AN IN VITRO KIT AND A METHOD FOR DIAGNOSTICS IN TRANSPLANTS AND/OR AUTOIMMUNE DISEASES AND/OR TUMOR DISEASES AND/OR VACCINATIONS AND A MESSENGER-SUBSTANCE-CONTAINING SUPERNATANT, CELL-CONTAINING SEDIMENT, AND A COMBINATION, FOR USE IN DIAGNOSTICS IN TRANSPLANTS AND/OR AUTOIMMUNE DISEASES AND/OR TUMOR DISEASES AND/OR VACCINATIONS

Abstract

The invention relates to the use in vitro of a kit for diagnostics, more particularly therapy-accompanying diagnostics, relating to a transplant and/or for diagnostics, more particularly therapy-accompanying diagnostics, relating to an autoimmune disease and/or for diagnostics, more particularly therapy-accompanying diagnostics, relating to a tumor disease and/or for diagnostics, more particularly therapy-accompanying diagnostics, relating to a vaccination, wherein the kit has the following components: a blood collection container, a blood collection set or blood collection system including a blood collection container, a nutrient medium for blood, and at least one activator for activating blood cells, more particularly immune cells, and/or at least one inhibitor for inhibiting blood cells, more particularly immune cells, and/or at least one modulator for modulating blood cells, more particularly immune cells.

The invention further relates to the use in vitro of a method for diagnostics in transplants and/or autoimmune diseases and/or tumor diseases and/or vaccinations, to a messenger-substance-containing supernatant for use in vitro in diagnostics in transplants and/or autoimmune diseases and/or tumor diseases and/or vaccinations, and to a combination for use in diagnostics in transplants and/or autoimmune diseases and/or tumor diseases and/or vaccinations.

Claims

1-16. (canceled)

17. An in vitro method for diagnostics including therapy-accompanying diagnostics, relating to a transplant, the prognostic assessment of prospects of success and/or predicting a clinical outcome of a transplant and/or determining suitability/effectiveness/dose of compounds, immunosuppressants, for a transplant, and/or therapy monitoring of a transplant, and/or for diagnostics including therapy-accompanying diagnostics, relating to an autoimmune disease, characterizing a stage of an autoimmune disease and/or prognostic assessment of the prospects of success and/or predicting a clinical outcome of a therapy of an autoimmune disease and/or determining the suitability and/or effectiveness and/or dose of compounds, immunosuppressants, for a therapy of an autoimmune disease, and/or therapy monitoring of an autoimmune disease, and/or for diagnostics including therapy-accompanying diagnostics, relating to a tumor disease, prognostic assessment of prospects of success and/or predicting a clinical outcome of a therapy of a tumor disease and/or determining suitability and/or effectiveness and/or dose of compounds for a therapy of a tumor disease and/or therapy monitoring of a tumor disease, and/or for diagnostics including therapy-accompanying diagnostics, relating to a vaccination, for prognostic assessment of the prospects of success and/or predicting a clinical outcome of a vaccination and/or therapy monitoring of a vaccination, comprising the step of using a kit, wherein the kit comprises: a blood collection container, a syringe, or a blood collection set or blood collection system including a blood collection container or a syringe barrel, a nutrient medium for blood, and at least one activator that activates blood cells or immune cells, and/or at least one inhibitor that inhibits blood cells or immune cells, and/or at least one modulator that modulates blood cells or immune cells.

18. The in vitro method as claimed in claim 17, wherein the at least one activator that activates blood cells or immune cells, is selected from the group consisting of cells from a transplant donor, antigen preparations of cells from a transplant donor, antigens, superantigens, autoantigens, tumor antigens, tumor vaccines, mitogens, ligands of blood cell surface receptors that have an activating or inhibitory effect on blood cells, ligands of intracellular receptors of blood cells that have an activating or inhibitory effect on blood cells, ligands of ion channels that have an activating or inhibitory effect on blood cells and are accessible via surface receptors or intracellular receptors of blood cells, ligands of enzymes that have an activating or inhibitory effect on blood cells and are accessible via surface receptors or intracellular receptors of blood cells, antibodies to blood cell surface receptors that have an activating or inhibitory effect on blood cells, antibodies to intracellular receptors of blood cells that have an activating or inhibitory effect on blood cells, antibodies to ion channels that have an activating or inhibitory effect on blood cells and are accessible via surface receptors or intracellular receptors of blood cells, antibodies to enzymes that have an activating or inhibitory effect on blood cells and are accessible via surface receptors or intracellular receptors of blood cells, adjuvants, and combinations or mixtures, of at least two of the activators.

