122positive.
[0082] The number of Tc+ lymphocytes can be calculated, for example, by multiplying the percentage of [CD8positive and CD11bnegative] and the percentage of [CD11bnegative and CD122positive] in the result of a quadruplestaining flowcytometry analysis for CD11b, CD122, CD3 and CD8, and multiplying this and the number of lymphocytes.
[0083] The number of Act.s/cT lymphocytes (Activated suppressor/cytotoxic T cells) can be calculated, for example, by multiplying the percentage of [CD8positive and HLADRpositive] in the result of a doublestaining flowcytometry analysis for HLADR and CD8 by the number of lymphocytes.
[0084] The number of Ts*DR lymphocytes (Activated suppressor T Cells) can be calculated, for example, by multiplying the number of Act.s/cT lymphocytes by [the number of Ts lymphocytes ÷ (the number of Tc lymphocytes + the number of Ts lymphocytes)].
[0085] The number of Tc*DR lymphocytes (Activated cytotoxic T cells) can be calculated, for example, by multiplying the number of Act.s/cT lymphocytes by [the number of Tc lymphocytes ÷ (the number of Tc lymphocytes + the number of Ts lymphocytes)].
[0086] NK cell (Natural killer Cell) means a lymphocyte that is CD16positive and CD56positive.
[0087] The number of NK cells can be calculated, for example, by multiplying the percentage of [CD16positive and CD56positive] in the results of triple staining analysis by flowcytometry for CD16, CD161 and CD56 by the number of lymphocytes.
[0088] NKT cells (Natural killer T cell) means a lymphocyte that is CD3positive and CD161positive.
[0089] The number of NKT cells can be calculated, for example, by multiplying the percentage of [CD3positive and CD161positive] in the result of a doublestaining flowcytometry analysis for CD3 and CD161 by the number of lymphocytes.
[0090] N3+ cell means a lymphocyte that is CD16positive, CD161positive and CD56positive.
[0091] The number of N3+ cell can be calculated, for example, by multiplying the percentage of [CD16positive, CD161positive and CD56positive] in the results of triple staining analysis by flowcytometry for CD16, CD161 and CD56 by the number of lymphocytes.
[0092] In one embodiment of the present invention, the multiple types of immunocompetent cells are three or more selected from a group consisting of, e.g., Th17+ lymphocyte, CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Th± lymphocyte, Th2 lymphocyte, Th+2 lymphocyte, Act.Th1 lymphocyte, Act.Th2 lymphocyte, Ti*DR lymphocyte, Ti± lymphocyte, Ti2 lymphocyte, Ti+2 lymphocyte, Tc*DR lymphocyte, Tc lymphocyte, Tc+ lymphocyte, Ts*DR lymphocyte, Ts lymphocyte, Ts+ lymphocyte, NK cell, NKT cell, N3+ cell, monocyte, basophil, eosinophils, neutrophil, Th1 lymphocyte, Th2 lymphocyte, Ti lymphocyte, Tc lymphocyte, Ts lymphocyte, Act.i/h T lymphocytes and Act.s/cT lymphocyte.
[0093] In one embodiment of the present invention, the multiple types of immunocompetent cells are three or more selected from a group consisting of, e.g., Th17+ lymphocyte, CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Th± lymphocyte, Th2 lymphocyte, Th+2 lymphocyte, Act.Th1 lymphocyte, Act.Th2 lymphocyte, Ti*DR lymphocyte, Ti± lymphocyte, Ti2 lymphocyte, Ti+2 lymphocyte, Tc*DR lymphocyte, Tc lymphocyte, Tc+ lymphocyte, Ts*DR lymphocyte, Ts lymphocyte, Ts+ lymphocyte, NK cell, NKT cell, N3+ cell, monocyte, basophil, eosinophil and neutrophil.
[0094] In one embodiment of the present invention, the multiple types of immunocompetent cells comprise, e.g., Th17+ lymphocyte.
[0095] In one embodiment of the present invention, the multiple types of immunocompetent cells are 17 types consisting of: CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Th1 lymphocyte, Th2 lymphocyte, Ti lymphocyte, Tc lymphocyte, Ts lymphocyte, Act.i/h T lymphocyte, Act.s/c T lymphocyte, NK cell, N3+ cell, monocyte, basophil, eosinophil and neutrophil.
[0096] In one embodiment of the present invention, preferably the multiple types of immunocompetent cells are 26 types consisting of: CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Th± lymphocyte, Th2 lymphocyte, Th+2 lymphocyte, Act.Th1 lymphocyte, Act.Th2 lymphocyte, Ti*DR lymphocyte, Ti± lymphocyte, Ti2 lymphocyte, Ti+2 lymphocyte, Tc*DR lymphocyte, Tc lymphocyte, Tc+ lymphocyte, Ts*DR lymphocyte, Ts lymphocyte, Ts+ lymphocyte, NK cell, NKT cell, N3+ cell, monocyte, basophil, eosinophil and neutrophil.
[0097] In one embodiment of the present invention, more preferably the multiple types of immunocompetent cells are 27 types consisting of: Th17+ lymphocyte, CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Th± lymphocyte, Th2 lymphocyte, Th+2 lymphocyte, Act.Th1 lymphocyte, Act.Th2 lymphocyte, Ti*DR lymphocyte, Ti± lymphocyte, Ti2 lymphocyte, Ti+2 lymphocyte, Tc*DR lymphocyte, Tclymphocyte, Tc+ lymphocyte, Ts*DR lymphocyte, Ts lymphocyte, Ts+ lymphocyte, NK cell, NKT cell, N3+ cell, monocyte, basophil, eosinophil and neutrophil.
[0098] In the present invention, immunodynamicsrelated information is obtained, for example, by the following (a) to (d), without being particularly limited:
 (a) performing a multiple regression analysis for data of the cellcounts of n types of immunocompetent cells that constitute one group, wherein one type of immunocompetent cell among the n types of immunocompetent cells is set as an objective variable, and n1 types of immunocompetent cells excluding the one type of immunocompetent cell that is set as the objective variable as explanatory variables, and wherein n is an integer of 4 or more;
 (b) ranking the n1 types of immunocompetent cells in descending order according to the magnitude of the absolute value of the standard partial regression coefficient obtained from the multiple regression analysis;
 (c) performing a regression analysis in which the one type of immunocompetent cell that is the objective variable in (a) above is set as an objective variable and the immunocompetent cell that is ranked as the first place in (b) above is set as explanatory variable, calculating the contribution ratio α_{1} which is considered as the influence degree of the firstplace ranked immunocompetent cell β_{1}; and
 (d) performing a multiple regression analysis in which the one type of immunocompetent cell that is the objective variable in (a) above is set as an objective variable and m types of immunocompetent cells from the first to the mth place ranked in (b) above are set as explanatory variables, calculating the contribution ratio α_{m}, and calculating the influence degree β_{m} of the immunocompetent cell ranked as mth place by the following formula:
for each of the immunocompetent cells ranked from the second to the mth place; wherein m is more than 3 and up to n1.
[0099] In the present invention, regression analysis not only encompasses a single regression analysis which involves one explanatory variable, but also encompasses a multiple regression analysis which involves two or more explanatory variables. Moreover, in the present invention, the description "performing multiple regression analyses repeatedly" may include embodiments in which the repeated regression analyses include a single regression analysis which involves one explanatory variable.
[0100] In the present invention, when performing a multiple regression analysis, a constant term may or may not be provided. Preferably, in the present invention, when performing a multiple regression analysis, no constant term is provided.
[0101] In the present invention, without performing a discriminant analysis, immunodynamicsrelated information can be obtained by performing regression analysis as described above on data of cellcounts of n types of immunocompetent cells which constitute the desired group.
[0102] In one embodiment of the present invention, the one type of immunocompetent cell that is set as the objective variable in (a) above can be selected from a group consisting of, e.g., Tc*DR lymphocyte, CD20*DR lymphocyte, NK cell, NKT cell, basophil, eosinophil and neutrophil.
[0103] The present invention, in one aspect, relates to a system for supplying immunodynamicsrelated information for use in determining therapy or prophylaxis for a disease and/or symptom of a subject.
[0104] The system comprises, for example:
 (i) a means to calculate a discriminant score by assigning the cellcount of each of multiple types of immunocompetent cells in blood collected from the subject into a discriminant function;
 (ii) a means to determine a group into which the subject is to be sorted by the calculated discriminant score; and
 (iii) a means to display immunodynamicsrelated information of the determined group; wherein:
the discriminant function is obtained by performing a discriminant analysis for a data cluster containing data of the condition of an individual and the cellcount of each of multiple types of immunocompetent cells in blood collected from the individual in a number that allows for the discriminant analysis, wherein the condition of the individual is set an objective variable and the multiple types of immunocompetent cells are set as explanatory variables; and
the group into which the subject is to be sorted is one of the multiple groups that are divided from the data cluster used for obtaining the discriminant function using discriminant scores as indicator.
[0105] The present invention, in one aspect, relates to a program to be run by a computer for supplying immunodynamicsrelated information for use in determining therapy or prophylaxis for a disease and/or symptom of a subject.
[0106] The program comprises, for example:
 (i) a step of calculating a discriminant score by assigning the cellcount of each of multiple types of immunocompetent cells in blood collected from the subject into a discriminant function;
 (ii) a step of determining a group into which the subject is to be sorted by the calculated discriminant score; and
 (iii) a step of displaying immunodynamicsrelated information of the determined group; wherein:
the discriminant function is obtained by performing a discriminant analysis for a data cluster containing data of the condition of an individual and the cellcount of each of multiple types of immunocompetent cells in blood collected from the individual in a number that allows for the discriminant analysis, wherein the condition of the individual is set an objective variable and the multiple types of immunocompetent cells are set as explanatory variables; and
the group into which the subject is to be sorted is one of the multiple groups that are divided from the data cluster used for obtaining the discriminant function using discriminant scores as indicator.
[0107] The present invention, in one aspect, immunodynamicsrelated information can be provided as an immunodynamics chart. By representing in an immunodynamics chart, the immunodynamics can generally be understood and evaluated. In the present invention, an immunodynamics chart can be generated based on immunodynamicsrelated information, for example, influence degrees of different immunocompetent cells.
[0108] In specific, examples include the immunodynamics charts shown in Figs. 640. The arrangement of various immunocompetent cells and arrows has been determined based on the process of differentiation and maturation of the immunocompetent cells and their interrelationships.
[0109] In order to draw a clear immunodynamics chart, for example, the area of a circle indicating an immunocompetent cell can be made proportional to the percentage (%) of the influence degree of the immunocompetent cell. Moreover, when the percentage of the influence degree (%) of immunocompetent cell is less than 0.00785 %, the immunocompetent cell can be represented by ×, and when the percentage of the influence degree (%) of immunocompetent cell is or more 0.00785 % and less than 0.0314 %, the immunocompetent cell can be represented by ●. Moreover, the line weight of the enclosing line can be made 1.0 pt when the percentage of the influence degree (%) of a immunocompetent cell is less than 0.5 %; 1.5 pt when the percentage of the influence degree (%) of a immunocompetent cell is 0.5 % or more and less than 1%; 2.5 pt when the percentage of the influence degree (%) of a immunocompetent cell is 1 % or more and less than 5 %; and 3.0 pt when the percentage of the influence degree (%) of a immunocompetent cell is 5 % or more.
[0110] Types of arrows and their meanings are as described in Table 1. The type of an arrow is determined by the interrelationship of immunocompetent cells (a relationship on the differentiation pathway, or a suppressing, facilitating or interacting relationship) and by whether the partial regression coefficient value of the immunocompetent cell obtained by the multiple regression analysis is either positive or negative. For example, when the partial regression coefficient of an immunocompetent cell that is to be explanatory variable is positive against that of an immunocompetent cell used as objective variable (i.e., Tc*DR lymphocyte, CD20*DR lymphocyte, NK cell, NKT cell, basophil, eosinophil or neutrophil), the relationship is active and an open white arrow will be applied. When the partial regression coefficient is negative, the relationship is inactive and a closed black arrow will be applied.
[0111] In an immunodynamics chart directed to Tc*DR, CD20*DR lymphocyte is in active or inactive interrelationship with Tc*DR; in an immunodynamics chart directed to NK cell, Tc*DR lymphocyte, CD20*DR lymphocyte and Ti*DR lymphocyte are in active or inactive interrelationships with NK cell; in an immunodynamics chart directed to NKT cell, Tc*DR lymphocyte, CD20*DR lymphocyte and N3+ cell are in active or inactive interrelationships with NKT cell; in an immunodynamics chart directed to CD20*DR lymphocyte, Tc*DR lymphocyte is in active or inactive interrelationship with CD20*DR lymphocyte; in an immunodynamics chart directed to basophil, Tc*DR lymphocyte and Act.Th2 lymphocyte are in active or inactive interrelationships with basophil; in an immunodynamics chart directed to eosinophil, Tc*DR lymphocyte and CD20*DR lymphocyte are active or inactive interrelationships with eosinophils; in an immunodynamics chart directed to neutrophil, Th17+ lymphocyte, Tc*DR lymphocyte and CD20*DR lymphocyte are in active or inactive interrelationship with neutrophil.
