TLR agonist-enhanced in vitro assay of cell mediated immune responsiveness

1. A method for measuring in vitro immune responsiveness to an antigen by lymphocytes from a test subject that is suspected of having been previously exposed to the antigen, comprising:

(A) incubating in vitro

(i) a sample that comprises lymphocytes from the test subject, with

(ii) one or more antigens associated with a disease or condition suspected of being present in the test subject, and

(iii) a limiting amount of a Toll-like receptor (TLR) agonist wherein the TLR agonist is selected from the group consisting of R848 (imidazoquinoline), lipomannan, and poly (I:C), said limiting amount of the TLR agonist being determined by a method that comprises a step selected from:

(a) identifying an amount of the TLR agonist that stimulates the lymphocytes to produce no background response or a minimal background response when incubated with no antigen present and thereby determining the limiting amount, and

(b) identifying an amount of the TLR agonist such that the TLR agonist is present at a ratio of TLR agonist to said one or more antigens of from 1:500 to 1:1.5 and thereby determining the limiting amount,

wherein said step of incubating the sample, the one or more antigens, and the limiting amount of TLR agonist is for a time and under conditions sufficient for:

(1) antigen responsiveness, by one or more T cells that are present in said lymphocytes, to one or more of said one or more antigens, thereby to stimulate the lymphocytes to produce an effector molecule, and

(2) TLR agonist responsiveness, by one or more T cells that are present in said lymphocytes, to said limiting amount of the TLR agonist to stimulate the lymphocytes to produce an enhanced level of the effector molecule when the limiting amount of the TLR agonist and said one or more antigens are present, relative to the level of the effector molecule that is produced when the TLR agonist is absent; and

(B) detecting the enhanced level of the effector molecule that is produced by the lymphocytes stimulated according to step (A), wherein the detected enhanced level of the effector molecule is greater than a detectable effector molecule level that is a sum of (i) the detectable effector molecule level that can be produced by said lymphocytes incubated only with said one or more antigens, plus (ii) the detectable effector molecule that can be produced by said lymphocytes incubated only with said limiting amount of the TLR agonist,

wherein if the enhanced level of the effector molecule that is detected is higher than a control level of the effector molecule that is detected following incubation of control lymphocytes from a healthy control subject with the limiting amount of the TLR agonist and the one more antigens, then the enhanced level indicates that the lymphocytes from the test subject have been previously exposed to the antigen associated with the disease or condition suspected of being present in the test subject, and wherein the effector molecule is selected from IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-6 (CXCL8), IL-10, IL-12, IL-13, IL-16 (LCF), IL-17, IL-1α(IL-1F1), IL-1β(IL-1F2), IL-1rα (IL-1F3), TGF-β, G-CSF, GM-CSF, complement component 5a (C5a), Groα (CXCL1), sICAM-1 (CD54), IP-10 (CXCL10), I-TAC (CXCL11), MCP-1 (CCL2), MIF (GIF), MIP-1α(CCL3), MIP-1β(CCL4), RANTES (CCL5) and MIG (CXCL9).

2. The method of claim 1 wherein the TLR agonist is R848.

3. The method of claim 1 wherein the test subject is a human.

4. The method of claim 1 wherein the sample is undiluted whole blood.

5. The method of claim 4 wherein the whole blood is collected in a tube comprising heparin.

6. The method of claim 1 wherein the effector molecule is interferon-γ (IFN-γ).

7. The method of claim 1 wherein the effector molecule is detected with antibodies specific for the effector molecule.

8. The method of claim 1 wherein the test subject has or has previously been exposed to an infection by a pathogenic agent selected from the group consisting of Mycobacterium species, Staphylococcus species, Streptococcus species, Borrelia species, Escherichia coli, Salmonella species, Clostridium species, Shigella species, Proteus species, Bacillus species, Herpes virus, Hepatitis B or C virus and Human immune deficiency virus (HIV).

9. The method of claim 8 wherein the test subject has or has previously been exposed to an infection by Mycobacterium tuberculosis.

10. The method of claim 1 wherein the subject has a disease condition selected from the group consisting of alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease multiple sclerosis, autoimmune disease of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue syndrome (CFIDS), chronic inflammatory demyelinating, chronic inflammatory polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, crest syndrome, cold agglutinin disease, Crohn's disease, dermatitis herpetiformis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia, glomerulonephritis, Grave's disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, insulin dependent diabetes (Type I), lichen planus, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, myocarditis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglancular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arrthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo and inflammatory bowel disease.