19. The in vitro method as claimed in claim 17, wherein the cells of the transplant donor are organ cells selected from the group consisting of blood cells, skin cells, kidney cells, liver cells, heart cells, lung cells, pancreas cells, small intestine cells, bone cells, bone marrow cells, and combinations or mixtures, of at least two of the cells from a transplant donor, and/or the antigen preparations of cells from a transplant donor are selected from the group consisting of intact cells, cell membrane fragments, microsomes, cell- and/or cell-fragment-free filtrates, ultrafiltrates, extracts, and combinations or mixtures, of at least two of the antigen preparations of cells from a transplant donor, and/or the superantigens are selected from the group consisting of staphylococcal enterotoxins such as staphylococcal enterotoxin A and/or staphylococcal enterotoxin B and/or staphylococcal enterotoxin C, toxic shock syndrome toxin, Streptococcus pyogenes exotoxin, and combinations or mixtures, of at least two of the superantigens, and/or the autoantigens are selected from the group consisting of myelin basic protein (MBP), outer surface protein (OspA), leukocyte function-associated antigen 1α (LFA-1α), acetylcholinesterase receptor antigen (AChR), and combinations or mixtures, of at least two of the autoantigens, and/or the tumor antigens are selected from the group consisting of MAGE, BAGE, GAGE, CTAG, NY-ESO, MUC-1, CDK4, beta-catenin, and combinations or mixtures, of at least two of the tumor antigens, and/or the tumor vaccines are combinations of a tumor antigen and an adjuvant, or a combination of PAP and GMCSF or a combination of MUC-1 and monophosphoryl lipid A, and/or the mitogens are selected from the group consisting of phytohemagglutinin, pokeweed mitogen, concanavalin A, and combinations or mixtures, of at least two of the mitogens, and/or the ligands of blood cell surface receptors that have an activating or inhibitory effect on blood cells are selected from the group consisting of ligands of CD2, ligands of CD3, ligands of CD11, ligands of CD16, ligands of CD18, ligands of CD32, ligands of CD64, and combinations or mixtures, of at least two of the ligands, and/or the ligands of intracellular receptors of blood cells that have an activating or inhibitory effect on blood cells are selected from the group consisting of TLR ligands, NOD ligands, RIG ligands, PPAR-γ ligands, and combinations or mixtures, of at least two of the ligands, and/or the antibodies to blood cell surface receptors that have an activating or inhibitory effect on blood cells are selected from the group consisting of antibodies to CD2, antibodies to CD3, antibodies to CD11, antibodies to CD16, antibodies to CD18, antibodies to CD32, antibodies to CD64, and combinations or mixtures, of at least two of the antibodies, and/or the antibodies to intracellular receptors of blood cells that have an activating or inhibitory effect on blood cells are selected from the group consisting of antibodies to TLR9, antibodies to NOD, antibodies to RIG, antibodies to PPAR-γ, and combinations or mixtures, of at least two of the antibodies.

20. The in vitro method as claimed in claim 17, wherein the at least one inhibitor that inhibits blood cells or immune cells, is selected from the group consisting of anti-inflammatory compounds/drugs, immunosuppressants, and combinations or mixtures, of at least two of the inhibitors, when the kit is used in vitro for diagnostics including therapy-accompanying diagnostics, relating to a transplant and/or an autoimmune disease, for the prognostic assessment of the prospects of success and/or predicting a clinical outcome of a transplant and/or a therapy of an autoimmune disease and/or determining the suitability and/or effectiveness and/or dose of compounds, immunosuppressants, for a transplant and/or of an autoimmune disease, and/or therapy monitoring of a transplant and/or an autoimmune disease.

21. The in vitro method as claimed in claim 20, wherein the anti-inflammatory compounds/drugs are selected from the group consisting of cortisones, inhibitors of the synthesis of arachidonic acid metabolites, kinase inhibitors, inhibitors of cyclooxygenases, inhibitors of lipoxygenases, receptor blockers, siRNA, ion-channel blockers, and combinations or mixtures, of at least two of the anti-inflammatory compounds/drugs, and/or the immunosuppressants are selected from the group consisting of glucocorticoids, mTOR inhibitors, cytostatics, antimetabolites, monoclonal antibodies, anti-T-lymphocyte globulin, mycophenolates, and combinations or mixtures, of at least two of the immunosuppressants.

22. The in vitro method as claimed in claim 17, wherein the at least one modulator that modulates blood cells or immune cells, is selected from the group consisting of methotrexate, azathioprine, 6-mercaptopurine, leflunomide, sulfasalazine, chloroquine, checkpoint inhibitors, calcineurin inhibitors, DMARDs (disease-modifying antirheumatic drugs), TLR agonists, and combinations or mixtures, of at least two of the modulators.

23. The in vitro method as claimed in claim 17, wherein the kit also includes at least one co-activator selected from the group consisting of ligands of CD (cluster of differentiation) surface molecules, antibodies to CD (cluster of differentiation) surface molecules, TLR ligands, NOD ligands, cytokines and combinations, more particularly mixtures, of at least two of the co-activators, and/or also at least one co-inhibitor selected from the group consisting of ligands of CD (cluster of differentiation) molecules, antibodies to CD (cluster of differentiation) molecules, and combinations or mixtures, of at least two of the co-inhibitors, and/or also at least one co-modulator selected from the group consisting of inhibitors of enzymes of tryptophan degradation such as TDO and/or IDO, modulating metabolites, ligands, antibodies of receptors, inhibitors or allosteric regulators of enzymes, enzymes from signal transduction cascades, inhibitors or activators of ion channels, and combinations or mixtures, of at least two of the co-modulators.

24. The in vitro method as claimed in claim 17, wherein the transplant is an organ transplant, more particularly a kidney transplant, liver transplant, heart transplant, lung transplant, pancreas transplant, small intestine transplant, bone marrow transplant, bone transplant or skin transplant.