[0112] In order to draw a clear immunodynamics chart, the line width of an arrow can be 6 pt when the percentage of the influence degree (%) of a immunocompetent cell is less than 0.1 %; 8 pt when the percentage of the influence degree (%) of a immunocompetent cell is or more 0.1 % and less than 0.5 %; 10 pt when the percentage of the influence degree (%) of a immunocompetent cell is 0.5 % or more and less than 1.0 %; 12 pt when the percentage of the influence degree (%) of a immunocompetent cell is 1.0 % or more and less than 5.0 %; and 14 pt when the percentage of the influence degree (%) of a immunocompetent cell is 5.0 % or more.
[0113] In an immunodynamics chart directed to Tc*DR, Monocyte > Act.Th1 > Tc*DR pathway is termed "Tcellular Antigen Recognition Mechanism" (TARM), which is also termed "killer cell activity". Monocyte > Act.Th2 > CD20*DR pathway is termed "Bcellular Antigen Recognition Mechanism" (BARM). If Monocyte > Act.Th2 > CD20*DR <=> Tc*DR pathway has been established, antibodydependent cellular cytotoxicity (ADCC) of killer Tcell will be in effect.
[0114] In an immunodynamics chart directed to NK cell, Monocyte > Act.Th1 > Tc*DR <=> NK pathway is termed NK activity, whereas Monocyte > Act.Th2 > CD20*DR <=> NK pathway is termed NKcell ADCC activity.
[0115] In an immunodynamics chart directed to NKT cell, Monocyte > Act.Th1 > Tc*DR <=> NKT pathway is termed NKT activity, whereas Monocyte > Act.Th2 > CD20*DR <=> NKT pathway is termed NKTcell ADCC activity.
[0116] In one aspect of the present invention, immunodynamicsrelated information can be used to calculate, e.g., NK activity index (NK Cell Activity Index: NKAI), ADCC activity index of NK (NK Cell Antibody Dependent Cellular Cytotoxicity Activity Index: NKADCCAI), NKT activity index (NKT Cell Activity Index: NKTAI), ADCC activity index of NKT (NKT Cell Antibody Dependent Cellular Cytotoxicity Activity Index: NKTADCCAI), killer Tcell activity index (Activated Cytotoxic T Cell Activity index) (Killer T Cell Activity Index: KillerAI), killer Tcell ADCC activity index (Killer T Cell Antibody Dependent Cellular Cytotoxicity Activity Index: KillerADCCAI). These indices can be used as indicator of immunodynamics, and are, in general, preferred at higher values. Moreover, using these indices, it is possible to predict the degree of tumor mass which can be destructed by an immunocytic therapy.
[0118] Here, contribution ratio in the regression analysis in which NK cell is set as objective variable and all other immunocompetent cells are set as explanatory variables is equal to the sum of respective influence degrees of all immunocompetent cells when the influence degrees are calculated using NK cell as the objective variable.
[0119] Also, contribution ratio in the regression analysis in which NKT cells is set as objective variable and all other immunocompetent cells are set as explanatory variables is equal to the sum of respective influence degrees of all immunocompetent cells when the influence degrees are calculated using NKT cells as the objective variable.
[0120] Also, contribution ratio in the regression analysis in which Tc*DR lymphocyte is set as objective variable and all other immunocompetent cells are set as explanatory variables is equal to the sum of respective influence degrees of all immunocompetent cells when the influence degrees are calculated using Tc*DR lymphocyte as the objective variable.
[0121] Note that, in multiple regression analysis in which NK cell is set as the objective variable, if the standard partial regression coefficient of monocyte, Act.Th1 lymphocyte or Tc*DR lymphocyte is negative, Tcellular antigen recognition mechanism (TARM) is yet to be established and therefore an NKcell activity index cannot be calculated. Moreover, in multiple regression analysis in which NK cell is set as the objective variable, if standard partial regression coefficient of monocyte, Act.Th2 lymphocyte or CD20*DR lymphocyte is negative, Bcellular Antigen Recognition Mechanism (BARM) is yet to be established and therefore an NKcell ADCC activity cannot be calculated.
[0122] Similarly, in a multiple regression analysis in which NKT cell is set as the objective variable, if the standard partial regression coefficient of monocyte, Act.Th1 lymphocyte or Tc*DR lymphocyte is negative, Tcellular antigen recognition mechanism (TARM) is yet to be established and therefore an NKTcell activity index cannot be calculated. Moreover, in multiple regression analysis in which NKT cell is set as the objective variable, if the standard partial regression coefficient of monocyte, Act.Th2 lymphocyte or CD20*DR lymphocyte is negative, Bcellular Antigen Recognition Mechanism (Bcellular Antigen Recognition Mechanism: BARM) is yet to be established and therefore an NKTcell ADCC activity cannot be calculated.
[0123] Similarly, in multiple regression analysis in which Tc*DR cell is set as the objective variable, if the standard partial regression coefficient of monocyte, Act.Th1 lymphocyte or Tc*DR lymphocyte is negative, Tcellular antigen recognition mechanism (TARM) is yet to be established and therefore a killer Tcell activity index cannot be calculated. Moreover, in multiple regression analysis in which Tc*DR cell is set as the objective variable, if the standard partial regression coefficient of monocyte, Act.Th2 lymphocyte or CD20*DR lymphocyte is negative, Bcellular Antigen Recognition Mechanism (Bcellular Antigen Recognition Mechanism: BARM) is yet to be established and therefore a killer Tcell ADCC activity cannot be calculated.
[0124] Hereinbelow, the present invention will be further explained in detail in reference to working examples, though the present invention is not to be limited by these working examples.
[Working Examples]
Example 1. Subject Sorting
[0125] Subjects consist of 177 cases undergoing hospital treatment for prostate cancer at various stages and levels of malignancy before treatment. Various treatments were given including hormonal therapy, radical operation and transurethral resection of prostate, etc.
[0126] Before starting treatment of prostate cancer, the numbers of monocytes, basophils, eosinophils and neutrophils, and the abundance ratio of different markers in different lymphocytes as well as PSA values were measured. The measurement was carried out by SRL, Inc. by request.
[0127] A multiple regression analysis was performed using PSA value as objective variable, and 17 types: CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Th1 lymphocyte, Th2 lymphocyte, Ti lymphocyte, Tc lymphocyte, Ts lymphocyte, Act.i/h T lymphocyte, Act.s/c T lymphocyte, NK cell, N3+ cell, monocyte, basophil, eosinophil and neutrophil as explanatory variables. Then, each expected value was subtracted from respective observed value to give a residual value, which was sorted in ascending order and divided into 3 groups containing 59 cases each, which were sorted into: GOOD group with low residual values; MODERATE group with middle residual values; and BAD group with high residual values.
[0128] GOOD group: Age between 54 and 89, median 73 years old. PSA value (ng/ml) between 0.006 and 218.1, median 11. Stages of advance by ABCD categorization: A1: 1 case, A2: 2 cases, B1: 12 cases, B2: 15 cases, C: 21 cases, D1: 4 cases, D2: 4 cases. Malignancy in Gleason score total: between 3 and 10, median 6. Followup between 84 and 5,260 days, median 885 days.
[0129] MODERATE group: Age between 53 and 90, median 72 years old. PSA value (ng/ml) between 0.1 and 426.92, median 11.54. Stages of advance: A2: 2 cases, B1: 11 cases, B2: 8 cases, C: 24 cases, D1: 5 cases, D2: 9 cases. Malignancy between 3 and 9, median 6. Followup between 118 and 2,305 days, median 745 days.
[0130] BAD group: Age between 51 and 89, median 71 years old. PSA value (ng/ml) between 0.983 and 6745, median 44.32. Stages of advance: A1: 4 cases, B1: 8 cases, B2: 4 cases, C: 16 cases, D1: 5 cases, D2: 23 cases. Malignancy between 3 and 10, median 7. Followup between 1 and 6308 days, median 901 days.
[0131] The survival rate curves of GOOD, MODERATE and BAD groups (59 cases each, total 177cases) are shown in Fig. 1. Sorting into groups by the residual values was correlated with the survival rate.
Example 2. Discriminant Analysis
[0132] A discriminant analysis was performed, in which three groups of GOOD, MODERATE and BAD groups were set as objective variables and 17 types of immunocompetent cells: CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Th1 lymphocyte, Th2 lymphocyte, Ti lymphocyte, Tc lymphocyte, Ts lymphocyte, Act.i/h Tlymphocyte, Act.s/c T lymphocyte, NK cell, N3+ cell, monocyte, basophil, eosinophil and neutrophil were set as explanatory variables, to give discriminant functions. The scatter plotting of discriminant scores plotted with first discriminant function values on Xaxis and second discriminant function values on Yaxis is shown in Fig. 2. The discrimination accuracy rate was 71.8 %.
Example 3. Categorization by Discriminant scores
[0133] Three hundred and fortyfour blood samples in total were collected from 66 subjects including 23 healthy individuals and 43 patients, and analyzed. Male/female ratio was 43:23, and the age was between 35 and 81 years old, median 65, mean ± S.D. 65.39 ± 7.70 years old. The number of measurements was between once and 47 times, the median was twice, and the mean ± S.D. was 5.2 ± 7.2 times. Healthy individuals are at the age between 44 and 73 years old, the median 60, the mean ± S.D. 59.21 ± 4.98 years old. The male/female ratio was 15/8, the number of measurements was between once and 12 times, the median was twice, and the mean ± S.D. was 3.13 ± 3.21 times. Cases for patients were as described in Table 2. A patient who had complication of gastric cancer and bladder cancer was recorded in duplicate.
[Table 2]
Identity of cancer  Number of cases  Notes  Female  Male 
Lung cancer 
5 
4 recurrence/metastasis cases being treated; 1 case indicating good postoperative course. 
2 
3 
Lingual cancer 
1 
Receiving followup. 

1 
Gastric cancer 
4 
1 case of complication of bladder cancer; only 1 case indicating good course. 
2 
2 
Liver cancer 
1 
Passed away due to disease progression. 

1 
Gallbladder cancer 
1 
Passed away due to disease progression. 

1 
Bile duct cancer 
1 
Receiving postoperative followup. 
1 

Pancreatic cancer 
1 
Treatment for liver metastasis; passed away due to disease progression. 

1 
Colon cancer 
3 
Only 1 case being treated for peritoneal metastasis. 

3 
Rectal cancer 
1 
Lymph node metastasis; radical cure after treatment with anticancer agent; postoperational peritoneal metastasis. 
1 

Breast cancer 
4 
Only 1 case indicating good postoperative course; others being treated with anticancer agent. 
4 

Ovary cancer 
3 
Treatment for intraperitoneal metastasis; only 1 case indicating good course. 
3 

Uterine body cancer 
1 
Being treated with anticancer agent. 
1 

Renal cancer 
2 
Molecular targeted therapy for metastasis; exacerbated. 

2 
Bladder cancer 
3 
Only 2 cases with pre/posttreatment observation; 1 case in treatment. 
1 
2 
Prostate cancer 
12 
11 cases of hormonal therapy for recurrence/relapse after treatment; only 1 receiving followup after treatment. 

12 
Testicular cancer 
1 
Indicating good postoperative course, no recurrence for 6 yrs. 

1 
Total 
44 

15 
29 
[0134] The numbers of monocytes, basophils, eosinophils and neutrophils, as well as the abundance ratios of different markers in different lymphocytes were measured by SRL, Inc. by request.
[0135] For each case, the discriminant scores were calculated by assigning cellcounts of immunocompetent cells into the discriminant functions obtained in Example 2, and each case was sorted into a group the center of which was the closest. 162 cases were sorted into GOOD group, 45 cases into MODERATE group and 137 cases into BAD group.
[0136] Fig. 3, 4 and 5 show scatter plots of discriminant scores of 162 cases sorted into GOOD group, 45 cases sorted into MODERATE group, and 137 cases sorted into BAD group, respectively.
Example 4. Regression Analysis and Immunodynamics Chart Generation
<GOOD/lower Xaxis group; n = 54/Tcell immunity>
[0137] 162 cases sorted in GOOD group are divided into three groups based on Xaxis value, and an analysis directed toward Tcell immunity was performed for cellcount data of 54 cases with low Xaxis values.
