Measuring frequency of pathogen-specific t cells in peripheral blood

Abstract

A method for detecting and quantifying of the frequency of T cells to multiple antigenic peptide epitopes comprising: measuring intracellular Ca2+ signaling in individual T cells that are labeled with Ca2+ sensitive fluorophore; wherein said T cells are placed on the glass bottom of a well-covered with antibodies or other capturing proteins specific for non-stimulatory T cells' surface receptors and wherein a peptide antigens are injected into the well and the peptide binds to MHC molecules on the T-cell surface, wherein an increase in the intracellular concentration of Ca2+ in responding T cells leads to rise in intracellular fluorescence that is detected by fluorescent microscope and wherein the response rate of said detected fluorescence can be utilized to determine the quantity of responding T cells and the efficiency of said cells.

1 - 6 . (canceled) 
     
     
         7 . A method to determine the frequency and functional activity of antigen-specific CD8 T cells from human PBMC through an assay based on measurement of T-cell intracellular Ca2+ signaling induced in response to antigen recognition by T-cell receptor comprising:
 immobilizing freshly isolated CD8 T cells from human PBMC either intact or labeled with Ca2+ sensitive fluorophore on the glass bottom of a well covered with capturing antibody or a capturing protein that bind to non-stimulatory T-cell surface receptor;   adding to the wells unlabeled or fluorescently labeled peptide-MHC proteins assembled on nanoparticles or any other peptide-MHC oligomers to detect antigen-specific T cells and/or to induce Ca2+ signaling in the responding T cells;   wherein the recognition of unlabeled stimulatory pMHC by the specific T cells labeled with Calcium fluorophore leads to increase of intracellular Ca2+ level and fluorescence intensity in the responding T cells, which is then identified after the subtracting fluorescence intensity for every T cell measured before the addition of the stimulatory peptide-MHC oligomers. The binding of fluorescently labeled cognate pMHC to unlabeled T cells will identify both responding and non-responding T cells specific for the same peptide-MHC ligands; and   calculating a fraction of responding T cells with the specificity of interest.   
     
     
         8 . (canceled) 
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 7 , wherein the frequency and efficiency of pathogen-specific or tumor-specific T cells are compared to a control. 
     
     
         11 . The method of  claim 10 , wherein the control is derived from a sample of T cells from a health patient. 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 11  wherein the frequency and efficiency of the responding T cells are compared to a control; wherein an efficiency is reduced by more than one standard deviation of the control indicates a compromised immune system. 
     
     
         15 . The method of  claim 11  wherein the frequency and efficiency of the responding T cells are compared to a control; wherein an efficiency within one standard deviation of the control indicates a functioning immune system and an efficiency reduced by more than one standard deviation indicates a compromised immune system; administering to a patient with a comprised immune system a composition suitable for treating a suspected virulent. 
     
     
         16 . The method of  claim 15  wherein the suspected virulent is CMV. 
     
     
         17 . (canceled)