25. The in vitro method as claimed in claim 17, wherein the autoimmune disease is selected from the group consisting of alopecia areata, autoimmune enteropathy, autoimmune hepatitis, APECED, bullous pemphigoid, chronic type A gastritis, eosinophilic granulomatosis with polyangiitis, CIDP, chronic active hepatitis, ulcerative colitis, dermatomyositis, type 1 diabetes mellitus, dermatitis herpetiformis (Duhring's disease), endocrine orbitopathy, epidermolysis bullosa acquisita, glomerulonephritis, Goodpasture syndrome, granulomatosis with polyangiitis, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic thrombocytopenic purpura, lichen sclerosus, lichen mucosae, linear IgA dermatosis, lupus erythematosus, microscopic polyangiitis, Graves' disease, Behcet's disease, Crohn's disease, ankylosing spondylitis, multiple sclerosis, myasthenia gravis, narcolepsy, PANDAS, pemphigus foliaceus, pemphigus seborrhoicus, pemphigus vulgaris, polychondritis, polymyalgia rheumatica, polymyositis, psoriasis, primary biliary cirrhosis, primary chronic polyarthritis, rheumatic fever, rheumatoid arthritis, giant cell arteritis, SAPHO syndrome, sarcoidosis (Boeck's disease), Sjögren's syndrome, scleroderma, stiff-person syndrome, sympathetic ophthalmia, systemic lupus erythematosus, Henoch-Schönlein purpura, Wegener's granulomatosis, vitiligo, and celiac disease, and/or the tumor disease is selected from the group consisting of carcinoma, melanoma, blastoma, lymphoma, leukemia, and sarcoma, and/or the vaccination is selected from the group consisting of antiviral vaccination, antibacterial vaccination, antifungal vaccination, antiparasitic vaccination, anti-autoimmunogical vaccination and antitumor vaccination.

26. The in vitro method as claimed in claim 17, wherein the at least one activator that activates blood cells or immune cells, and/or the at least one inhibitor that inhibits blood cells or immune cells, and/or the at least one modulator that modulates blood cells or immune cells, are/is present in liquid or dissolved form, and/or the at least one activator that activates blood cells or immune cells, and/or the at least one inhibitor that inhibits blood cells or immune cells, and/or the at least one modulator that modulates blood cells or immune cells, are/is present in the blood collection container, and/or the nutrient medium is present in the blood collection container.

27. The in vitro method as claimed in claim 17, wherein the blood collection container or the syringe, has a hollow cylinder in which a plunger is arranged such that it can be displaced with a plunger rod to create a seal, and a closure cap for closing the hollow cylinder, and the closure cap has an attachment socket for a cannula, and/or the blood collection set/blood collection system comprises, in addition to the syringe, a cannula or a butterfly cannula, and/or blood collection tubing, and/or the kit also comprises a component selected from the group consisting of anticoagulant, thermostatically controllable device such as a thermoblock, a number of cooling blocks, a refrigerator, a freezer, a container with dry ice, a nitrogen tank, and combinations of at least two of the components.

28. The in vitro method as claimed in claim 17, wherein the kit also comprises a separating component associated with the blood collection container for the separation of a supernatant and sediment formed in the blood collection container.

29. The in vitro method as claimed in claim 28, wherein the separating component comprises a valve or a valve plunger that has a valve and a pusher, wherein the valve is open when the valve or the valve plunger is connected to the pusher, and the valve is closed when the valve or the valve plunger are separated from one another.

30. The in vitro method for diagnostics including therapy-accompanying diagnostics, relating to a transplant, prognostic assessment of prospects of success and/or predicting a clinical outcome of a transplant and/or determining suitability and/or effectiveness and/or dose of compounds, immunosuppressants, for a transplant, and/or therapy monitoring of a transplant, and/or for diagnostics including therapy-accompanying diagnostics, relating to an autoimmune disease characterizing a stage of an autoimmune disease and/or prognostic assessment of prospects of success and/or for predicting a clinical outcome of a therapy of an autoimmune disease and/or determining suitability and/or effectiveness and/or dose of compounds, immunosuppressants, for a therapy of an autoimmune disease, and/or therapy monitoring of an autoimmune disease, and/or for diagnostics including therapy-accompanying diagnostics, relating to a tumor disease for prognostic assessment of prospects of success and/or predicting a clinical outcome of a therapy of a tumor disease and/or determining suitability and/or effectiveness and/or dose of compounds for a therapy of a tumor disease and/or therapy monitoring of a tumor disease, and/or for diagnostics including therapy-accompanying diagnostics, relating to a vaccination for prognostic assessment of prospects of success and/or predicting a clinical outcome of a vaccination and/or therapy monitoring of a vaccination, wherein the method comprises: a) preparing a mixture comprising whole blood, a nutrient medium for whole blood, and at least one activator that activates blood cells or immune cells, in addition to any activators present in the whole blood, and/or at least one inhibitor that inhibits blood cells or immune cells, in addition to any inhibitors present in the whole blood, and/or at least one modulator that modulates blood cells or immune cells, in addition to any modulators present in the whole blood, b) incubating the prepared mixture, c) separating a messenger-substance-containing supernatant formed through incubation of the prepared mixture from a cell-containing sediment formed through incubation of the prepared mixture, or a messenger-substance-containing supernatant formed through centrifugation of the incubated prepared mixture from a cell-containing sediment formed through centrifugation of the incubated prepared mixture, and d) detecting and/or determining messenger substances present in the supernatant and/or determining cell surface molecules and/or intracellular activation markers of blood cells or immune cells, present in the sediment.

