Comparative ligand mapping from MHC positive cells

Abstract

The present invention relates generally to a methodology for the isolation, purification and identification of peptide ligands presented by MHC positive cells. In particular, the methodology of the present invention relates to the isolation, purification and identification of these peptide ligands from soluble class I and class II MHC molecules which may be uninfected, infected, or tumorgenic. The methodology of the present invention broadly allows for these peptide ligands and their comcomittant source proteins thereof to be identified and used as markers for infected versus uninfected cells and/or tumorgenic versus nontumorgenic cells with said identification being useful for marking or targeting a cell for therapeutic treatment or priming the immune response against infected cells.

1 . A method for identifying at least one endogenously loaded peptide ligand that distinguishes an infected cell from an uninfected cell, comprising the steps of: 
 providing an uninfected cell line containing a construct that encodes an individual soluble MHC class I heavy chain molecule, the uninfected cell line being able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of MHC class I molecules;    infecting a portion of the uninfected cell line with at least one of a microorganism, a gene from a microorganism or a tumor gene, thereby providing an infected cell line;    culturing the uninfected cell line and the infected cell line under conditions which allow for expression of individual soluble MHC class I heavy chain molecules from the construct, such conditions also allowing for endogenous loading of a peptide ligand in the antigen binding groove of each individual soluble MHC class I molecule prior to secretion of the individual soluble MHC class I molecules from the cell;    isolating the secreted individual soluble MHC class I molecules having the endogenously loaded peptide ligands bound thereto from the uninfected cell line and the infected cell line;    separating the endogenously loaded peptide ligands from the individual soluble MHC class I molecules from the uninfected cell line and separating the endogenously loaded peptide ligands from the individual soluble MHC class I molecules from the infected cell line;    isolating the endogenously loaded peptide ligands from the uninfected cell line and the endogenously loaded peptide ligands from the infected cell line;    comparing the endogenously loaded peptide ligands isolated from the infected cell line to the endogenously loaded peptide ligands isolated from the uninfected cell line; and    identifying at least one endogenously loaded peptide ligand presented by the individual soluble MHC class I molecule on the infected cell line that is not presented by the individual soluble MHC class I molecule on the uninfected cell line.    
     
     
         2 . The method of  claim 1  wherein, in the step of providing an uninfected cell line containing a construct that encodes an individual soluble MHC class I heavy chain molecule, the construct further encodes a tag which is attached to the individual soluble MHC class I heavy chain molecule and aids in isolating the individual soluble MHC class I heavy chain molecule.  
     
     
         3 . The method of  claim 1  wherein, the uninfected cell line is class I negative.  
     
     
         4 . The method of  claim 1  wherein, the uninfected cell line expresses endogenous class I molecules.  
     
     
         5 . The method of  claim 1  wherein, in the step of providing an uninfected cell line containing a construct that encodes an individual soluble MHC class I heavy chain molecule, the uninfected cell line containing the construct that encodes the individual soluble MHC class I heavy chain molecule is produced by a method comprising the steps of: 
 obtaining genomic DNA or cDNA encoding at least one class I molecule;    identifying an allele encoding an individual class I molecule in the genomic DNA or cDNA;    PCR amplifying the allele encoding the individual class I molecule in a locus specific manner such that a PCR product produced therefrom encodes a truncated, soluble form of the individual class I molecule;    cloning the PCR product into an expression vector, thereby forming a construct that encodes the individual soluble MHC class I heavy chain molecule; and    transfecting the construct into an uninfected cell line.    
     
     
         6 . The method of  claim 5 , wherein the construct further encodes a tag which is attached to the soluble class I molecule and aids in isolating the individual soluble MHC class I molecule.  
     
     
         7 . The method of  claim 6 , wherein at least one of: 
 (a) the tag is selected from the group consisting of a HIS tail and a FLAG tail;    (b) the tag is encoded by a PCR primer utilized in the step of PCR amplifying an allele encoding the individual class I molecule; and    (c) the tag is encoded by the expression vector into which the PCR product is cloned.    
     
     
         8 . The method of  claim 1 , further comprising the step of identifying a source protein from which the at least one endogenously loaded peptide ligand presented by the individual soluble MHC class I molecule on the infected cell line and not presented by the individual soluble MHC class I molecule on the uninfected cell line is obtained.  
     
