Multiparametric method for assessing immune system status

Abstract

The invention provides a multiparametric method of assessing the reaction of a patient's immune system to a test subject. The invention compares a patient sample reacted with a test sample and a third party sample and combines the assessments of the multiple parameters to correlate the test reaction with a clinical event.

1 .- 53 . (canceled) 
     
     
         54 . A multiparametric method of assessing the reaction of a patient's immune system to a test stimulant, the method comprising:
 a) obtaining a first sample and a second sample from the patient, wherein the first sample and the second sample comprise lymphocytes, peripheral blood mononuclear cells (PBMCs), or a mixture of lymphocytes and peripheral blood mononuclear cells, and optionally labeling the cells with a first marker or first set of markers,   b) subjecting the patient's first sample to a stimulant in a first vessel under conditions sufficient for the patient's cells to react with the stimulant,   c) placing the patient's second sample into a second vessel under conditions similar to those of the first vessel, measuring multiple parameters of the reaction in the first vessel to obtain a test measurement for each parameter,   d) measuring multiple parameters of the reaction in the second vessel to obtain a control measurement for each parameter,   e) comparing each test measurement of a parameter to each control measurement of the same parameter, whereby the test measurement is expressed as a fraction of the control measurement to provide an assessment of the parameter, and   f) combining the assessments of the multiple parameters to correlate the test reaction with a clinical event.   
     
     
         55 . The method of  claim 54  wherein the stimulant is an antigen, autoantigen, viral peptide, bacterial peptide, antigen-attached to tetramer, or another form infectious agent. 
     
     
         56 . The method of  claim 55  wherein the clinical event is the patient's allergy status, autoimmune status, or status of an infectious disease. 
     
     
         57 . A method of assaying the immune response of a test subject to a viral antigen, the method comprising
 a) obtaining a sample from the test subject, wherein the sample comprises CD8+ cells,   b) labeling a viral antigen with dye conjugated MHC-tetramer,   c) exposing CD8+ cells within the sample to the labeled viral antigen,   d) assaying for cells which are both CD8+ and bound to the viral antigen, whereby the presence of CD8+ cells that bind viral antigen are indicative that the test subject has developed an immune response to the viral antigen.   
     
     
         58 . (canceled) 
     
     
         59 . (canceled) 
     
     
         60 . The method of  claim 54 , wherein a first marker is CD154 or CD28. 
     
     
         61 . The method of  54 , wherein the first set of markers comprise CD154 and CD28. 
     
     
         62 . The method of  claim 54 , wherein the first set of markers comprise the markers selected from the group consisting of 7-AAD, Annexin V, CD 154, active caspases, CCR4, CCR5, CD11c, CD 123, CD14, CD16/56, CD19, CD25, CD27, CD28, CD3, CD4, CD45RA, CD45RO, CD54, CD62 ligand, CD69, CD71, CD8, CD86, CTLA4, HLA-DR, IFN-gamma, IL10, IL-12, IL-2, ILT3, TGF-beta, and TNF-alpha. 
     
     
         63 . The method of  54 , wherein the multiple parameters of the reaction in the first vessel and of the reaction in the second vessel are measured using a flow cytometer. 
     
     
         64 . The method of  claim 54 , wherein a clinical event is monitoring the severity of bacterial or viral infections. 
     
     
         65 . The method of  claim 54 , wherein a clinical event is monitoring the response of the patient to an antiviral or antibacterial agent. 
     
     
         66 . The method of  claim 54 , wherein at least 7 parameters are measured in each vessel. 
     
     
         67 . The method of  claim 54 , wherein at least one of the multiple parameters comprises at least one physical characteristic, such as cell size and complexity or degree of aggregation of the cells. 
     
     
         68 . The method of  claim 54 , wherein at least one of the multiple parameters comprises cell type or cell function. 
     
     
         69 . The method of  57 , wherein the viral antigen is Epstein-Barr, adenovirus, and/or cytomegalovirus. 
     
     
         70 . The method of  claim 68 , wherein the cell function state is death, apoptosis, proliferation, cytokine production, or stage in the cell cycle, degree of activity (e.g., highly activated, activated, or inactive), whether the cells have memory or are naive and whether the memory is effector memory or central memory. 
     
     
         71 . The method of  claim 71 , wherein the cell function is the production of one or more pro-inflammatory cytokines. 
     
     
         72 . The method of  claim 72 , wherein the pro-inflammatory cytokine is interferon gamma, tumor necrosis factor alpha, interleukin-2, interleukin-12, or a mixture of two or more these. 
     
     
         73 . The method of  claim 70 , wherein a cell function is the production of one or more suppressive cytokines. 
     
     
         74 . The method of  claim 73 , wherein the suppressive cytokine is interleukin-10, scurfin, transforming growth factor beta, CTLA4, or a mixture of two or more of these. 
     
     
         75 . The method of  claim 54 , wherein at least one of the multiple parameters comprises the presence, absence, amount, or relative expression of a predetermined protein, such as a cytokine, such as interferon gamma, tumor necrosis factor alpha,interleukin-2, interleukin-10, interleukin-12, scurfin, transforming growth factor beta, CTLA4, or a mixture of two or more these.