31. An in vitro method for diagnostics including therapy-accompanying diagnostics, relating to a transplant in prognostic assessment of prospects of success and/or in predicting a clinical outcome of a transplant and/or in determining the suitability and/or effectiveness and/or dose of compounds, immunosuppressants, for a transplant, and/or in therapy monitoring of a transplant, and/or diagnostics including therapy-accompanying diagnostics, relating to an autoimmune disease, characterizing a stage of an autoimmune disease and/or in prognostic assessment of prospects of success and/or in predicting a clinical outcome of a therapy of an autoimmune disease and/or in determining suitability and/or effectiveness and/or dose of compounds or immunosuppressants, for a therapy of an autoimmune disease, and/or in therapy monitoring of an autoimmune disease, and/or diagnostics including therapy-accompanying diagnostics, relating to a tumor disease, in prognostic assessment of prospects of success and/or in predicting a clinical outcome of a therapy of a tumor disease and/or in determining suitability and/or effectiveness and/or dose of compounds for a therapy of a tumor disease, and/or in therapy monitoring of a tumor disease, and/or diagnostics including therapy-accompanying diagnostics, relating to a vaccination in prognostic assessment of prospects of success and/or in predicting a clinical outcome of a vaccination and/or in therapy monitoring of a vaccination, comprising using a messenger-substance-containing supernatant or cell-containing sediment, wherein the messenger-substance-containing supernatant or the cell-containing sediment is produced or producible by a method comprising: a) preparing a mixture comprising whole blood, a nutrient medium for whole blood, and at least one activator that activates blood cells or immune cells, in addition to any activators present in the whole blood, and/or at least one inhibitor that inhibits blood cells or immune cells, in addition to any inhibitors present in the whole blood, and/or at least one modulator that modulates blood cells or immune cells, in addition to any modulators present in the whole blood, b) incubating the prepared mixture, and c) separating a messenger-substance-containing supernatant formed through incubation of the prepared mixture from a cell-containing sediment formed through incubation of the prepared mixture, or a messenger-substance-containing supernatant formed through centrifugation of the incubated prepared mixture from a cell-containing sediment formed through centrifugation of the incubated prepared mixture.

32. An in vitro method for diagnostics including therapy-accompanying diagnostics, relating to a transplant in prognostic assessment of prospects of success and/or in predicting a clinical outcome of a transplant and/or in determining suitability and/or effectiveness and/or dose of compounds or immunosuppressants, for a transplant, and/or in therapy monitoring of a transplant, and/or diagnostics including therapy-accompanying diagnostics, relating to an autoimmune disease characterizing the stage of an autoimmune disease and/or in prognostic assessment of prospects of success and/or in predicting a clinical outcome of a therapy of an autoimmune disease and/or in determining suitability and/or effectiveness and/or dose of compounds or immunosuppressants, for a therapy of an autoimmune disease, and/or in therapy monitoring of an autoimmune disease, and/or diagnostics including therapy-accompanying diagnostics, relating to a tumor disease in prognostic assessment of prospects of success and/or in predicting a clinical outcome of a therapy of a tumor disease and/or in determining suitability and/or effectiveness and/or dose of compounds for a therapy of a tumor disease, and/or in therapy monitoring of a tumor disease, and/or diagnostics including therapy-accompanying diagnostics, relating to a vaccination in prognostic assessment of prospects of success and/or in predicting a clinical outcome of a vaccination and/or in therapy monitoring of a vaccination, comprising using a combination or a mixture, comprising whole blood, a nutrient medium for whole blood, and at least one activator that activates blood cells or immune cells, in addition to any activators present in the whole blood, and/or at least one inhibitor that inhibits blood cells or immune cells, in addition to any inhibitors present in the whole blood, and/or at least one modulator that modulates blood cells or immune cells, in addition to any modulators present in the whole blood.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0198] The following is shown schematically in the drawings:

[0199] FIG. 1: Dose-response curves of four TLR agonists (stimulated release of IL-6) using a kit; activating active substances developed for tumor therapy (TLR agonists); activation of immune cells from six healthy donors quantified on the basis of release of IL-6 (characteristic cytokine for macrophage activation).

[0200] FIG. 2: Result of titration of a checkpoint inhibitor (CPI-1, developed for adjuvant immune cell stimulation in tumor patients) in SEB-stimulated cultures (release of IL-2/four healthy donors) using a kit; IL-2 is here specific for T-cell activation.

[0201] FIG. 3a: Dose-response curves of two immunosuppressants developed for autoimmune diseases (donor A) using a kit.

[0202] FIG. 3b: Dose-response curves of two immunosuppressants developed for autoimmune diseases (donor B) using a kit. Suppressant active substances developed for autoimmune diseases were used. To investigate the inhibitory effect on immune cells, these were stimulated using a TLR agonist. The results of two healthy donors quantified on the basis of release of various inflammation-relevant cytokines are shown.

[0203] FIG. 4a: Influence of various additives on SEB-induced T-cell activity (with release of IFNg) using a kit.

[0204] FIG. 4b: Influence of various additives on SEB-induced T-cell activity (release of IL-2) using a kit.

[0205] FIG. 4c: Influence of various additives on SEB-induced T-cell activity (release of IL-4) using a kit.

[0206] FIG. 4d: Influence of various additives on SEB-induced T-cell activity (release of IL-5) using a kit.