     
         9 . The method of  claim 1  wherein, in the step of identifying at least one endogenously loaded peptide ligand presented by the individual soluble MHC class I molecule on the infected cell line but not on the uninfected cell line, the at least one endogenously loaded peptide ligand is obtained from a protein encoded by a gene from a microorganism with which the cell line was infected to form the infected cell line.  
     
     
         10 . The method of  claim 1  wherein, in the step of identifying at least one endogenously loaded peptide ligand presented by the individual soluble MHC class I molecule on the infected cell line but not on the uninfected cell line, the at least one endogenously loaded peptide ligand is obtained from a protein encoded by the tumor gene with which the cell line was infected to form the infected cell line.  
     
     
         11 . The method of  claim 1  wherein, in the step of identifying at least one endogenously loaded peptide ligand presented by the individual soluble MHC class I molecule on the infected cell line but not on the uninfected cell line, the at least one endogenously loaded peptide ligand is obtained from a protein encoded by the uninfected cell line.  
     
     
         12 . The method of  claim 11 , wherein the protein encoded by the uninfected cell line from which the at least one endogenously loaded peptide ligand is obtained has increased expression in a tumor cell line.  
     
     
         13 . The method of  claim 1  wherein, in the step of infecting a portion of the uninfected cell line, the portion of the uninfected cell line is infected with HIV.  
     
     
         14 . A method for identifying at least one endogenously loaded peptide ligand that distinguishes an infected cell from an uninfected cell, comprising the steps of: 
 providing an uninfected cell line containing a construct that encodes an individual soluble MHC class I heavy chain molecule, the uninfected cell line being able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of class I molecules;    infecting a portion of the uninfected cell line with at least one of a microorganism, a gene from a microorganism or a tumor gene, thereby providing an infected cell line;    culturing the uninfected cell line and the infected cell line under conditions which allow for expression of the individual soluble MHC class I heavy chain molecules from the construct, such conditions also allowing for endogenous loading of a peptide ligand into the antigen binding groove of each individual soluble MHC class I molecule prior to secretion of the individual soluble MHC class I molecules from the cell;    isolating the secreted individual soluble MHC class I molecules having the endogenously loaded peptide ligands bound thereto from the uninfected cell line and the infected cell line;    separating the endogenously loaded peptide ligands from the individual soluble MHC class I molecules from the uninfected cell line and separating the endogenously loaded peptide ligands from the individual soluble MHC class I molecules from the infected cell line;    isolating the endogenously loaded peptide ligands from the uninfected cell line and the endogenously loaded peptide ligands from the infected cell line;    comparing the endogenously loaded peptide ligands isolated from the uninfected cell line to the endogenously loaded peptide ligands isolated from the infected cell line; and    identifying at least one endogenously loaded peptide ligand presented by the individual soluble MHC class I molecule on the uninfected cell line that is not presented by the individual soluble MHC class I molecule on the infected cell line.    
     
     
         15 . The method of  claim 14  wherein, in the step of providing an uninfected cell line containing a construct that encodes an individual soluble MHC class I heavy chain molecule, the construct further encodes a tag which is attached to the individual soluble MHC class I heavy chain molecule and aids in isolating the individual soluble MHC class I molecule.  
     
     
         16 . The method of  claim 14 , wherein the uninfected cell line is class I negative.  
     
     
         17 . The method of  claim 14 , wherein the uninfected cell line expresses endogenous class I molecules.  
     
     
         18 . The method of  claim 14  wherein, in the step of providing an uninfected cell line containing a construct that encodes an individual soluble MHC class I heavy chain molecule, the uninfected cell line containing the construct that encodes the individual soluble MHC class I heavy chain molecule is produced by a method comprising the steps of: 
 obtaining genomic DNA or cDNA encoding at least one class I molecule;    identifying an allele encoding an individual class I molecule in the genomic DNA or cDNA;    PCR amplifying the allele encoding the individual class I molecule in a locus specific manner such that a PCR product produced therefrom encodes a truncated, soluble form of the individual class I molecule;    cloning the PCR product into an expression vector, thereby forming a construct that encodes the individual soluble MHC class I heavy chain molecule; and    transfecting the construct into an uninfected cell line.    
     
     
         19 . The method of  claim 18 , wherein the construct further encodes a tag which is attached to the individual soluble MHC class I heavy chain molecule and aids in isolating the individual soluble MHC class I molecule.  
     
