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