[0138] A multiple regression analysis was performed using Tc*DR lymphocyte as objective variable, and CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Th± lymphocyte, Th2 lymphocyte, Th+2 lymphocyte, Act.Th1 lymphocyte, Act.Th2 lymphocyte, Ti*DR lymphocyte, Ti± lymphocyte, Ti2 lymphocyte, Ti+2 lymphocyte, Tc lymphocyte, Tc+ lymphocyte, Ts*DR lymphocyte, Ts lymphocyte, Ts+ lymphocyte, NK cell, NKT cell, N3+ cell, monocyte, basophil, eosinophil and neutrophil as explanatory variables. The contribution ratio was 96.473 %. The standard partial regression coefficients of 25 types of immunocompetent cells obtained by multiple regression analysis are shown in Table 3.
[0139] The standard partial regression coefficients of the immunocompetent cells obtained in this analysis were sorted in descending order according to their absolute values. Namely, in descending order from the highest absolute value: CD4positive lymphocyte, Th± lymphocyte, Ti+2 lymphocyte, Ts*DR lymphocyte, Th2 lymphocyte, CD3positive lymphocyte, Ti2 lymphocyte, Th+2 lymphocyte, Tc+ lymphocyte, N3+ cell, basophil, Ti± lymphocyte, monocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Tc lymphocyte, NKT cell, NK cell, Act.Th2 lymphocyte, Act.Th1 lymphocyte, eosinophil, Ts lymphocyte, Ts+ lymphocyte, neutrophil and Ti*DR lymphocyte.
[Table 3]
Immunocompetent Cell  Standard Partial Regression Coefficient 
CD4positive lymphocyte 
2.9130 
Th± lymphocyte 
0.8605 
Ti+2 lymphocyte 
0.7539 
Ts*DR lymphocyte 
0.6438 
Th2 lymphocyte 
0.6067 
CD3positive lymphocyte 
0.5657 
Ti2 lymphocyte 
0.3586 
Th+2 lymphocyte 
0.3487 
Tc+ lymphocyte 
0.3439 
N3+ cell 
0.3185 
Basophil 
0.2636 
Ti± lymphocyte 
0.2249 
Monocyte 
0.2238 
CD8positive lymphocyte 
0.2191 
CD20*DR lymphocyte 
0.2149 
Tc lymphocyte 
0.2020 
NKT cell 
0.1689 
NK cell 
0.1418 
Act.Th2 lymphocyte 
0.1185 
Act.Th1 lymphocyte 
0.1028 
Eosinophil 
0.0938 
Ts lymphocyte 
0.0850 
Ts+ lymphocyte 
0.0421 
Neutrophil 
0.0387 
Ti*DR lymphocyte 
0.0132 
[0140] Next, a regression analysis was performed using Tc*DR lymphocyte as objective variable, and CD4positive lymphocyte that was ranked as the first place for the magnitude of the absolute value of the standard partial regression coefficient as explanatory variable, and the contribution ratio was calculated. The contribution ratio thus obtained was 63.411 %, which was considered as the influence degree of the CD4positive lymphocyte.
[0141] Then, a multiple regression analysis was performed using Tc*DR lymphocyte as objective variable, and two types of immunocompetent cells: CD4positive lymphocyte that was ranked as the first place for the magnitude of the absolute value of the standard partial regression coefficient and Th± lymphocyte that was ranked as the second place as explanatory variables, and the contribution ratio was calculated as 65.381 %. Subtracting from this value the contribution ratio 63.411 % in the case when only CD4positive lymphocyte was used as explanatory variable gave a value 1.970 %, which was considered as the influence degree of Th± lymphocyte.
[0142] Next, a multiple regression analysis was performed using three types of immunocompetent cells that were ranked as the first to the third places, i.e., CD4positive lymphocyte, Th± lymphocyte and Ti+2 lymphocyte as explanatory variable, and the contribution ratio was calculated to be 70.130 %. Subtracting from this value the contribution ratio 65.381 % in the case when two types of immunocompetent cells: CD4positive lymphocyte and Th± lymphocyte were used as explanatory variables gave a value 4.749 %, which was considered as the influence degree of Ti+2 lymphocyte.
[0143] A series of multiple regression analyses was performed with increasing number of explanatory variables, starting from CD4positive lymphocyte that was ranked as the first place to the last (25th) immunocompetent celltype. The calculated influence degrees of 25 types of immunocompetent cells are shown in Table 4.
[Table 4]
Immunocompetent Cell  Influence Degree (%) 
CD4positive lymphocyte 
63.411 
Th± lymphocyte 
1.970 
Ti+2 lymphocyte 
4.749 
Ts*DR lymphocyte 
13.385 
Th2 lymphocyte 
0.290 
CD3positive lymphocyte 
2.158 
Ti2 lymphocyte 
2.101 
Th+2 lymphocyte 
0.962 
Tc+ lymphocyte 
2.777 
N3+ cell 
1.420 
Basophil 
0.010 
Ti± lymphocyte 
0.480 
Monocyte 
0.117 
CD8positive lymphocyte 
0.167 
CD20*DR lymphocyte 
0.670 
Tc lymphocyte 
0.893 
NKT cell 
0.170 
NK cell 
0.429 
Act.Th2 lymphocyte 
0.038 
Act.Th1 lymphocyte 
0.041 
Eosinophil 
0.138 
Ts lymphocyte 
0.065 
Ts+ lymphocyte 
0.015 
Neutrophil 
0.017 
Ti*DR lymphocyte 
0.000 
[0144] The percentage of the influence degree was reflected to the area of a circle to generate an immunodynamics chart. × denotes an influence degree below 0.00785 %, whereas ● denotes the influence degree at or above 0.00785 % and less than 0.0314 %. The generated immunodynamics chart is shown in Fig. 6.
[0145] Antitumor effect is generally exerted as follows. An antigen is loaded by an antigenpresenting cell (such as monocytes/macrophages) onto an MHC (Major Histocompatibility Complex) Class II molecule, presented to MHC Class II molecules on Act.Th1 lymphocyte. Act.Th1 lymphocyte produces cytokines, etc. based on information of the antigen, promoting activation of activated cytotoxic T cells (Tc*DR), and at the same time presents the cancer (tumor) specific antigen on MHC Class I, thereby exerting antitumor effect. Therefore, in order to obtain a persistent antitumor effect, it is necessary that Monocyte > Act.Th1 > Tc*DR pathway (Tcellular antigen recognition mechanism (TARM)) has been established.
[0146] In the immunodynamics chart, Act.Th1 is in negative relationship to Tc*DR and the pathway from Act.Th1 to Tc*DR has been disrupted. Thus, no antitumor effect can be expected.
[0147] Activation of TARM can be achieved by therapies for stimulating the pathway from monocyte to Act.Th1 by dendritic cell vaccine therapy, or alternatively by administrating interleukin1β (IL1β) inhibitor (e.g., canakinumab, etc.) or functional foods or supplements such as fungal glycoproteins. In addition, therapies of enhancing Tc*DR by activating Act.Th1 using interferon α, etc. can also be employed.
[0148] Moreover, since Ts*DR is at a level as high as 13.385 %, the suppressing of Ts*DR using monoclonal antibodies, etc. would relieve Tc*DR from the suppression by Ts*DR and successfully activate/enhance CD3 > CD8 > Tc > Tc+ > Tc*DR pathway, which might in turn open the TARM pathway.
[0149] Moreover, since differentiation and proliferation are stagnated at CD4, stimulating it with interleukin2 (IL2), a Tcell growth factor, would activate helper system, which might in turn open the TARM pathway.
[0150] Moreover, since Active Regulatory Thelper Cells (Th+2) which serve in immunosuppression is at a slightly high level as 0.962 %, suppressing them with an antibody medicine such as ipilimumab (Trade name: Yervoy®) and mogamulizumab would relieve the antigenpresenting cells of the monocyte/macrophage system, which might bring TARM to function.
[0151] CD20*DR lymphocyte goes through Monocyte > Act.Th2 > CD20*DR pathway (Bcellular Antigen Recognition Mechanism (BARM)) to produce antigens in cancer (tumor) antigendependent manner, whereas NK cell and NKT cell exert an antibodydependent cellular cytotoxicity (ADCC).
[0152] In the immunodynamics chart, the BARM pathway has been opened, and thus antibodydependent cellular cytotoxicity might be weak but still be expected.
<GOOD/middle Xaxis group; n = 54/Tcell immunity>
[0153] 162 cases sorted into GOOD group are divided into three groups based on Xaxis value, and an analysis directed toward Tcell immunity was performed for cellcount data of 54 cases that had middle Xaxis values in a similar way as in GOOD/lower Xaxis group/Tcell immunity. The influence degrees were calculated and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 7.
[0154] In the immunodynamics chart, both TARM and BARM have been established. Note that, unlike Fig. 6, differentiation and proliferation are stagnated at CD8, and the pathway from CD8 to Tc*DR has been disrupted. Possible treatments include activation/enhancement of CD3 > CD8 > Tc > Tc+ > Tc*DR pathway by IL2 administration, etc. Moreover, monocyte/macrophage system is at an extremely low level as 0.007 %, which also needs to be activated. Th+2 is at a low level and it therefore does not need to be suppressed. Ts*DR is at a level as high as 13.324, and therefore, in case if there is no improvement from the aforementioned treatment, a therapy for suppressing Ts*DR with antibody medicine may be employed.
<GOOD/higher Xaxis group; n = 54/NK cell>
[0155] 162 cases sorted in GOOD group are divided into three groups based on Xaxis value, and an analysis directed toward NK cell was performed for cellcount data of 54 cases that had high Xaxis values.
[0156] A multiple regression analysis was performed using NK cell as objective variable, and CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Th± lymphocyte, Th2 lymphocyte, Th+2 lymphocyte, Act.Th1 lymphocyte, Act.Th2 lymphocyte, Ti*DR lymphocyte, Ti± lymphocyte, Ti2 lymphocyte, Ti+2 lymphocyte, Tclymphocyte, Tc+ lymphocyte, Tc*DR lymphocyte, Ts*DR lymphocyte, Ts lymphocyte, Ts+ lymphocyte, NKT cell, N3+ cell, monocyte, basophil, eosinophil and neutrophil as explanatory variables. The standard partial regression coefficients of the immunocompetent cells obtained in this analysis were ranked in descending order according to their absolute values, and a multiple regression analysis using NK cell as objective variable was repeatedly performed in similar way as in GOOD/lower Xaxis group/Tcell immunity, the influence degrees were calculated for all immunocompetent cells and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 8.
[0157] Differentiation and proliferation are stagnated at CD8, and the pathway has been disrupted. Although TARM has been established, Tc*DR is at a level as low as 0.172 %, and it is necessary to activate/enhance the pathway from CD8 to Tc*DR with IL2 administration, etc. Namely, it can be said that this immune condition is just appropriate for applying NKcell adoptive immunotherapy. Because BARM has also been established and ADCC is expected, antitumor immunity can be maintained and continued by activating TARM and BARM and enhancing NK cells.
<GOOD/middle Xaxis group; n = 26/NK cell>
[0158] An analysis directed to NK cell was performed for cellcount data of 26 cases out of 54 cases sorted in GOOD/middle Xaxis group in a similar way as in <GOOD/higher Xaxis group; n = 54/NK cell>. The influence degrees were calculated and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 9.
[0159] Monocyte > Act.Th1 > Tc*DR <=> NK pathway has been established and monocyte is at 10.272 %, Act.Th1 is at 3.248 %, though Tc*DR is at extremely low value as 0.003 %. Moreover, since Th2 pathway has been disrupted and Th+2 is at a level as high as 16.921 %, antitumor effect cannot be expected. Therapies of activating Th2 pathway with IL2 administration, etc. and of suppressing Th+2 are required.
[0160] Besides, since Act.Th2 is at extremely low value as 0.000 %, ADCC cannot be expected. NKcell adoptive immunotherapy is considered to be promising.
<GOOD group; n = 26/Tcell immunity>
[0161] For cellcount data of 26 cases out of 162 cases sorted in GOOD group, an analysis directed toward Tcell immunity was performed in a similar way as in <GOOD/lower Xaxis group; n = 54/Tcell immunity>. The influence degrees were calculated and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 10.
[0162] TARM has been established though being weak. However, the differentiation/activation/enhancement pathway has been disrupted at CD3, and in this case, no effect can be expected by carrying out dendritic cell vaccine therapy.
[0163] The influence degree of CD3 is 94.528 %, and thus the influence degree of pathways for other immunocompetent cells including granulocyte system are 5.471 %, which is only approx. 5 %. Treatment may include dissolving of the stagnation at CD3 by IL2 administration, etc.
[0164] BARM has also been established, though CD20*DR is at a level as low as 0.010 %, and neither antigen production effect nor ADCC can be expected.