     
         20 . The method of  claim 19 , wherein at least one of: 
 (a) the tag is selected from the group consisting of a HIS tail and a FLAG tail;    (b) the tag is encoded by a PCR primer utilized in the step of PCR amplifying an allele encoding the individual class I molecule; and    (c) the tag is encoded by the expression vector into which the PCR product is cloned.    
     
     
         21 . The method of  claim 14 , further comprising the step of identifying a source protein from which the at least one endogenously loaded peptide ligand presented by the individual soluble MHC class I molecule on the uninfected cell line and not presented by the individual soluble MHC class I molecule on the infected cell line is obtained.  
     
     
         22 . The method of  claim 14  wherein, in the step of infecting a portion of the uninfected cell line, the portion of the uninfected cell line is infected with HIV.  
     
     
         23 . A method for identifying a self protein that is processed into at least one peptide fragment, wherein the at least one peptide fragment is endogenously loaded in an individual class I molecule and presented by the individual class I molecule on an infected cell but not on an uninfected cell, the method comprising the steps of: 
 providing an uninfected cell line containing a construct that encodes an individual soluble MHC class I heavy chain molecule, the uninfected cell line being able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of class I molecules;    infecting a portion of the uninfected cell line with at least one of a microorganism, a gene from a microorganism or a tumor gene, thereby providing an infected cell line;    culturing the uninfected cell line and the infected cell line under conditions which allow for expression of the individual soluble MHC class I heavy chain molecules, such conditions also allowing for endogenous loading of a peptide ligand in the antigen binding groove of each individual soluble MHC class I molecule prior to secretion of the individual soluble MHC class I molecules from the cell;    isolating the secreted individual soluble MHC class I molecules having endogenously loaded peptide ligands bound thereto from the uninfected cell line and the infected cell line;    separating the endogenously loaded peptide ligands from the individual soluble MHC class I molecules from the uninfected cell line and the infected cell line;    isolating the endogenously loaded peptide ligands from the uninfected cell line and the endogenously loaded peptide ligands from the infected cell line;    comparing the endogenously loaded peptide ligands isolated from the infected cell line to the endogenously loaded peptide ligands isolated from the uninfected cell line;    identifying at least one endogenously loaded peptide ligand presented by the individual soluble MHC class I molecule on the infected cell line that is not presented by the individual soluble MHC class I molecule on the uninfected cell line;    determining the source protein from which the at least one endogenously loaded peptide ligand is obtained; and    identifying the source protein as a self protein if the source protein is not encoded by the microorganism, gene from a microorganism or tumor gene with which the infected cell line is infected but is encoded by the uninfected cell line.    
     
     
         24 . A method for identifying a self protein that is processed into at least one peptide fragment, wherein the at least one peptide fragment is endogenously loaded in an individual class I molecule presented by the individual class I molecule on an uninfected cell but not on an infected cell, the method comprising the steps of: 
 providing an uninfected cell line containing a construct that encodes an individual soluble MHC class I heavy chain molecule, the uninfected cell line being able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of class I molecules;    infecting a portion of the uninfected cell line with at least one of a microorganism, a gene from a microorganism or a tumor gene, thereby providing an infected cell line;    culturing the uninfected cell line and the infected cell line under conditions which allow for expression of the individual soluble MHC class I heavy chain molecules, such conditions also allowing for endogenous loading of a peptide ligand in the antigen binding groove of each individual soluble MHC class I molecule prior to secretion of the individual soluble MHC class I molecules from the cell;    isolating the secreted individual soluble MHC class I molecules having endogenously loaded peptide ligands bound thereto from the uninfected cell line and from the infected cell line;    separating the endogenously loaded peptide ligands from the soluble class I molecules from the uninfected cell line and from the infected cell line;    isolating the endogenously loaded peptide ligands from the uninfected cell line and the endogenously loaded peptide ligands from the infected cell line;    comparing the endogenously loaded peptide ligands isolated from the uninfected cell line to the endogenously loaded peptide ligands isolated from the infected cell line;    identifying at least one endogenously loaded peptide ligand presented by the individual soluble MHC class I molecule on the uninfected cell line that is not presented by the individual soluble MHC class I molecule on the infected cell line; and    determining the source protein from which the at least one endogenously loaded peptide ligand is obtained.