11. The method of claim 10 wherein the disease is Celiac's disease.

12. The method of claim 1 wherein the subject has a cancer selected from the group consisting of ABL1 protooncogene, AIDS related cancers, acoustic neuroma, acute lymphocytic leukaemia, acute myeloid leukaemia, adenocystic carcinoma, adrenocortical cancer, agnogenic myeloid metaplasia, alopecia, alveolar soft-part sarcoma, anal cancer, angiosarcoma, aplastic anaemia, astrocytoma, ataxia-telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancers, bowel cancer, brain stem glioma, brain and CNS tumors, breast cancer, CNS tumors, carcinoid tumors, cervical cancer, childhood brain tumors, childhood cancer, childhood leukaemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukaemia, chronic myeloid leukaemia, colorectal cancers, cutaneous T-Cell lymphoma, dermatofibrosarcoma-protuberans, desmoplastic-small-round-cell-tumor, ductal carcinoma, endocrine cancers, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extra-hepatic bile duct cancer, eye cancer, eye: melanoma, retinoblastoma, fallopian tube cancer, fanconi anemia, fibrosarcoma, gall bladder cancer, gastric cancer, gastrointestinal cancers, gastrointestinal-carcinoid-tumor, genitourinary cancers, germ cell tumors, gestational-trophoblastic-disease, glioma, gynaecological cancers, hematological malignancies, hairy cell leukaemia, head and neck cancer, hepatocellular cancer, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, hydatidiform mole, hypercalcemia, hypopharynx cancer, intraocular melanoma, islet cell cancer, Kaposi's sarcoma, kidney cancer, Langerhan's-cell-histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, Li-Fraumeni syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, male breast cancer, malignant-rhabdoid-tumor-of-kidney, medulloblastoma, melanoma, merkel cell cancer, mesothelioma, metastatic cancer, mouth cancer, multiple endocrine neoplasia, mycosis fungoides, myelodysplastic syndromes, myeloma, myeloproliferative disorders, nasal cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, nijmegen breakage syndrome, non-melanoma skin cancer, non-small-cell-lung-cancer-(NSCLC), ocular cancers, oesophageal cancer, oral cavity cancer, oropharynx cancer, osteosarcoma, ostomy ovarian cancer, pancreas cancer, paranasal cancer, parathyroid cancer, parotid gland cancer, penile cancer, peripheral-neuroectodermal-tumors, pituitary cancer, polycythemia vera, prostate cancer, rare-cancers-and-associated-disorders, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome, salivary gland cancer, sarcoma, schwannoma, Sezary syndrome, skin cancer, small cell lung cancer (SCLC), small intestine cancer, soft tissue sarcoma, spinal cord tumors, squamous-cell-carcinoma-(skin), stomach cancer, synovial sarcoma, testicular cancer, thymus cancer, thyroid cancer, transitional-cell-cancer-(bladder), transitional-cell-cancer-(renal-pelvis-/-ureter), trophoblastic cancer, urethral cancer, urinary system cancer, uroplakins, uterine sarcoma, uterus cancer, vaginal cancer, vulva cancer, Waldenstrom's-macroglobulinemia and Wilms' tumor.

13. The method of claim 1 wherein the subject was exposed to a toxicant.

14. A method for measuring in vitro immune responsiveness to an antigen by lymphocytes from a test subject that is suspected of having been previously exposed to the antigen, comprising:

(A) incubating in vitro

(i) a sample that comprises lymphocytes from the test subject, with

(ii) one or more antigens associated with a disease or condition suspected of being present in the test subject wherein the antigen is selected from an Epstein-Barr Virus (EBV) antigen, a cytomegalovirus (CMV) antigen, and a tuberculosis (TB) antigen, and

(iii) a limiting amount of a Toll-like receptor (TLR) agonist wherein the TLR agonist is selected from the group consisting of R848 (imidazoquinoline), lipomannan, and poly (I:C), said limiting amount of the TLR agonist being determined by a method that comprises a step selected from:

(a) identifying an amount of the TLR agonist that stimulates the lymphocytes to produce no background response or a minimal background response when incubated with no antigen present and thereby determining the limiting amount, and

(b) identifying an amount of the TLR agonist such that the TLR agonist is present at a ratio of TLR agonist to said one or more antigens of from 1:500 to 1:1.5 and thereby determining the limiting amount,

wherein said step of incubating the sample, the one or more antigens, and the limiting amount of TLR agonist is for a time and under conditions sufficient for:

(1) antigen responsiveness, by one or more T cells that are present in said lymphocytes, to one or more of said one or more antigens, thereby to stimulate the lymphocytes to produce an effector molecule, and

(2) TLR agonist responsiveness, by one or more T cells that are present in said lymphocytes, to said limiting amount of the TLR agonist to stimulate the lymphocytes to produce an enhanced level of the effector molecule when the limiting amount of the TLR agonist and said one or more antigens are present, relative to the level of the effector molecule that is produced when the TLR agonist is absent; and

(B) detecting the enhanced level of the effector molecule that is produced by the lymphocytes stimulated according to step (A), wherein the detected enhanced level of the effector molecule is greater than a detectable effector molecule level that is a sum of (i) the detectable effector molecule level that can be produced by said lymphocytes incubated only with said one or more antigens, plus (ii) the detectable effector molecule that can be produced by said lymphocytes incubated only with said limiting amount of the TLR agonist,

wherein if the enhanced level of the effector molecule that is detected is higher than a control level of the effector molecule that is detected following incubation of control lymphocytes from a healthy control subject with the limiting amount of the TLR agonist and the one more antigens, then the enhanced level indicates that the lymphocytes from the test subject have been previously exposed to the antigen associated with the disease or condition suspected of being present in the test subject, and wherein the effector molecule is interferon-gamma (IFN-γ).