[0165] In the immunodynamics chart, Act.Th2 (0.056%) > Act.Th1 (0.020%) and Bcell immunity are dominant. Therefore, it should be noted that a sudden start of therapy with an immune checkpoint inhibitor would cause autoimmune diseaselike side effects.
<GOOD group; n = 26/Bcell immunity>
[0166] For cellcount data of 26 cases out of 162 cases sorted in GOOD group, an analysis directed to Bcell immunity was performed.
[0167] A multiple regression analysis was performed using CD20*DR lymphocyte as objective variable, and CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, Th± lymphocyte, Th2 lymphocyte, Th+2 lymphocyte, Act.Th1 lymphocyte, Act.Th2 lymphocyte, Ti*DR lymphocyte, Ti± lymphocyte, Ti2 lymphocyte, Ti+2 lymphocyte, Tclymphocyte, Tc+ lymphocyte, Tc*DR lymphocyte, Ts*DR lymphocyte, Ts lymphocyte, Ts+ lymphocyte, NK cell, NKT cell, N3+ cell, monocyte, basophil, eosinophil and neutrophil as explanatory variables. The standard partial regression coefficients of the immunocompetent cells obtained in this analysis were ranked in descending order according to their absolute values, and a multiple regression analysis using CD20*DR lymphocyte as objective variable was repeatedly performed in similar way as in GOOD/lower Xaxis group/Tcell immunity, and the influence degrees were calculated for all immunocompetent cells and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 11.
[0168] Similar to Fig. 10, differentiation and proliferation are stagnated at CD3, and neither TARM nor BARM have been established. In order to drive the immunodynamics, it is necessary to dissolve the stagnation at CD3 by IL2 administration, etc.
<GOOD group; n = 26/basophil>
[0169] For cellcount data of 26 cases out of 162 cases sorted in GOOD group, an analysis directed to basophil was performed.
[0170] A multiple regression analysis was performed using basophil as objective variable, and CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Th± lymphocyte, Th2 lymphocyte, Th+2 lymphocyte, Act.Th1 lymphocyte, Act.Th2 lymphocyte, Ti*DR lymphocyte, Ti± lymphocyte, Ti2 lymphocyte, Ti+2 lymphocyte, Tclymphocyte, Tc+ lymphocyte, Tc*DR lymphocyte, Ts*DR lymphocyte, Ts lymphocyte, Ts+ lymphocyte, NK cell, NKT cell, N3+ cell, monocyte, eosinophil and neutrophil as explanatory variables. The standard partial regression coefficients of the immunocompetent cells obtained in this analysis were ranked in descending order according to their absolute values, and a multiple regression analysis using basophil as objective variable was repeatedly performed in similar way as in GOOD/lower Xaxis group/Tcell immunity, and the influence degrees were calculated for all immunocompetent cells and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 12.
[0171] An immunodynamics chart directed to basophil shows an immunity related to immediate allergy, etc. When Th1immunity (Monocyte > Act.Th1 > Tc*DR <=> Basophil) or Th2immunity (Monocyte > Act.Th2 > CD20*DR <=> Basophil) has been established and activated, it is a severe case with immediate allergic reaction that would be an indication of an adrenaline intramuscular injection.
[0172] Although TARM has been weakly established, Tc*DR is in a negative relationship to basophil and the pathway has been disrupted.
[0173] BARM has been established, and furthermore, in Monocyte > Act.Th2 > CD20*DR  > Basophil <=> Act.Th2 pathway, the loop circuit from Act.Th2 to Act.Th2 has been established. Therefore, it should be noted that carrying out a therapy such as an immune checkpoint inhibitor such as nivolumab (Trade name: Opdivo®) from the first would induce immediate allergic symptoms, etc., and require emergency treatment such as adrenaline intramuscular injection.
<GOOD group; n = 26/eosinophil>
[0174] For cellcount data of 26 cases out of 162 cases sorted in GOOD group, an analysis directed to eosinophil was performed.
[0175] A multiple regression analysis was performed using eosinophil as objective variable, and CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Th± lymphocyte, Th2 lymphocyte, Th+2 lymphocyte, Act.Th1 lymphocyte, Act.Th2 lymphocyte, Ti*DR lymphocyte, Ti± lymphocyte, Ti2 lymphocyte, Ti+2 lymphocyte, Tclymphocyte, Tc+ lymphocyte, Tc*DR lymphocyte, Ts*DR lymphocyte, Ts lymphocyte, Ts+ lymphocyte, NK cell, NKT cell, N3+ cell, monocyte, basophil and neutrophil as explanatory variables. The standard partial regression coefficients of the immunocompetent cells obtained in this analysis were ranked in descending order according to their absolute values, and a multiple regression analysis using eosinophil as objective variable was repeatedly performed in similar way as in GOOD/lower Xaxis group/Tcell immunity, and the influence degrees were calculated for all immunocompetent cells and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 13.
[0176] Although TARM has been established, Tc*DR is in a negative relationship to eosinophil and the pathway has been disrupted.
[0177] However, BARM has been established, and furthermore, in Monocyte > Act.Th2 > CD20*DR > Basophil <=> Act.Th2 pathway, the loop circuit from Act.Th2 to Act.Th2 has been established. Therefore, it should be noted that carrying out a therapy such as an immune checkpoint inhibitor such as nivolumab (Trade name: Opdivo®) from the first would induce immediate allergic symptoms, etc., and require emergency treatment such as adrenaline intramuscular injection.
<GOOD group; n = 26/neutrophil>
[0178] For cellcount data of 26 cases out of 162 cases sorted in GOOD group, an analysis directed to neutrophil was performed.
[0179] A multiple regression analysis was performed using neutrophil as objective variable, and CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Th± lymphocyte, Th2 lymphocyte, Th+2 lymphocyte, Act.Th1 lymphocyte, Act.Th2 lymphocyte, Ti*DR lymphocyte, Ti± lymphocyte, Ti2 lymphocyte, Ti+2 lymphocyte, Tclymphocyte, Tc+ lymphocyte, Tc*DR lymphocyte, Ts*DR lymphocyte, Ts lymphocyte, Ts+ lymphocyte, NK cell, NKT cell, N3+ cell, monocyte, basophil and eosinophil as explanatory variables. The standard partial regression coefficients of the immunocompetent cells obtained in this analysis were ranked in descending order according to their absolute values, and a multiple regression analysis using neutrophil as objective variable was repeatedly performed in similar way as in GOOD/lower Xaxis group/Tcell immunity, and the influence degrees were calculated for all immunocompetent cells and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 14.
[0180] Immunodynamics has been stagnated at CD3 almost completely. The influence degree of CD3 is 97.302 %, and thus the influence degree for other pathway is less than 2.7 %. Tcell immunity (anticancer immunity) cannot be expected. TARM has barely been maintained, though Tc*DR is 0.000 % and has been completely obstructed. Moreover, Tc*DR is in negative relationship to neutrophil, and in this case, the antitumor effect by neutrophils such as with fractalkines can hardly be expected.
[0181] Furthermore, in a similar was as in Fig. 12 and 13, in Monocyte > Act.Th2 > CD20*DR > Neutrophil <=> Act.Th2 pathway, the loop circuit from Act.Th2 to Act.Th2 has been established. Therefore, a caution should be taken for inflammatory diseases.
<MODERATE group; n = 45/NK cell>
[0182] For cellcount data of 45 cases sorted in MODERATE group, an analysis directed to NK cell was performed in a similar way as in <GOOD/higher Xaxis group/NK cell>. The influence degrees were calculated and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 15.
[0183] Since there is stagnation at CD8, differentiating/multiplying/enhancing CD8 > Tc > Tc+ > Tc*DR pathway by IL2 administration, etc. will also actively enhances Monocyte > Act.Th1 > Tc*DR <=> NK pathway, exerting an anticancer effect. ADCC activity can also be sufficiently expected, and in a case of such immunodynamics chart, an adoptive immunotherapy should be performed. If possible, continuous administration of IL2 by subcutaneous injection or intravenous infusion for a few days after NKcell autotransfusion will lead to an enhancement of the effect.
<MODERATE group; n = 35/Tcell immunity>
[0184] For cellcount data of 35 cases out of 45 cases sorted in MODERATE group, an analysis directed toward Tcell immunity was performed in a similar way as in <GOOD/lower Xaxis group; n = 54/Tcell immunity>. The influence degrees were calculated and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 16.
[0185] There is stagnation at Ts*DR, indicating considerable suppression by Ts*DR. Although Tcell immunity has been established, Ts*DR is considerably dominant and required to be controlled somehow. Therapies include a method of suppressing Ts*DR with monoclonal antibodies. NKcell adoptive immunotherapy or NKTcell adoptive immunotherapy may also be promising in some cases.
<BAD group; n = 40/Tcell immunity>
[0186] For cellcount data of 40 cases out of 137 cases sorted in BAD group, an analysis directed toward Tcell immunity was performed in a similar way as in <GOOD/lower Xaxis group; n = 54/Tcell immunity>. The influence degrees were calculated and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 17.
[0187] There is stagnation at CD3 and Tc+. Bringing this pathway to flow smoothly will induce anticancer (antitumor) immunity for a great deal, contributing to the reduction of cancer. To this end, allowing them to differentiate, proliferate, or be increased by IL2 administration, etc. is considered to be effective. Furthermore, immune checkpoint inhibitors such as nivolumab (Trade name: Opdivo®) are considered to be effective.
[0188] Besides, since Monocyte > Act.Th2 > CD20*DR <=> Tc*DR pathway has been established, ADCC activity may also be expected. Note that when using an immune checkpoint inhibitor, it is necessary to keep monitoring and observing the dynamics such that Bcell immunity will not become dominant.
<BAD group; n = 26/eosinophil>
[0189] For cellcount data of 26 cases out of 137 cases sorted in BAD group, an analysis directed to eosinophil was performed in a similar way as in <GOOD group; n = 26/eosinophil>. The influence degrees were calculated and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 18.
[0190] There is stagnation at CD4, and neither TARM nor BARM have been established. Moreover, no loop circuit from Act.Th2 to Act.Th2 has been established in Monocyte > Act.Th2 > CD20*DR > Eosinophil <=> Act.Th2 pathway.
<BAD group; n = 26/basophil>
[0191] For cellcount data of 26 cases out of 137 cases sorted in BAD group, an analysis directed to basophil was performed in a similar way as in <GOOD group; n = 26/basophil>. The influence degrees were calculated and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 19.
[0192] There is stagnation at CD4, and neither TARM nor BARM have been established. Moreover, since a loop circuit from Act.Th2 to Act.Th2 has been established in Monocyte > Act.Th2 > CD20*DR > Basophil <=> Act.Th2 pathway, a care has to be taken in carrying out immunotherapy.
<BAD group; n = 26/neutrophil>
[0193] For cellcount data of 26 cases out of 137 cases sorted in BAD group, an analysis directed to neutrophil was performed in a similar way as in <GOOD group; n = 26/neutrophil>. The influence degrees were calculated and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 20.
[0194] There is stagnation at CD4, and neither TARM nor BARM have been established. Moreover, since a loop circuit from Act.Th2 to Act.Th2 has been established in Monocyte > Act.Th2 > CD20*DR > Neutrophil <=> Act.Th2 pathway, a care has to be taken in carrying out immunotherapy.
<BAD group; n = 26/Bcell immunity>
[0195] For cellcount data of 26 cases out of 137 cases sorted in BAD group, an analysis directed to Bcell immunity was performed in a similar way as in <GOOD group; n = 26/Bcell immunity>. The influence degrees were calculated and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 21.
[0196] There is stagnation at CD4, and neither TARM nor BARM have been established.
<BAD group; n = 26/NKT cell>
[0197] For cellcount data of 26 cases out of 137 cases sorted in BAD group, an analysis directed to NKT cell was performed.
[0198] A multiple regression analysis was performed using NKT cell as objective variable, and CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Th± lymphocyte, Th2 lymphocyte, Th+2 lymphocyte, Act.Th1 lymphocyte, Act.Th2 lymphocyte, Ti*DR lymphocyte, Ti± lymphocyte, Ti2 lymphocyte, Ti+2 lymphocyte, Tclymphocyte, Tc+ lymphocyte, Tc*DR lymphocyte, Ts*DR lymphocyte, Ts lymphocyte, Ts+ lymphocyte, NK cell, N3+ cell, monocyte, basophil, eosinophil and neutrophil as explanatory variables. The standard partial regression coefficients of the immunocompetent cells obtained in this analysis were ranked in descending order according to their absolute values, a multiple regression analysis using NKT cell as objective variable was repeatedly performed in similar way as in GOOD/lower Xaxis group/Tcell immunity, and the influence degrees were calculated for all immunocompetent cells and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 22.