[0207] FIG. 4e: Influence of various additives on SEB-induced T-cell activity (release of IL-10) using a kit.

[0208] FIGS. 4a-e are exemplary depictions of five cytokines measured in the culture supernatants of whole blood cultures by multiplex analysis after different culture times. Stimulation of T-cells by SEB (=“superantigen”) was however carried out with addition of LPS for activation of monocytes/macrophages or of co-stimulus 1. Superantigens activate T cells like normal antigens, but activate a higher number of T cells at the same time than with standard antigens. FIG. 5: Antigen-specifically induced release of IFNg by the influenza vaccine Fluzone using a kit.

[0209] FIG. 5 shows clearly that the release of the messenger substance interferon gamma (IFNg), which is characteristic for T-cell activation, took place almost independently of the dose. FIG. 6: Antigen-specifically induced release of IL-2 by the influenza vaccine Fluzone using a kit.

[0210] FIG. 6 showing clearly that the release of the messenger substance interleukin-2 (IL-2), which is characteristic for T-cell activation, took place almost independently of the dose.

[0211] FIG. 7: A kit after incubation of whole blood, nutrient medium, and the at least one activator for activating blood cells, more particularly immune cells, and/or the at least one inhibitor for inhibiting blood cells, more particularly immune cells, and/or the at least one modulator for modulating blood cells, more particularly immune cells.

DETAILED DESCRIPTION

[0212] FIG. 7 shows a kit 100.

[0213] The kit 100 comprises a blood collection container in the form of a syringe 1, a nutrient medium (not shown), and at least one activator that activates blood cells, more particularly immune cells (not shown) and/or at least one inhibitor that inhibits blood cells, more particularly immune cells (not shown) and/or at least one modulator that modulates blood cells, more particularly immune cells (not shown). With regard to suitable activators/inhibitors/modulators, reference is made in full to the prior description.

[0214] The syringe 1 has a hollow cylinder 2 in which a syringe plunger 3 is arranged such that it can be displaced with the aid of a plunger rod 4 to create a seal. The interior of the hollow cylinder 2 is preferably sterile. The hollow cylinder 2 has a taper 5 in the region of the plunger rod 4 that guides the plunger rod 4. The plunger rod 4 has a defined break point 6 close to the syringe plunger 3 that comes to rest in the region of the taper 5 when the plunger is pulled backwards and more particularly enables the plunger rod 4 to be snapped off (easily) when the syringe plunger 3 is guided backwards. The end 7 of the hollow cylinder 2 pointing away from the plunger rod 4 is open over the entire cross section and can be closed with a closure cap, more particularly a screw cap 8. The closure cap 8 preferably has an attachment socket 9 for a cannula or syringe needle 10. The cannula 10 can preferably be locked in place with a bayonet closure. The attachment socket 9 preferably has on the inside a pierceable seal 11, more particularly made of rubber, that is pierced when the cannula 10 is attached and forms a seal again when the cannula 10 is removed. The pierceable seal 11 particularly advantageously makes it possible to ensure the sterility of the interior of the hollow cylinder 2 before, during and after collection of blood.

[0215] The kit 100 also includes a separating component, more particularly a valve plunger 12. The separating component 12 is preferably associated with the syringe 1. In the unused state, the separating component 12 is preferably arranged outside the syringe 1. A pusher (not shown in the drawing) is advantageously associated with the separating component 12. After removing the closure cap 8, the pusher can be used to insert the separating component 12 into the syringe 1. Insertion takes place at the end of incubation of a whole blood sample contained in the syringe 1 that has separated into a supernatant 13 and a sediment 14 containing deposited blood cells. For permanent separation of supernatant 13 and sediment 14, the separating component 12 is pushed in as far as the interface between supernatant 13 and sediment 14, wherein the supernatant 13 flows through a valve 15 of the separating component 12 until the separation process is completed on reaching the interface between supernatant 13 and sediment 14. On completion of the pressing-in process and retraction of the pusher of the separating component 12, the valve 15 preferably closes automatically. The syringe plunger 3 can then be tightly resealed by attaching the closure cap 8, and the syringe plunger 3 used as a storage, cooling, and transport container, which means that the blood or separated parts thereof can be stored between blood collection and the actual performance of the detection and/or determination of the cytokines present in the supernatant 13 without any need to be decanted. It is, for example, possible to use, as syringe 1, a syringe commercially available under the name “Monovette” from Sarstedt and, as separating component 12, a valve plunger available under the name “Seraplas” from Sarstedt.

Examples Section

Performance of Whole Blood Cultures (General)

[0216] Syringes of the kit were frozen immediately after being filled with the appropriate substances dissolved in proprietary nutrient medium and stored at −20° C. until use. After thawing and inverting, a puncture cannula/butterfly or similar hollow needle system was used with the aid of an adapter to puncture a vein of a blood donor/patient. This was followed by the collection of 1 ml of blood by carefully drawing it up directly into the syringe. The contents (blood and nutrient medium with or without additives: activators, inhibitors, modulators or the like) were immediately mixed several times by inverting the syringes and incubated at 37° C. in a block thermostat for the desired culture time of e.g., 24 or 48 hours.

[0217] After removing the syringes from the block thermostat at the end of the culture time, the reaction was stopped by carefully inserting a separating valve (“Seraplas filter”), which was accompanied by the permanent separation of the sedimented cells from the culture supernatant.

