Isolation and identification of cross-reactive t cells

Abstract

Cross-reactive T cells recognizing both MBP 93-105 and HHV-6 1-13 peptides represent a significant subset of T cells with some degree of TCR degeneracy. It appears that the recognition of the cross-reactive T cells has a less stringent requirement for the flanking residues of the two peptides. In contrast, these flanking residues are critical for the T cell recognition of mono-specific T cells. The association between HHV-6 and autoreactive immune responses to MBP indicates that cross-reactive T cells, peptides from the V-D-J region of the T cell receptor from autoreactive T cells, and antiviral agents may prevent or treat MS.

1 . A method for isolating one or more T cells that cross-react with a self-antigen and a foreign antigen comprising: 
 (a) incubating a sample comprising T cells with an antigen that comprises an epitope present in the self-antigen and the foreign antigen, and wherein said sample optionally comprises one or more autoantigens; and    (b) isolating the one or more cross-reactive T cells by cloning or direct expansion,    wherein the self-antigen is myelin basic protein, and wherein the foreign antigen is human herpesvirus-6 U24.    
     
     
         2 . The method of  claim 1 , wherein the self-antigen comprises a sequence of residues 96-102 of myelin basic protein or residues 93-105 of myelin basic protein, and wherein the foreign antigen comprises a sequence of residues 4-10 of human herpesvirus-6 U24, residues 1-13 of human herpesvirus-6 U24.  
     
     
         3 . The method of  claim 1 , wherein the autoantigen is selected from the group consisting of myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, collagen type II peptides, heat shock protein, MAGE, PSA, CA125, GAD protein, and tumor associated antigen.  
     
     
         4 . The method of  claim 1 , wherein the autoantigen comprises an immunodominant epitope of a member selected from the group consisting of myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, collagen type II peptides, heat shock protein, MAGE, PSA, CA125, GAD protein, and tumor associated antigen.  
     
     
         5 . The method of  claim 4 , wherein said immunodominant epitope is selected from the group consisting of residues 83-99 of myelin basic protein and residues 151-170 of myelin basic protein.  
     
     
         6 . The method of  claim 1 , further comprising 
 selecting one or more T cells that express one or more first markers selected from the group consisting of CD69, CD4, CD25, CD36 and HLADR and one or more second markers selected from the group consisting of IL-2, IFNγ, TNFα, IL5, IL-10 and IL-13.    
     
     
         7 . The method of  claim 6 , wherein the self-antigen comprises a sequence of residues 96-102 of myelin basic protein or residues 93-105 of myelin basic protein and wherein the foreign antigen comprises a sequence of residues 4-10 of human herpesvirus-6 U24, residues 1-13 of human herpesvirus-6 U24.  
     
     
         8 . The method of  claim 6 , wherein the autoantigen is selected from the group consisting of myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, collagen type II peptides, heat shock protein, MAGE, PSA, CA125, GAD protein, and tumor associated antigen.  
     
     
         9 . (canceled)  
     
     
         10 . (canceled)  
     
     
         11 . The method of  claim 7 , wherein the cells expressing said first and said second markers are selected using antibodies to said first and second markers respectively, or optionally a bi-specific antibody which binds both first and second markers in combination with an antibody which binds said second marker.  
     
     
         12 . The method of  claim 11 , wherein one or more of said antibodies is fluorescently labeled and wherein said T cell is selected by fluorescent activated cell sorting.  
     
     
         13 . (canceled)  
     
     
         14 . The method of  claim 11 , wherein said first antibody is conjugated to a magnetic microbead and wherein said T cell is selected by magnetic activated cell sorting.  
     
     
         15 . (canceled)  
     
     
         16 . A composition comprising one or more T cells that cross-react with a self antigen and a foreign antigen, 
 wherein the self-antigen is myelin basic protein,    wherein the foreign antigen is human herpesvirus-6 U24    and wherein the cross-reacting T cells are enriched with respect to other T cells that react with the self-antigen.    
     
     
         17 . The composition of  claim 16 , wherein the self-antigen comprises a sequence of residues 96-102 of myelin basic protein or residues 93-105 of myelin basic protein, and wherein the foreign antigen comprises a sequence of residues 4-10 of human herpesvirus-6 U24, residues 1-13 of human herpesvirus-6 U24.  
     
     
         18 . A method for quantifying the number of T cells in a sample that cross-react with an self-antigen and a foreign antigen comprising: 
 (a) incubating a sample comprising T cells with an antigen that comprises an epitope present in the self-antigen and the foreign antigen, and wherein said sample optionally comprises one or more autoantigens;    (b) selecting one or more T cells that express one or more first markers selected from the group consisting of CD69, CD4, CD25, CD36 and HLADR and one or more second markers selected from the group consisting of IL-2, IFNγ, TNFα, IL5, IL-10 and IL-13; and    (c) determining the number of T cells selected by step (b).    wherein the self-antigen is myelin basic protein, and wherein the foreign antigen is human herpesvirus-6 U24.    
     
     
         19 . A method for diagnosing an autoimmune disease in a patient, comprising: 
 (a) quantifying the number of cross-reactive T cells according to the method of  claim 18;  and    (b) comparing the number of cross-reactive T cells and optionally other autoreactive T cells to a control.    
     
     
         20 . A method for monitoring an autoimmune disease in a patient, comprising: 
 (a) quantifying the number of cross-reactive T cells according to the method of  claim 18;  and    (b) comparing the number of cross-reactive T cells and optionally other autoreactive T cells to a control.    (c)    
     
     
         21 . A method for treating an autoimmune disease in a patient, comprising administering the composition of  claim 16  to a patient in need thereof.  
     
     
         22 . A method for producing the composition of  claim 16 , comprising: 
 (a) incubating a sample derived from said patient comprising T cells with an antigen that comprises an epitope present in a self-antigen and a foreign antigen, wherein said sample optionally comprises one or more autoantigens;    (b) selecting one or more T cells that express one or more first markers selected from the group consisting of CD69, CD4, CD25, CD36 and HLADR and one or more second markers selected from the group consisting of IL-2, IFNγ, TNFα, IL5, IL-10 and IL-13; and    (c) inactivating the T cells selected by step (b),    wherein the self-antigen is myelin basic protein, and wherein the foreign antigen is human herpesvirus-6 U24.    
     
     
         23 . The method of  claim 22  further comprising expanding the number of T cells selected in step (b).  
     
     
         24 . (canceled)  
     
     
         25 . A method for isolating a nucleic acid encoding a T cell receptor, or a portion thereof, wherein said T cell receptor is specific for a self-antigen and a foreign antigen, comprising: 
 (a) isolating one or more T cells according to the method of  claim 6;  and    (b) amplifying the nucleic acid encoding said T cell receptor from a T cell isolated by step (a) using at least one first primer specific for the variable region of the T cell receptor gene and a second primer specific for the constant region of the T cell receptor gene,    
     
     
         26 . (canceled)  
     
     
         27 . (canceled)