[0199] There is stagnation and disruption at CD4. Therapies require differentiation, proliferation or enhancement of CD4 system by IL2 administration, etc. Then, therapies should be reconsidered according to the changes in immunodynamics.
<BAD group; n = 26/NK cell>
[0200] For cellcount data of 26 cases out of 137 cases sorted in BAD group, an analysis directed to NKcell immunity was performed in a similar way as in <GOOD/higher Xaxis group; n = 54/NK cell>. The influence degrees were calculated and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 23.
[0201] Neither TARM nor BARM have been established. There is stagnation and disruption at CD4, which should be dissolved by IL2 administration, etc. Although there is a possibility that TARM and BARM will be recovered by NKcell adoptive immunotherapy, this is not an appropriate therapy in the light of the immunodynamics chart.
<BAD group; n = 26/Tcell immunity>
[0202] For cellcount data of 26 cases out of 137 cases sorted in BAD group, an analysis directed toward Tcell immunity was performed in a similar way as in <GOOD/lower Xaxis group; n = 54/Tcell immunity>. The influence degrees were calculated and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 24.
[0203] Neither TARM nor BARM have been established. Since there are stagnations at CD4 and CD3, it is at first necessary to activate the pathways by IL2 administration, etc. to recover these stopped pathways. Then, therapies should be reconsidered according to the changes in immunodynamics.
<BAD/higher Xaxis group; n = 46/Tcell immunity>
[0204] One hundred thirtyseven cases that have been sorted into BAD group are divided into three groups based on Xaxis value, and for the cellcount data of 46 cases with high Xaxis values, an analysis directed toward Tcell immunity was performed in a similar way as in <GOOD/lower Xaxis group; n = 54/Tcell immunity>. The influence degrees were calculated and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 25.
[0205] Both TARM and BARM have been established. However, since CD3 > CD8 > Tc  > Tc+ pathway has been stagnated and stopped, the stagnation at CD3 and Tc+ should be improved by IL2 administration, etc. in order for the pathway to smoothly go around. A therapy by administering an immune checkpoint inhibitor can also be expected to be effective. There is no need to be concerned about any autoimmune diseaselike side effect since Act.Th1 is dominant (Act.Th1 (0.599%) > Act.Th2 (0.000%)). Note that although BARM has been established, CD20*DR is in negative relationship to Tc*DR and therefore ADCC activity may not be expected.
<BAD/middle Xaxis group; n = 46/NKT cell>
[0206] One hundred and thirtyseven cases that have been sorted into BAD group are divided into three groups based on Xaxis value, and for the cellcount data of 46 cases with middle Xaxis values, an analysis directed to NKT cell was performed in a similar way as in <BAD group; n = 26/NKT cell>. The influence degrees were calculated and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 26.
[0207] Although TARM has been established, there is significant stagnation at CD8. First of all, this stagnation should be improved by IL2 administration, etc. Tc*DR is in positive relationship to NKT and is maintained. Since BARM has not been established, no ADCC activity can be expected.
[0208] Moreover, although Act.Th2 is dominant (Act.Th1 (0.001%) <Act.Th2 (0.020%)), Act.Th2 > CD20*DR > NKT pathway has been disrupted and thus there is no need to be concerned about an autoimmunitylike side effect. Still, a discreet administration is desired when carrying out immunotherapy using an immune checkpoint inhibitor, etc. Monitoring by an immunodynamics chart monitor will be necessary.
<BAD/middle Xaxis group; n = 26/Tcell immunity>
[0209] Among 137 cases that have been sorted into BAD group, for 26 cases of cellcount data with middle Xaxis values, an analysis directed toward Tcell immunity was performed in a similar way as in <GOOD/lower Xaxis group; n = 54/Tcell immunity>. The influence degrees were calculated and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 27.
[0210] TARM has barely been established, but there is significant stagnation at CD4. It is presumed that by relieving this stagnation by IL2 administration, etc., the helper system will differentiate, proliferate or be enhanced, and the immunity will smoothly be turn around.
[0211] Moreover, although Act.Th2 is dominant (Act.Th1 (0.001%) <Act.Th2 (0.015%)), Act.Th2 > CD20*DR pathway has been disrupted and thus there is no need to be concerned about an autoimmunitylike side effect. However, in order to carry out immunotherapy with discreetness, monitoring by an immunodynamics chart monitor will be necessary.
<BAD/lower Xaxis group; n = 45/Bcell immunity>
[0212] One hundred and thirtyseven cases that have been sorted into BAD group are divided into three groups based on Xaxis value, and for the cellcount data of 45 cases with low the Xaxis values, an analysis directed to Bcell immunity was performed in a similar way as in <GOOD group; n = 26/Bcell immunity>. The influence degrees were calculated and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 28.
[0213] BARM has been established, whereas TARM has not. Moreover, there is significant stagnation at CD4 and the pathway has been disrupted againstAct.Th1 (0.005 %). It is presumed that promoting of differentiation and proliferation by IL2 administration, etc. will increase the influence degree of Act.Th1, which brings TARM to be established. Because Th2 is dominant (Act.Th1 (0.005 %) <Act.Th2 (0.017 %)), it is necessary to be careful when using an immune checkpoint inhibitor.
<BAD/lower Xaxis group; n = 45/Tcell immunity>
[0214] One hundred and thirtyseven cases that have been sorted into BAD group are divided into three groups based on Xaxis value, and for the cellcount data of 45 cases with low Xaxis values, an analysis directed toward Tcell immunity was performed in a similar way as in <GOOD/lower Xaxis group; n = 54/Tcell immunity>. The influence degrees were calculated and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 29.
[0215] Neither TARM nor BARM have been established. Moreover, there is significant stagnation at CD4. It is essential to dissolve this stagnation by IL2 administration, etc. to promote differentiation and proliferation of helper T cells. Since Ts*DR is at a considerably high level (17.621%), immunotherapy for suppressing it is also promising.
<BAD/middle Yaxis group; n = 46/Tcell immunity>
[0216] One hundred and thirtyseven cases that have been sorted into BAD group were divided into three groups based on Yaxis value, and for the cellcount data of 46 cases with middle Yaxis value , an analysis directed toward Tcell immunity was performed in a similar way as in <GOOD/lower Xaxis group; n = 54/Tcell immunity>. The influence degrees were calculated and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 30.
[0217] Both TARM and BARM have been established. However, there is significant stagnation at CD8, it is essential to promote differentiation and proliferation by IL2 administration, etc. Regardless of the stagnation at CD8, the pathway to Tc*DR has not been disrupted and is still open, and therefore it is considered that the use of an immune checkpoint inhibitor from the beginning will be considerably effective. Moreover, since Act.Th1 is dominant (Act.Th1 (2.666%) > Act.Th2 (0.019%)), there would be no concern about an autoimmune diseaselike side effect.
<BAD/middle Yaxis group; n = 46/NKT cell>
[0218] One hundred and thirtyseven cases that have been sorted into BAD group were divided into three groups based on Yaxis value, and for the cellcount data of 46 cases with middle Yaxis value, an analysis directed to NKT cell was performed in a similar way as in <BAD group; n = 26/NKT cell>. The influence degrees were calculated and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 31.
[0219] Both TARM and BARM have been established, and NKT activity and ADCC activity have also been established. However, since there is stagnation and disruption at Ti*DR, immunity will be more effective by any therapy for dissolving the stagnation of Ti*DR and promoting differentiation and proliferation of helper lymphocytes. It is also possible that Ti *DR will be suppressed by NKcell adoptive immunotherapy.
[0220] This is suitable for an application of an immune checkpoint inhibitor or NKTcell adoptive immunotherapy. Since Th1 is dominant (Act.Th1 (7.431%) > Act.Th2 (0.065%)), there is presumably no concern about side effects.
<BAD/higher Yaxis group; n = 45/Bcell immunity>
[0221] One hundred and thirtyseven cases that have been sorted into BAD group were divided into three groups based on Yaxis value, the cellcount data of 45 cases with high Yaxis values, an analysis directed to Bcell immunity was performed in a similar way as in <GOOD group; n = 26/Bcell immunity>. The influence degrees were calculated and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 32.
[0222] There is significant stagnation at CD4. BARM has been established, whereas TARM has not. Because BARM has been established, it is necessary to be careful about a side effect which may be caused by using an immune checkpoint inhibitor.
<BAD/lower Yaxis group; n = 45/Bcell immunity>
[0223] One hundred and thirtyseven cases that have been sorted into BAD group were divided into three groups based on Yaxis value, and for the cellcount data of 45 cases with low Yaxis values, an analysis directed to Bcell immunity was performed in a similar way as in <GOOD group; n = 26/Bcell immunity>. The influence degrees were calculated and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 33.
[0224] There is significant stagnation at CD4. BARM has been established, whereas TARM has not. ADCC (Monocyte > Act.Th2 > CD20*DR <=> Tc*DR) has also been established. Dissolving of this stagnation at CD4 and activating of ADCC will turn the anticancer (antitumor) immunity effective. Since Th1 is dominant (Act.Th1 (0.476%) > Act.Th2 (0.001%)), there is presumably no concern about side effects by immune checkpoint inhibitor.
<n=29/Tcell immunity>
[0225] A multiple regression analysis was performed for the cellcount data of 29 cases, using Tc*DR lymphocyte as objective variable, and Th17+ lymphocyte, CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Th± lymphocyte, Th2 lymphocyte, Th+2 lymphocyte, Act.Th1 lymphocyte, Act.Th2 lymphocyte, Ti*DR lymphocyte, Ti± lymphocyte, Ti2 lymphocyte, Ti+2 lymphocyte, Tc lymphocyte, Tc+ lymphocyte, Ts*DR lymphocyte, Ts lymphocyte, Ts+ lymphocyte, NK cell, NKT cell, N3+ cell, monocyte, basophil, eosinophil and neutrophil as explanatory variables. The standard partial regression coefficients of the immunocompetent cells obtained in this analysis were ranked in descending order according to their absolute values, a multiple regression analysis using Tc*DR lymphocyte as objective variable was repeatedly performed in similar way as in GOOD/lower Xaxis group/Tcell immunity, and the influence degrees were calculated for all immunocompetent cells and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 34.
[0226] TARM has been established, whereas BARM has not. There is significant stagnation at CD8, and in order to further activate the pathway from CD3 to Tc*DR and make this pathway open without any stagnation, therapy such as administering a cytokine such as IL2 can be considered. After observing any changes in immunodynamics by this therapy, mainly changes in the degree of activation of Tcell immunity, the next therapeutic means shall be considered.
[0227] Moreover, in the immunodynamics chart, Monocyte > Th17+ > Tc*DR pathway has been established, which is weak but still contributing to anticancer (antitumor) immunity. Note that the influence degree of neutrophil is as low as 0.013 % and it may have a low influence. Here, if interleukin17A/F (IL17A/F), etc. is produced by being stimulated by various treatment such as fractalkines (either administered or produced in the body), and a pathway of:
is further activated, an enhanced differentiation, activation or proliferation towards Tc*DR, i.e., an enhancement of anticancer (antitumor) effect can be expected.
[0228] Besides, in the context of anticancer (antitumor) immunity, it is necessary that Tcell immunity has been established, though Bcell immunity is better if not established. This is because it may cause an allergic or autoimmunitylike side effect. Note that this shall not apply where antibody dependent cellular cytotoxicity (ADCC) is to be expected.
<n=27/Bcell immunity>
[0229] A multiple regression analysis was performed for the cellcount data of 27 cases using CD20*DR lymphocyte as objective variable, and Th17+ lymphocyte, CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, Th± lymphocyte, Th2 lymphocyte, Th+2 lymphocyte, Act.Th1 lymphocyte, Act.Th2 lymphocyte, Ti*DR lymphocyte, Ti± lymphocyte, Ti2 lymphocyte, Ti+2 lymphocyte, Tc lymphocyte, Tc+ lymphocyte, Tc*DR lymphocyte, Ts*DR lymphocyte, Ts lymphocyte, Ts+ lymphocyte, NK cell, NKT cell, N3+ cell, monocyte, basophil, eosinophil and neutrophil as explanatory variables. The standard partial regression coefficients of the immunocompetent cells obtained in this analysis were ranked in descending order according to their absolute values, a multiple regression analysis using CD20*DR lymphocyte as objective variable was repeatedly performed in similar way as in GOOD/lower Xaxis group/Tcell immunity, and the influence degrees were calculated for all immunocompetent cells and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 35.
[0230] There is stagnation of differentiation and proliferation at CD4. Although the pathways of helper T cells are open, they are hindered and tapering off. TARM has barely been established.