[0218] Both the cells and the culture supernatant could then be sent for appropriate methods of detection for determination of the appropriate endpoints. The cells could be investigated to detect intracellular factors, for example, by flow cytometry or RNA analysis, for example, by PCR. For cellular investigations, it could be necessary first to separate the culture supernatant from the cells and then to add to the cells appropriate solutions for stabilization, fixation and/or preservation. The messenger substances released into the culture supernatant could be quantified, for example, by chromatographic methods (for example, HPLC) or by immunoassay such as ELISA, Simoa, Luminex, or other methods. If the cells or the culture supernatant were not analyzed immediately, they could be stored in the syringes at −20° C. until further investigation. Performance of whole blood cultures—Description of the method for the experiment shown in FIG. 1

Tumor Therapy: Pre-Screening and Therapy Monitoring

[0219] The proprietary nutrient medium for these whole blood cultures was supplemented with the TLR activators “compound A,” “compound B,” “VTX” and gardiquimod that were developed for the activation of macrophages in the course of tumor therapies. In addition, a combination of LPS (0.1 μg/ml)+SEB (0.4 μg/ml) was used as a positive control. The concentration of the four TLR activators was in a serial 1:5 dilution between 0.00064 and 2 μM final concentration. The correspondingly freshly loaded syringes were stored at −20° C. until use.

[0220] After the syringes had been thawed, they were after venipuncture each filled with 1 ml of blood from the blood donor and incubated as described above at 37° C. in a block thermostat for the desired culture time of 24 hours.

[0221] At the end of the incubation, the separating valves were inserted and the syringes stored at −20° C. until testing for cytokine release. IL-6, which is a characteristic cytokine for macrophage activation, was determined as the endpoint by ELISA.

[0222] The data presented in FIG. 1 show clear activation of macrophages, the expression of which depended on both the activators and the dosage thereof.

Performance of Whole Blood Cultures—Description of the Method for the Experiment Shown in FIG. 2

Tumor Therapy: Pre-Screening and Therapy Monitoring

[0223] The proprietary nutrient medium for these whole blood cultures was supplemented with a checkpoint inhibitor developed for the activation of T cells in the course of tumor therapies.

[0224] (CPI-1). The activity of such active substances can be demonstrated only in the presence of a T-cell-specific basal stimulus. The superantigen SEB was chosen for this purpose and was also used as a positive control. The correspondingly freshly loaded syringes were stored at −20° C. until use.

[0225] After the syringes had been thawed, they were after venipuncture each filled with 1 ml of blood from the blood donor and incubated as described above at 37° C. in a block thermostat for the desired culture time of 48 h.

[0226] At the end of the incubation, the separating valves were inserted and the tubes stored at −20° C. until testing for cytokine release. IL-2, which is a characteristic cytokine for T-lymphocyte activation, was determined as the endpoint by ELISA.

[0227] The data presented in FIG. 2 show clear activation of T-lymphocytes, which exhibits a clear improvement in its expression. The strength of the activating effect is moreover individual-specific, as can be seen from the different courses in the cultures of the individual donors. Performance of whole blood cultures—Description of the method for the experiment shown in FIGS. 3a and 3b

Autoimmune Diseases/Organ Transplantation:

Individual Pre-Testing of the Suitability of Particular Preparations and Therapy Monitoring

[0228] The proprietary nutrient medium for these whole blood cultures was supplemented with the TLR7/TLR8 ligand R848, to induce a relatively broad immune cell response. In addition, two inhibitors newly developed for the treatment of autoimmune diseases (“compound A,” “compound B”) were each added in a dilution series. R848 served as a positive control. The correspondingly freshly loaded syringes were stored at −20° C. until use.

[0229] After the syringes had been thawed, they were after venipuncture each filled with 1 ml of blood from the blood donor and incubated as described above at 37° C. in a block thermostat for the desired culture time of 24 hours.

[0230] At the end of the incubation, the separating valves were inserted and the tubes stored at −20° C. until testing for cytokine release. A number of different cytokines that are characteristic messenger substances for broader inflammatory responses were determined as the endpoint by Luminex.

[0231] The data shown in FIGS. 3a-b reveal very strong, dose-dependent inhibition of various immune cells, with the two compounds showing no appreciable differences. The dose-response curves of the two donors tested were likewise very similar.

Performance of Whole Blood Cultures—Description of the Method for the Experiment Shown in FIGS. 4a-e

Antigen-Specific Responses (Autoimmune Diseases, Vaccinations):

[0232] The proprietary nutrient medium for these whole blood cultures was supplemented with the T-cell-specific superantigen SEB, to induce a response in these cells that corresponds to that of normal antigens except that, rather than activating just a few T-lymphocytes, it stimulates the majority of these cells. In addition, two reagents were used (LPS and “Co-Stim. 1,” the latter having been developed in house) to test the extent to which antigen-induced activations can be enhanced. This will be of particular importance when normal antigens are used that, although inducing the same activation pathways in T cells, are able to achieve a response in just a few of these lymphocytes. To control the signal-to-noise ratio of, for example, cytokine assays, it is important in such situations that each individual activatable cell releases as much messenger substance as possible. SEB was titrated to examine whether the co-stimulating agents have different positive effects with different antigen signal strengths. The correspondingly freshly loaded syringes were stored at −20° C. until use.