[0231] In order to improve this to be further effective, it is necessary to either activate Tcellular antigen recognition mechanism by interferon α, dendritic cell vaccine therapy, or fungal supplements for enhancing Tc*DR, or to carry out immunotherapy for activating and enhancing CD3 > CD8 > Tc > Tc+ > Tc*DR pathway by IL2 or immune checkpoint inhibitors, etc.
[0232] Since BARM pathway has been established, there is concern for development of an allergic or autoimmunitylike side effect. However, Tcell immunity is dominant (Act.Th1 (1.132%) > Act.Th2 (0.193%)), the possibility of developing a side effect is presumably low. Nevertheless, because the dominance may be altered to Act.Th1 < Act.Th2 by some treatment, treatment should be carried out under strict observation while generating and monitoring immunodynamics charts as appropriate.
[0233] In addition, although Monocyte > Th17+ > CD20*DR pathway has also been established, Th17+lymphocyte is 0.000 % and extremely low, and is in a negative relationship with Th17+, the possibility of developing an allergic or autoimmunitylike side effect is low, with very little concern for such side effects. However, depending on the treatment, Neutrophil > Th17+ > CD20*DR pathway may be activated and the side effects such as fever or allergic skin disorders may be developed. Therefore, a similar strict observation is essential.
[0234] Furthermore, since basophil is at relatively high level as 1.662 %, which may cause an unexpected immediate allergic response. Therefore, immunotherapy should be carried out with discretion under a sufficient observation. If the influence degree of basophil towards Act.Th2 is further increased by a treatment, the treatment must be stopped.
<n=27/NKT cells>
[0235] A multiple regression analysis was performed for the cellcount data of 27 cases using NKT cell as objective variable, and Th17+ lymphocyte, CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Th± lymphocyte, Th2 lymphocyte, Th+2 lymphocyte, Act.Th1 lymphocyte, Act.Th2 lymphocyte, Ti*DR lymphocyte, Ti± lymphocyte, Ti2 lymphocyte, Ti+2 lymphocyte, Tc lymphocyte, Tc+ lymphocyte, Tc*DR lymphocyte, Ts*DR lymphocyte, Ts lymphocyte, Ts+ lymphocyte, NK cell, N3+ cell, monocyte, basophil, eosinophil and neutrophil as explanatory variables. The standard partial regression coefficients of the immunocompetent cells obtained in this analysis were ranked in descending order according to their absolute values, a multiple regression analysis using NKT cell as objective variable was repeatedly performed in similar way as in GOOD/lower Xaxis group/Tcell immunity, and the influence degrees were calculated for all immunocompetent cells and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 36.
[0236] There is stagnation of differentiation and proliferation at CD4positive lymphocyte, though TARM has been established.
[0237] In order to bring Tc*DR to differentiate, be activated or proliferate to enhance antitumor immunity, it is necessary to enhance the pathway towards Tc*DR by, e.g., administering αgalactosylceramide (αGalCer) or NKT cellactivating adoptive immunotherapy.
[0238] The influence degree of each immunocompetent cell in Monocyte > Th17+ > Tc*DR pathway is extremely low, and the influence degree of neutrophil is also 0.000 % and a negative factor, and it is therefore not an enhancing factor. Here, if IL17 A/F, etc. is produced by stimulation by various treatment such as fractalkines (either administered or produced in the body) and a pathway of Neutrophil > Th17+ > Tc*DR pathway is established, the differentiation, activation and proliferation towards Tc*DR will be enhanced, and an antitumor effect can be expected.
[0239] The influence degree of basophil is slightly high as 0.987 %, and there is a concern about development of immediate allergy which needs to be noted. If the influence degree of basophil is increased by a treatment, it is necessary to consider stopping or discontinuing the treatment.
<n=28/NK cell>
[0240] A multiple regression analysis was performed for the cellcount data of 28 cases using NK cell as objective variable, and Th17+ lymphocyte, CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Th± lymphocyte, Th2 lymphocyte, Th+2 lymphocyte, Act.Th1 lymphocyte, Act.Th2 lymphocyte, Ti*DR lymphocyte, Ti± lymphocyte, Ti2 lymphocyte, Ti+2 lymphocyte, Tc lymphocyte, Tc+ lymphocyte, Tc*DR lymphocyte, Ts*DR lymphocyte, Ts lymphocyte, Ts+ lymphocyte, NKT cell, N3+ cell, monocyte, basophil, eosinophil and neutrophil as explanatory variables. The standard partial regression coefficients of the immunocompetent cells obtained in this analysis were ranked in descending order according to their absolute values, a multiple regression analysis using NK cell as objective variable was repeatedly performed in similar way as in GOOD/lower Xaxis group/Tcell immunity, and the influence degrees were calculated for all immunocompetent cells and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 37.
[0241] There is stagnation of differentiation and proliferation at CD4positive lymphocyte. The influence degree of Tc*DR is extremely low at 0.003 %, and no anticancer (antitumor) immunity can be expected. Although TARM has weakly been established, Tc*DR lymphocyte is in negative relationship to NK and socalled NK cell activity (Monocyte > Act.Th1 > Tc*DR <=> NK) has not been established. Bcellular antigen recognition mechanism (Monocyte > Act.Th2 > CD20*DR <=> NK) has been disrupted between Act.Th2 and CD20*DR, and CD20*DR is also in negative relationship to NK, antibody dependent cellular cytotoxicity (ADCC: Monocyte > Act.Th2 > CD20*DR <=> NK) has not be established.
[0242] In order to activate and enhance anticancer (antitumor) immunity, it is necessary to recover these stopped/disrupted pathways by, e.g., IL2 administration or activated NKcell adoptive immunotherapy. Here, it is important to confirm the increase in Tc*DR influence degree derived from the treatment by generating and monitoring immunodynamics chart.
[0243] With respect to the influence degrees of Act.Th1 and Act.Th2, Act.Th2 is dominant (Act.Th1 (0.121%) <Act.Th2 (2.142%)), and thus it is important to take strict care not to facilitate Bcell antigenproducing immunity and thereby cause an onset of an allergic or autoimmunitylike disease. To this end, it becomes essential to monitor immunodynamics.
[0244] Besides, the pathway:
has weakly been established, a care needs to be taken for development of psoriasiform dermatitis and other skin allergylike diseases.
[0245] In various immunotherapies, in particular, a single or combined therapy with an immune checkpoint inhibitor, a care should be taken when eosinophil, basophil, etc. are in positive relationship to NK and the influence degree is increased. In particular, when basophil is in positive relationship and the influence degree is increased, a special care is required because there is a concern for development of immediate allergic reaction.
<n=28/basophil>
[0246] A multiple regression analysis was performed for the cellcount data of 28 cases using basophil as objective variable, and Th17+ lymphocyte, CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Th± lymphocyte, Th2 lymphocyte, Th+2 lymphocyte, Act.Th1 lymphocyte, Act.Th2 lymphocyte, Ti*DR lymphocyte, Ti± lymphocyte, Ti2 lymphocyte, Ti+2 lymphocyte, Tc lymphocyte, Tc+ lymphocyte, Tc*DR lymphocyte, Ts*DR lymphocyte, Ts lymphocyte, Ts+ lymphocyte, NK cell, NKT cell, N3+ cell, monocyte, eosinophil and neutrophil as explanatory variables. The standard partial regression coefficients of the immunocompetent cells obtained in this analysis were ranked in descending order according to their absolute values, a multiple regression analysis using basophil as objective variable was repeatedly performed in a similar way as in <GOOD group; n = 26/basophil>, and the influence degrees were calculated for all immunocompetent cells and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 38.
[0247] As mentioned with respect to Fig. 12, in an immunodynamics chart directed to basophil, when Th1immunity (Monocyte > Act.Th1 > Tc*DR <=> Basophil) or Th2immunity (Monocyte > Act.Th2 > CD20*DR <=> Basophil) is established, it is a severe case with immediate allergic reaction that would be an indication of an adrenaline intramuscular injection. A immunodynamics chart directed to basophil is an essential immunodynamics chart, because any severe allergic side effect can be predicted by generating it.
[0248] In Fig. 38, Th1immunity (Monocyte > Act.Th1 > Tc*DR <=> Basophil), Th2immunity (Monocyte > Act.Th2 > CD20*DR <=> Basophil), and Monocyte > 17+ > Basophil pathway are disrupted, and thus there would be very little risk of an allergic reaction being developed.
<n=27/eosinophil>
[0249] A multiple regression analysis was performed for the cellcount data of 27 cases using eosinophil as objective variable, and Th17+ lymphocyte, CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Th± lymphocyte, Th2 lymphocyte, Th+2 lymphocyte, Act.Th1 lymphocyte, Act.Th2 lymphocyte, Ti*DR lymphocyte, Ti± lymphocyte, Ti2 lymphocyte, Ti+2 lymphocyte, Tc lymphocyte, Tc+ lymphocyte, Tc*DR lymphocyte, Ts*DR lymphocyte, Ts lymphocyte, Ts+ lymphocyte, NK cell, NKT cell, N3+ cell, monocyte, basophil and neutrophil as explanatory variables. The standard partial regression coefficients of the immunocompetent cells obtained in this analysis were ranked in descending order according to their absolute values, a multiple regression analysis using eosinophil as objective variable was repeatedly performed in a similar way as in <GOOD group; n = 26/eosinophil>, and the influence degrees were calculated for all immunocompetent cells and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 39.
[0250] There is stagnation of differentiation and proliferation at CD4. Specific immunity that consists of Th1antigen recognition mechanism (Monocyte > Act.Th1 > Tc*DR) to induce activated cytotoxic T cell (Tc*DR) has been established. Moreover, the influence degrees of Act.Th1 and Act.Th2 are Act.Th1 (8.212%) > Act.Th2 (0.041%), and Tcell immunity is dominant over Bcell immunity.
[0251] Activated regulatory T cell (Th+2), which is involved in immune suppression, is at relatively high level as 1.682 %, which suppresses monocyte to an extremely low level. When the use of Tc*DRinducing immunity is considered, it is also considered to use Th+2suppressing agent such as ipilimumab (Trade name: Yervoy®) while taking sufficient care about following points:
[0252] The pathway in which Th17+ is involved (Monocyte > Th17+ > Eosinophil) has also been established, though the influence degree of Th17+ is 0.000 %, and Th17+ is in negative relationship to Neutrophil. Thus, Th17+ is poorly involved.
[0253] It needs to be noted here that the influence degree of basophil is relatively high as 1.003 %. It needs to be noted that, because Monocyte > Act.Th2 > CD20*DR pathway is open, treatment might bring CD20*DR into positive relationship to eosinophil, which will increase the risk for the onset of severe allergy.
<n=27/neutrophil>
[0254] A multiple regression analysis was performed the cellcount data of 27 cases using neutrophil as objective variable Th17+ lymphocyte, CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Th± lymphocyte, Th2 lymphocyte, Th+2 lymphocyte, Act.Th1 lymphocyte, Act.Th2 lymphocyte, Ti*DR lymphocyte, Ti± lymphocyte, Ti2 lymphocyte, Ti+2 lymphocyte, Tc lymphocyte, Tc+ lymphocyte, Tc*DR lymphocyte, Ts*DR lymphocyte, Ts lymphocyte, Ts+ lymphocyte, NK cell, NKT cell, N3+ cell, monocyte, basophil and eosinophil as explanatory variables. The standard partial regression coefficients of the immunocompetent cells obtained in this analysis were ranked in descending order according to their absolute values, a multiple regression analysis using neutrophil as objective variable was repeatedly performed in a similar way as in <GOOD group; n = 26/neutrophil>, and the influence degrees were calculated for all immunocompetent cells and an immunodynamics chart was generated. The generated immunodynamics chart is shown in Fig. 40.
[0255] There is stagnation of differentiation and proliferation at CD4. Anticancer (antitumor) immunity (Monocyte > Act.Th1 > Tc*DR <=> Neutrophil) has weakly been established. In this immunodynamics chart, the influence degree of Th+2 as high as 1.788 % is noticeable. In order to activate this pathway, not only a vaccine therapy, but also Th+2surressing therapy may be effective.
[0256] Bcell immunity (Monocyte > Act.Th2 > CD20*DR), which is a Th2system, is open, and CD20*DR is so far in negative relationship to neutrophil, and thus there seems no issue of side effect. However, if it turns to positive relationship due to a treatment, serious side effects (e.g., inflammatory autoimmune diseases) may be developed. Therefore, monitoring by an immunodynamics chart is essential. In pathways in which Th17+ is involved, Th17+ is in negative relationship to neutrophil, and thus there seems no concern about an inflammatory autoimmune dermatitis, etc. being developed.
Example 5. Providing Subjects with ImmunodynamicsRelated Information (1)
[0257] Certain cellcount of each of multiple types of immunocompetent cells in blood collected from a subject was assigned into the discriminant function obtained in Example 2, the given discriminant score belonged to GOOD/middle Xaxis group. Therefore, as the immunodynamicsrelated information for the subject about for Tcell immunity, the immunodynamics chart described in Fig. 7 is presented.
[0258] With reference to Fig. 7, it can be found that although both TARM and BARM have been established, differentiation and proliferation are stagnated at CD8, and the pathway from CD8 to Tc*DR has been disrupted. Treatments for this subject may include activation/enhancement of CD3 > CD8 > Tc > Tc+ > Tc*DR pathway by IL2 administration, etc. Moreover, because the monocyte/macrophage system is at an extremely low level, this also needs to be activated. Th+2 is at a low level and thus does not need to be suppressed. Ts*DR is at a high level, and, if no improvement is achieved by the aforementioned treatment, Ts*DRsuppressing therapy with an antibody medicine can be considered.
Example 6. Providing Subjects with ImmunodynamicsRelated Information (2)
[0259] Certain cellcount of each of multiple types of immunocompetent cells in blood collected from the subject was assigned into the discriminant function obtained in Example 2, the given discriminant score belonged to <GOOD/higher Xaxis group; n = 54 > which is expressed as an immunodynamics chart in Fig. 8. Moreover, the average number of NK cells per 1 µL blood of GOOD/higher Xaxis group was 135.2 (cells/µL).
[0260] Therefore, the NK activity index and NKcell ADCC activity index of the subject was calculated as follows:
Example 7. Providing Subjects with ImmunodynamicsRelated Information (3)
[0261] In a subject suffering from lingual cancer (male, born in 1947), the NK activity index, NKADCCactivity index, NKT activity index, ADCC activity index of NKT (NKTADCCActivity Index), killer Tcell activity index (Killer Activity Index), and killer Tcell ADCC activity index (KillerADCCActivity Index) were observed over time. The results are shown in Fig. 41.
[0262] In the beginning of May, 1997, the subject received preoperational radiotherapy with irradiation of 30Gy, then experienced a radical operation of the cancer in the left radix linguae by incision of mandible in midline. After that, the cancer relapsed repeatedly in oral mucosal epithelium every three years, approximately. The inventor has been his attending physician since 2004, started inspection and observation over time. Immunity examination also stated in the middle of July, 2007.
[0263] As therapy, the subject was received an instruction to take supplements and Chinese herbal medicines that was said to increase immunocompetence. In the beginning of October, 2007, a few white spots (major axis; 12 mm) was pointed out, and followup was required. In the middle of September, 2010, the subject had chest pain during night which persists to the next morning. Late in September, the subject consulted to a general hospital, and admitted thereto for close examination. The subject was diagnosed with angina with coronary vasospasm, and treatment started. A strict instruction was given for antianginal agents and nutritional therapy, lipid restriction, in particular. The instruction for prohibiting alcohol was not strictly followed, and intemperance lasted.
[0264] The subject caught a cold in April, 2012, made it worse and suffered from bad canker sores for one month. In the immunological examination in the beginning of May, 2015, NK activity index (NKActivityIndex) was absent and a relapse was suspected. In the immunological examinations in beginning of August, and in beginning of October, same year, NK activity index had been recovered, but killer Tcell activity index (KillerActivityIndex) and killer Tcell ADCC activity index were absent in both two examinations. NKT activity index (NKTActivityIndex) was also absent in the beginning of October. Clinically, several locations containing small cauliflowershaped tumor masses that were more evident than white spot were observed. Since their number and size were increased, the subject consulted again to the hospital where he had the radical operation of cancer in the beginning of November, 2012, and the relapse was confirmed. Although an ablative surgery was given late in November, same year, there are too many small tumor masses to ablate all, leaving some unablated. It is considered that cooperation of killer activity and NK activity had suppressed recurrence to this point.
[0265] In the beginning of April, 2013, NKcell activity index, etc. was still absent after the operation, though both NKTcell activity index and killer activity index had recovered. Fortunately, NKT activity index, killer Tcell activity index, and killer Tcell ADCC activity index had been established, and a followup examination was an acceptable treatment in immunological viewpoint, too.
[0266] In the examination in the middle of March, 2016, NKcell activity index had recovered, but killer Tcell activity index was still absent, while NKTcell activity index had been maintained. In the end of September, 2017, the subject received the third operation. Fortunately, there was no metastasis, with only local recurrence of squamous cell carcinoma. In two postoperational examinations, NKcell activity index had been maintained former value, but killer Tcell activity index was still absent, and in the examination in the beginning of March, 2018, NKTcell activity index had recovered. Most recent examination in the middle of July, 2018, although NKcell activity index was at a low value, a desired NK cell ADCC activity index had been recovered.
[0267] It is considered that, if the subject strictly follows diet cure and avoids intemperance in future and thereby keep NKcell activity index and NKcell ADCC activity index or induce both killer Tcell activity and killer Tcell ADCC activity, it would be possible to prevent recurrence or metastasis. If immunological condition goes bad in future, immunocytic therapy should be considered, although it is an expensive therapy. In the examination in the beginning of November, 2018, only killer Tcell activity had been induced, which is undependable. The followup examination in the general hospital was once a month at first, but it is now once in two months, and it is concerned that any recurrence or metastasis might be overlooked. Besides, this subject also suffers from severe pollinosis, and should be recommended to take medicines for pollinosis during severe pollinotic seasons. Anyway, immunological transition should be followed by future care and examinations.
[0268] In the first place, in antitumor immunity, it is ideal to be able to induce a high level of killer Tcell activity index, which is the leading role in adoptive immunity. Moreover, when immunity by NK cell is the main constituent, induction of NK cell ADCC activity is important in antitumor immunity. In this subject, even though immunity by NK cell is the main constituent, NK cell ADCC activity index was absent, and furthermore, killer Tcell activity index, killer Tcell ADCC activity index and NKTcell activity index were at low levels. In future, it is planned to monitor care such that both killer Tcell activity index and killer Tcell ADCC activity index would appear.
Example 8. Providing Subjects with ImmunodynamicsRelated Information (4)
[0269] In a subject suffering from prostate cancer (male, born in 1940), PSA value, NK activity index , NKADCCactivity index, NKT activity index, ADCC activity index of NKT (NKTADCCActivity Index), killer Tcell activity index (Killer Activity Index), and killer Tcell ADCC activity index (KillerADCCActivity Index) were observed over time. The results are shown in Fig. 4244. Fig. 42 shows transition of all indices, Fig. 43 shows transition of killer Tcell activity index and killer Tcell ADCC index in this subject, Fig. 44 shows transition of NKcell activity index and NKcellADCC index in this subject, and Fig. 45 shows transition of NKTcell activity index and NKTcellADCC index in this subject.
[0270] The subject received a prostatectomy in 2002 as radical cure for prostate cancer. He has been receiving followup examination because PSA value had been elevated from December, 2003 to January, 2004. Here, PSA (prostatespecific antigen) has been used as a prostate cancer marker, and it is considered that PSA value is elevated by 2.2 ng/ml for 1 gram of cancer (
Lerner SE, Seay TM, Blute ML, Bergstralh EJ, Barrett D, Zincke H: Prostate specific antigen detected prostate cancer (clinical stage T1c): an interim analysis. J Urol 155:821826, 1996), and the ultrasensitive measurement limit of PSA value is 0.001 ng/ml. The subject has past history of hypertension and hypothyroidism, and has been taking Amlodin, Tanatril, and Thyradin S. Hormonal therapy with dihydrotestosterone (DHT) inhibitor started in 2009.
[0271] The inventor has been the attending physician since January, 2010. Immunotherapy has been considered since February, 2010, but has not been introduced, because the subject is categorized into GOOD group in the immunological examination in February 9, same year and seem to be in immunologically good condition regardless of an rising tendency of PSA value. The subject fell into BAD group in the immunological examination in May 11, same year, and instructed to receive vaccine therapy. It became clear after two years that the subject fell into BAD group during the season of Japanese cedar pollinosis. After this, treatment for pollinosis has been given in combination. Furthermore, the subject was instructed to strictly follow diet cure.
[0272] On May 22, 2012, the immunological condition went extremely worse and was categorized into BAD group. This is considered to be because of pollinosis. After that, the subject was instructed to take medicine for perennial allergy throughout a year. Basically, Allegra (fexofenadine) was taken. In the immunological examination on December 4, same year, the immunological condition transferred from GOOD to BAD group, indicating exacerbation of immunological condition. Therefore, the subject was instructed to avoid overeating and strictly keep abstinence.
[0273] Hormone therapy, Casodex therapy started on December 18, 2014. From July 1, 2016, in addition to Allegra, Claritin started to be administered in combination. By these treatments, it was expected that the eosinophil count in peripheral blood can be suppressed under 100/mm
^{3}. After that, the eosinophil count in peripheral blood started to decrease.
[0274] Immunity was kept in GOOD group to February, 2017, but fell in MODERATE group in the examination in March, same year. Therefore, vaccine therapy was terminated and changed to NKcell adoptive immunotherapy from April. Although vaccine maintenance therapy has been carried out once a month, no effect was observed as vaccine. Although folk medicines, Chinese herbal medicines, etc. were used in combination, no obvious effect was observed.
[0275] The subject changed clinic in April, 2018, but continued NKcell therapy. By NKcell therapy, PSA value decreased to 0.002, which is closed to measurement limit, i.e., 0.001. However, although the activity of NK cells was 96.2 % (effector target ratio; 12: 1) and the cultured NKcell count (hereinafter, "cultured" means the lymphocytes collected from 40µL of peripheral blood of the subject and cultured) was 2.49 billion and the cultured CD8 + αβT cell count was 4.024 billion on July 10, 2018, NK cell activity extremely decreased to 4.1 % (effector target ratio; 12:1) on August 10, 2018. Moreover, an increase of αβT cells was confirmed by a simple test. Therefore, NKcell therapy was replaced with αβT cell therapy from August 10, 2018. Moreover, the subject changed clinic.
Illustration of Course and Transition of Each Activity Index of KillerT Cell, NK Cell and NKT Cell
[0276] For antitumor immunity, maintaining high levels of the indices is essential. Particularly, induction of both killer Tcell activity and killer antibodydependent cellular cytotoxicity at high level is essential. This is also evident from the relationship with PSA value.
[0277] The cultured living cell count was extremely low on April 6, 2017, April 24, same year, and May 11, same year for unknown reason. It is presumably because of systemic physical deconditioning of the subject.
[0278] Appearance of all six indices on the day of immunological examination is the condition for inducing a potent antitumor immunity. However, barely three indices hardly appeared. For indices other than killer Tcell ADCC activity index, connecting lines were drawn where the index was absent. In the case of killer Tcell ADCC activity index, it appeared only four times and no connecting line was drawn. In cases where the index was absent, it is presumed to decrease to 10
^{9}  10
^{10} level. Dendritic cell vaccine therapy had been performed until February, 2017, but PSA value had never been under 0.008 ng/ml. It was therefore switched to NKcell therapy, which was carried out once a month from March 9. It was then changed to αβT cell therapy from August 9, 2018, and PSA value further decreased.
[0279] Besides, Iatrogenic Hematemesis on October 6, 2018 indicates an incidence of major bleeding (approximately 600 ml) and emergency operation after drinking small amount of alcohol on the day of gastric mucosal biopsy. Although the killer activity had successfully increased until this and further improvement had been expected, immunity dropped at once after this day. It is considered to be because of this incidence. The result of biopsy was not a cancer.
Transition of Killer TCell Activity Index and Killer Tcell ADCC Index
[0280] Killer Tcell activity index (Killer Cell Activity Index: KillerAI) is in inverse correlation with PSA value, and thus when killer Tcell activity index is high, PSA value is low, while killer Tcell activity index is low or is not induced, PSA value is high.
[0281] On April 20, 2017, killer Tcell activity index (KillerActivityIndex) was 0.00978821, and killer Tcell antibodydependent cellular cytotoxicity index (KillerADCCActivityIndex) was 0.000104608. Both indices appeared together only on May 29, 2018. PSA value was further decreased presumably due to the appearance of both indices.
[0282] The treatment was changed to αβT cell adoptive immunotherapy from August 10, 2018, in expectation of further enhancement and induction of killer activity.
[0283] In order to measure influences and effects by the cellular therapy, an examination was performed for subsets of peripheral blood lymphocytes.
[0284] On April 3, 2018, the activity of the peripheral blood NK cells (uncultured) was 21 %. On April 24, same year, the cultured living cell count was 2.210 billion, and the activity of cultured NK cells was 95.5 %, the cultured NKcell count was 1.210 billion, the CD3+CD8+αβT cell count was 0.890 billion.