[0233] After the syringes had been thawed, they were after venipuncture each filled with 1 ml of blood from the blood donor and incubated as described above at 37° C. in a block thermostat for the desired culture time of 24 h and 48 h.

[0234] At the end of the incubation, the separating valves were inserted and the tubes stored at −20° C. until testing for cytokine release. Five cytokines (IFN-gamma, IL-2, IL-4, IL-5, and IL-10) considered to be relevant in T-cell activations were determined as the endpoint by Luminex.

[0235] The data shown in FIGS. 4a-e exhibit clear differences between donors in the overall strength of their immune cell response to SEB stimulation. Clear dose dependencies were in addition observed and this run too demonstrated that T cells benefit from a longer incubation time (48 h). The two co-stimulatory additives, LPS and Co-Stim 1, elicited moderate and also characteristic changes in the cytokine levels of these cultures, which means that further use of these substances may—depending on the situation—be useful. Co-Stim 1 was noteworthy in that it showed clearly enhancing effects for IL-2 and IL-5: IL-2 in respect of activation of Th1 cells and IL-5 in respect of activation of Th2 cells.

Performance of Whole Blood Cultures—Description of the Method for the Experiment in Tables 1-4

[0236] The proprietary nutrient medium for these whole blood cultures was supplemented with individual or combined “recall antigens” (tetanus toxoid, TeTo, or peptides from various prominent viruses such as cytomegalovirus, Epstein Barr, and influenza, CEF) on the one hand, and also with various co-stimulants (Co-Stim. 1, Co-Stim. 2, CM 2, CM 3, and Stim. 2) in various combinations. Whereas the CEF peptides were used in different concentrations, the very good response obtained with tetanus toxoid meant that this could be used with just one final concentration (2 μg/mL). Syringes filled just with medium served as negative controls (Neg. Co). Immediately after filling, the syringes were frozen and stored at −20° C. until use.

[0237] After the syringes had been thawed, they were after venipuncture each filled with 1 ml of blood from the blood donor and incubated as described above at 37° C. in a block thermostat for 48 hours.

[0238] At the end of the incubation, the separating valves were inserted and the syringes stored at −20° C. until testing for cytokine release. As the endpoints, either the cytokines IFN-gamma and IL-2, which are considered to be relevant in T-cell activation, were determined (by Simoa/Quanterix), or a spectrum of messenger substances was quantified in a multiplex assay (Luminex; see Table 4).

[0239] The data presented in Tables 1-4 below clearly demonstrate, on the basis of the reliably measurable tetanus-toxoid-induced or CEF-peptide-induced release of characteristic cytokines, the highly specific detection of functional antigen-specific activation of T cells using the kit.

TABLE-US-00001 TABLE 1 IFNg IL-2 CEF Conc Conc [μg/ml] [pg/mL] SI [pg/mL] SI CM 2 Donor A 0  365  0.126 1  1022  2.80  1.76 14.1  2  1515  4.15  3.07 24.5  4  1870  5.12  4.53 36.1  Donor B  8189  0.272 1 17986  2.20  6.85 24.1  2 23106  2.82  8.38 30.9  4 26953  3.29  8.25 30.4  Stim. 2 Donor A 38435  0.109 1 29038  0.755  1.28 11.8  2 28511  0.742  1.27 11.6  4 22252  0.579  1.46 13.4  Donor B 42685  0.447 1 38192  0.895  0.533  1.19 2 33448  0.784  0.672  1.50 4 31714  0.743  0.388  0.868
Cytokine release in whole blood culture supernatants of differently co-activated whole blood cultures under stimulation with CEF peptides (mixture of viral antigens that triggers an immunological memory response in approx. 90% of all blood donors).

[0240] The supernatants from 48 h cultures were analyzed using highly sensitive Simoa assays. CM=cytokine mix

TABLE-US-00002 TABLE 2 IFNg IL-2 Conc Conc [pg/mL] mean [pg/mL] mean Donor A Neg. Co. <0.10 <0.10 <0.116 <0.116 <0.10 <0.116 TeTo 9.18 9.61 32.9 33.7 10.0 34.4 TeTo + 7.62 7.64 26.2 26.4 Co-Stim. 1 7.66 26.6 Donor B Neg. Co. <0.10 <0.10 <0.116 <0.116 <0.10 <0.116 TeTo 8.87 9.90 32.6 34.6 10.9 36.6 TeTo + 8.13 8.58 27.0 25.9 Co-Stim. 1 9.02 24.8 Donor C Neg. Co. 0.309 0.427 <0.116 <0.116 0.545 <0.116 TeTo 129 137 142 146 145 149 TeTo + 77.sub..9 86.3 86.4 98.0 Co-Stim. 1 94.7 110
Cytokine release in the supernatants of tetanus-toxoid-activated whole blood cultures. The supernatants from 48 h cultures were analyzed using the Simoa assay.