[0285] On May 29, same year, the cultured living cell count was 3.470 billion, the activity of cultured NK cells was 96.5 %, cultured NKcell count was 2.110 billion, and the CD3+CD8+αβ cell count was 1.2 billion.
[0286] On June 19, same year, the cultured living cell count was 3.240 billion, the activity of peripheral blood NK cells (uncultured) was 14 %, the activity of cultured NK cells was not measured, cultured NKcell count was 1.07 billion, and the CD3+CD8+αβ cell count was 2.210 billion.
[0287] On July 10, same year, the cultured living cell count was 6.640 billion, the activity of cultured NK cells was 97.4 %, cultured NKcell count was 2.490 billion, and the CD3+CD8+αβ cell count was 4.020 billion.
[0288] On August 10, same year, the activity of cultured NK cells was extremely decreased to 4.1 %. This was the reason for determining that it was the time to change to αβT cell therapy.
[0289] In the examination on October 7, same year, the cultured living cell count was 4.020 billion, the activity of cultured killer Tcells was 84.4 %, the cultured NKcell count was 0.670 billion, the CD3+CD8+αβ cell count was 2.870 billion, and the CD3+CD4+αβT cell count was 0.380 billion.
[0290] On October 27, same year, the cultured living cell count was 9.750 billion, the activity of cultured killer Tcells was 73.7 %, the cultured NKcell count was 0.670 billion, the activity of peripheral blood NK cells (uncultured) was extremely decreased to 7.2 %, the CD3+CD8+αβ cell count was 8.290 billion, and the CD3+CD4+αβT cell count was 0.310 billion.
[0291] On November 17, same year, the cultured living cell count was 8.730 billion, the activity of cultured killer Tcells was 91.1 %, the cultured NKcell count was 1.960 billion, the CD3+CD8+αβT cell count was 5.450 billion, and the CD3+CD4+αβT cell count was 0.750 billion.
[0292] On December 8, same year, the cultured living cell count was 6.810 billion, the activity of cultured killer Tcells was 72.3 %, the cultured NKcell count was 1.360 billion, the CD3+CD8+αβT cell count was 4.2 billion, and the CD3+CD4+αβT cell count was 0.790 billion. Note that the listing of indices is omitted.
Transition of NKCell Activity Index and NKCell ADCC Index
[0293] The treatment was changed to NKcell therapy from March 9, 2017. NKcell activity index (NKActivityIndex) and NKcellADCC activity index (NKADCCActivityIndex) did not appear before NKcell therapy, but the treatment induced their appearance.
[0294] Approximately five month later, on July 28, same year, NKcell activity index was as high as 2.397, and indicated 2.463 which was the highest value on December 27, 2017. In a similar way as in killer Tcell activity index, when NKcell activity index was high, PSA value decreased, indicating that they are in inverse correlation. NKTcellADCC activity index decreased, in particular, from August 10, 2018, and aforementioned killer Tcell ADCC activity index appeared on May 29, 2018, but has been disappeared since then. Therefore, αβT cell adoptive immunotherapy was considered to be appropriate, and the treatment was changed from NKcell therapy to αβT cell adoptive immunotherapy. It is natural that NKcell activity index and NKcellADCC activity index last appeared on September 11, 2018 and disappeared until December 10, presumably due to the change in cell therapy.
Transition of NKTCell Activity Index and NKTCell ADCC Index
[0295] NKTcell activity index (NKTActivityIndex) is in inverse correlation with PSA value in a similar way as the NKcell activity index and the killer Tcell activity index.
[0296] On June 19, 2018, NKTcell activity index and NKTcellADCC activity index (NKTADCCActivityIndex) were both induced at the highest level. It is considered that PSA value was further decreased because of this. However, it last appeared in the beginning of October, 2018 and has not appeared thereafter.
[0297] From August 10, 2018, the treatment was changed to αβT cell adoptive immunotherapy in expectation of induction of the appearance of killer Tcell activity index. In the analysis of peripheral blood lymphocyte subset on December 10, 2018, a desired high level of KillerActivityIndex could be induced. Although NKT activity could not be induced, the appearance of NKTADCCActivityIndex could be induced. It is considered that due to the appearance of these indices the desired PSA value <0.001 could be achieved in 1 year and 10 months.
Prediction of Tumor Mass Destruction
[0298] By using each of the activity indices, the degree of tumor mass that can be destroyed by an immunocytic therapy can be predicted.
[0299] Usually, it is said that 1 gram of tumor mass contains 10
^{9} cancer cells. In prostate cancer, it is said that 1 gram of tumor mass exist if there is an increase in PSA value by 2.2 ng/ml.
[0300] Accordingly, based on the killer Tcell activity index,
wherein killer Tcell activity is introduced in decimal percentage, but not in integer percentage.
[0301] Moreover, based on the killer Tcell ADCC activity index,
wherein killer Tcell activity is introduced in decimal percentage, but not in integer percentage.
[0302] In October 29, 2018, the number of cultured killer Tcells (CD3+CD8+αβT cell) was 8.29 x 10
^{9}, the activity of cultured killer Tcells (effector target ratio; 12:1) was 73.7 %, the killer Tcell activity index was 0.015619, and the killer Tcell ADCC activity index was 0.053472.
[0303] By assigning into the above formula,
based on the killer Tcell activity index, weight of tumor mass to be decreased is:
and the weight of tumor mass to be decreased can be predicted to be 0.00795 g.
[0304] This can be converted into PSA value:
indicating that the PSA value is predicted to decrease by 0.0175 ng/ml at a time.
[0305] Moreover, based on the killer Tcell ADCC activity index, weight of tumor mass to be decreased was:
and this was converted into the PSA value:
[0306] When both of these are added, it means that 0.03517 g of tumor mass will be destroyed. 0.077395 ng/ml of decrease in PSA value is predicted.
[0307] On December 10, 2018, KillerActivityIndex was 1.168043, and no KillerADCCActivityIndex appearance was induced or evoked.
of destruction and reduction were predicted.
[0308] When this is converted into PSA value, a reduction of 0.58341 x 2.2= 1.28350 (ng/ml) is predicted. A strong activity was induced and evoked as predicted, and PSA value could be decreased, as desired, to below 0.001 ng/ml which is an ultrasensitive measurement limit.
[0309] Similarly, by performing a similar calculation for NK cell, the weight of tumor mass that was destroyed by NKcell therapy can be predicted.
[0310] Based on the NKcell activity index,
wherein the activity of NK cells is introduced in decimal percentage, but not in integer percentage.
[0311] Moreover, based on the NKcell ADCC activity index,
wherein the activity of NK cells is introduced in decimal percentage, but not in integer percentage.
[0312] On July 10, 2018, the number of cultured NK cells was 2.49 x 10
^{9}, the activity of cultured NK cells (effector target ratio; 12:1) was 96.2 %, NKcell activity index was 2.102970, and NKcell ADCC activity index was 2.484033.
[0313] By assigning into the above formula,
the NKcell activity index was predicted to reduce weight of tumor mass by:
and the NKcell ADCC activity index was predicted to reduce weight of tumor mass by:
and in total, it was predicted to reduce weight of tumor mass by 0.91563 g. Thus, it was presumed that NKcell therapy was sufficiently effective, although the PSA value was still 0.002 ng/ml. Thereafter, the PSA value remained to be 0.002 and was not reduced, and in addition to it, NK activity and NKcell count were reduced. Therefore, no effect seems to be expected by NKcell therapy and the treatment was changed to αβT cell therapy.
[0314] Similarly, by performing a similar calculation for NKTcell, the weight of tumor mass that was destroyed by NKTcell therapy can be predicted.
[0315] Based on the NKTcell activity index,
wherein NKTcell activity is introduced in decimal percentage, but not in integer percentage.
[0316] Moreover, based on the NKTcell ADCC activity index,
wherein NKTcell activity is introduced in decimal percentage, but not in integer percentage.
[0317] In data of October 9, 2018, the NKTcell activity index was 0.23397 and the NKTcell ADCC activity index was 0.000219. NKT cells were cultured separately; although the activity was not measured, it can be predicted that:
assuming the number of cultured NKT cells to be Z x 10
^{9}, and the activity of cultured killer Tcells (effector target ratio; 12:1) to be Znkt;
for the NKTcell activity index:
of tumor mass can be destroyed. When this is converted into PSA value, a reduction of 0.019498 x Z x Znkt x 2.2 ≒ 0.0429 x Z x Znkt (ng/ml) is predicted.
[0318] For NKTcell ADCC activity index, it can be predicted that:
of tumor mass can be destroyed. When this is converted into PSA value, a reduction of 0.00001825 x 2.2 x Z x Znkt (ng/ml) is predicted. As a result, the sum of these, i.e., 0.019516 x Z x Znkt (g) will be destroyed.
[0319] In the analysis of peripheral blood lymphocyte on December 10, 2018, no appearance of NKT activity index was induced, though the NKTADCCActivityIndex was 0.0475174, which was higher as compared to that on October 9, same year.
[0320] It is considered that the tumor mass destruction calculated by the indices continues during the period before the administration of next therapy (23 weeks). Therefore, the weight of tumor mass which would be destroyed will be the product of the number of grams predicted to be destroyed as mentioned above being multiplied by the number of days (i.e., 1421). Considering in this way, an effective immunotherapy should be carried out appropriately and precisely such that each index will be increased. An inappropriate immunotherapy might cause an exacerbation, and it is important and essential to keep monitoring immunodynamics. Although the examination takes costs, it should be done if necessary, regardless of the costs.
[0321] Besides, the volume of tumor mass that would be destroyed can be presumed if data can be obtained for the respective activity of each lymphocyte in peripheral blood, without culturing each lymphocyte. Moreover, the volume of tumor mass of the subject's cancer that would be destroyed can be presumed by using cancer cells of the subject him/herself for the activity evaluation, not K562 cells which are usually used for activity evaluation.
[0322] From the equation: the body weight x (1/13) x each peripheral blood cell count (killer αβT, NK or NKT) (/mm
^{3}), the total number of effector cells (unit: 10
^{9} cells) is obtained. The percentage of the activity of each effector cell can be examined, and the weight of tumor mass which would be destroyed can be predicted using indices obtained from an analysis of the chart of lymphocyte subset immunodynamics and calculating as mentioned above.
Example 9. Generating Improved Discriminant Function
[0323] The subjects were 344 cases that were sorted into GOOD group, MODERATE group in Example 3. A discriminant analysis was performed using 3 groups of GOOD, MODERATE and BAD groups as objective variables and 26 types of immunocompetent cells: CD3positive lymphocyte, CD4positive lymphocyte, CD8positive lymphocyte, CD20*DR lymphocyte, Th± lymphocyte, Th2 lymphocyte, Th+2 lymphocyte, Act.Th1 lymphocyte, Act.Th2 lymphocyte, Ti*DR lymphocyte, Ti± lymphocyte, Ti2 lymphocyte, Ti+2 lymphocyte, Tc*DR lymphocyte, Tclymphocyte, Tc+ lymphocyte, Ts*DR lymphocyte, Ts lymphocyte, Ts+ lymphocyte, NK cell, NKT cell, N3+ cell, monocyte, basophil, eosinophil and neutrophil as explanatory variables to give a discriminant function. Cases which could not be sorted well in scatter plotting of discriminant scores were excluded, and the discriminant analysis was performed repeatedly. As a result, a discriminant function with 100 % discrimination accuracy rate among 267 cases. A scatter plot of discriminant scores in which the first discriminant function values is on Xaxis and the second discriminant function values is on Yaxis is shown in Fig. 46. The discriminant score obtained by assigning the cellcount of each of immunocompetent cells of subjects into this discriminant function can be used as an indicator to divide the subjects into multiple groups.
[0324] As above, the present invention comprises methods of predicting prognosis of a subject from the cellcounts of multiple types of immunocompetent cells. For instance, for data cluster containing the values of levels of cancerous markers such as prostatespecific antigen (PSA) of individuals and data of the cellcount of each of multiple types of immunocompetent cells in blood collected from the individuals in a number that allows for the discriminant analysis, a multiple regression analysis is performed using PSA value as objective variable and the multiple types of immunocompetent cells are set as explanatory variables, subtracting the expected value from the observed value to give a residual value, and the individuals can be sorted into any number of groups in such a way that the smaller the residual value is, the better the expected prognosis would be. By performing a discriminant analysis using said groups as objective variables and the multiple types of immunocompetent cells are set as explanatory variables, a discriminant function can be obtained. By assigning the cellcount of the multiple types of immunocompetent cells of a subject into the discriminant function and calculating a discriminant score, the group to which the subject belongs can be determined and the prognosis can be predicted. Moreover, by obtaining immunodynamicsrelated information in each of the sorted groups, immunodynamicsrelated information of the subject is obtained, and therapy or prophylaxis according to the immunodynamics of the subject can be determined with that information.