TABLE-US-00003 TABLE 3 IFNg IL-2 Conc Conc [pg/mL] SI [pg/mL] SI Donor A Medium 0.333 0.023 TeTo 1069 3208 NQ nb CM 3 Medium 309 0.131 TeTo 7105 23.0 212 1617 CM 2 Medium 347 0.130 TeTo 7833 22.6 153 1176 Stim. 2 Medium 33400 0.130 TeTo 148332 4.44 43.0 331 Donor B Medium 0.732 0.039 TeTo 11.3 15.4 12.1 314 CM 3 Medium 4669 0.320 TeTo 41010 8.78 6.70 20.9 CM 2 Medium 5993 0.178 TeTo 46177 7.71 4.93 27.7 Stim. 2 Medium 50768 0.161 TeTo 252303 4.97 5.50 34.2
Cytokine release in differently co-activated whole blood cultures under stimulation with tetanus toxoid. In this example, subjects recently vaccinated against tetanus were used as blood donors (hence the high IFNg levels). The supernatants from 48 h cultures were analyzed using the Simoa assay. For comparison of the various co-activators, the stimulation indices (SI) were additionally calculated, for which the ratio of the value for the unstimulated culture (“medium”) to that of the respective stimulated (TeTo) culture was calculated. nd=not determinable

TABLE-US-00004 TABLE 4 Analy GM-CSF IFN-g IL-10 IL-18 IL-2 IL-3 IL-4 IL-5 Units pg/mL pg/mL pg/mL pg/mL pg/mL ng/mL pg/mL pg/mL LLOQ 29.4 2.28 4.24 16.7 12.2 0.00186 18.9 7.28 Donor A Medium Med. <LOW> <LOW> <LOW> 21.9 4.35 <LOW> <LOW> <LOW> TeTo 144 204 8.24 37.5 580 0.0185 197 58.8 CM 3 Med. <LOW> 74.2 35.5 29.8 8.74 0.00248 80.4 <LOW> TeTo 425 1750 187 43.1 369 0.0295 151 25.4 CM 2 Med. <LOW> 75.2 31.4 33.7 6.62 0.00339 77.4 <LOW> TeTo 426 2020 203 41.3 310 0.0304 189 51.4 Co- Med. 157 6450 979 69.1 40.5 0.0199 79.4 <LOW> Stim. 2 TeTo 939 28800 430 212 166 0.0399 181 8.57 Donor B Medium Med. <LOW> 0.749 2.27 78.2 <LOW> <LOW> <LOW> <LOW> TeTo <LOW> 3.55 8.47 83.7 27.8 0.00099 24.8 <LOW> CM 3 Med. <LOW> 1190 3.63 92.7 <LOW> 0.00309 41.4 <LOW> TeTo <LOW> 10600 6.85 85.5 32 0.0131 82.5 <LOW> CM 2 Med. <LOW> 1540 2.5 78.2 6.62 0.00445 47.6 <LOW> TeTo <LOW> 11400 6.85 90.9 21.4 0.0156 90.6 <LOW> Co- Med. 14.6 14200 1580 125 53.1 0.0268 76.3 <LOW> Stim. 2 TeTo 62.8 52900 2230 249 93.9 0.0418 148 <LOW> IL-6 IL-7 IL-8 MCP-1 MIP-1 a MIP-1 a TNF-a TNF-b pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL 3.27 10.7 3.88 26.9 17.3 28.3 10.4 5.76 Donor A Medium <LOW> <LOW> 91.1 111 7.11 133 <LOW> <LOW> 228 190 >5700 >25680 2450 6200 745 50.1 CM 3 111 85 >5700 >25680 194 >11550 6180 7.93 13500 75.5 >228000 139000 24200 260000 9840 71.6 CM 2 109 92 >5700 >25680 207 >11550 6070 6.44 16200 79.6 >228000 158000 26600 269000 9990 75.7 Co- 8480 69.9 6260 29000 15800 404000 9340 73.2 Stim. 2 9820 71.3 7390 4960 25400 325000 22100 159 Donor B Medium 1.16 <LOW> 51.5 126 7.11 429 <LOW> <LOW> 21.2 31.8 833 5420 33 7050 28.3 2.15 CM 3 14.4 37.3 1860 18100 186 29400 3840 6.44 302 110 15400 103000 1380 111000 5280 41.8 CM 2 14.1 36.4 1760 21300 219 33100 4760 7.93 328 86.3 11900 90400 1490 107000 5990 50.9 Co- 11800 55.6 5810 38300 24600 >462000 8210 69.9 Stim. 2 22100 81 10000 17600 57300 >462000 42800 135
Cytokine release in the supernatants of differently co-activated whole blood cultures under stimulation with tetanus toxoid (same test run as in Tab. 3). In this example, the supernatants from 48 h cultures were however determined by a multiplex cytokine analysis. CM=cytokine mix

[0241] Table 5 below shows the result of a whole blood analysis from two donors using a kit. The whole blood from the two donors was then analyzed to see whether the immune system specifically recognizes tetanus toxoid, the standard tetanus vaccine. The release of the messenger substances IFN-gamma (IFNg) and interleukin-2 (IL-2), which are regarded as typical for T-cell activation, was examined. The extent to which the signal-to-noise ratio can be improved through co-stimulating additives (such as cytokines) was at the same time examined.

TABLE-US-00005 TABLE 5 IFNg IL-2 Spender Costim Antigen [pg/mL] [pg/mL] A ohne — 0.333 0.023 TetTox 1069 NQ A — 309 0.131 TetTox 7105 212 B — 347 0.130 TetTox 7833 153 C — 33400 0.130 TetTox 148332 43.0 B ohne — 0.732 0.039 TetTox 11.3 12.1 A — 4669 0.320 TetTox 41010 6.70 B — 5993 0.178 TetTox 46177 4.93 C — 50768 0.161 TetTox 252303 5.50
Result of whole blood analysis of two donors (TetTox=tetanus toxoid)