Comparative ligand mapping from mhc class i 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 a transfected cell from a non-transfected cell, comprising the steps of:
 providing a non-transfected cell line containing a construct that encodes an individual soluble class I MHC molecule, the non-transfected cell line being able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of class I MHC molecules;   transfecting a portion of the non-transfected cell line with at least one of a gene from a microorganism and a tumor gene, thereby providing a transfected cell line;   culturing the non-transfected cell line and the transfected 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 non-transfected cell line and the transfected cell line;   separating the endogenously loaded peptide ligands from the individual soluble class I MHC molecules from the non-transfected cell line and separating the endogenously loaded peptide ligands from the individual soluble class I MHC molecules from the transfected cell line;   isolating the endogenously loaded peptide ligands from the non-transfected cell line and the endogenously loaded peptide ligands from the transfected cell line;   comparing the endogenously loaded peptide ligands isolated from the transfected cell line to the endogenously loaded peptide ligands isolated from the non-transfected cell line; and   identifying at least one individual, endogenously loaded peptide ligand presented by the individual soluble class I MHC molecule on the transfected cell line that is not presented by the individual soluble class I MHC molecule on the non-transfected 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 transfected cell line and not presented by the individual soluble class I MHC molecule on the non-transfected 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 transfected cell line but not on the non-transfected cell line, the at least one individual, endogenously loaded peptide ligand is obtained from a protein encoded by at least one of the gene from a microorganism and a tumor gene with which the cell line was transfected to form the transfected 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 transfected cell line but not on the non-transfected cell line, the at least one individual, endogenously loaded peptide ligand is obtained from a protein encoded by the non-transfected cell line. 
     
     
         5 . The method of  claim 4 , wherein the protein encoded by the non-transfected 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 a non-transfected 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 non-transfected cell line is class I MHC negative. 
     
     
         8 . The method of  claim 1 , wherein the non-transfected cell line expresses endogenous class I MHC molecules. 
     
     
         9 . The method of  claim 1  wherein, in the step of providing a non-transfected cell line containing a construct that encodes an individual soluble class I MHC molecule, the non-transfected 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 a non-transfected cell line. 
 
 
     
     
         10 . The method of  claim 9  wherein, in the step of providing a non-transfected 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 transfecting a portion of the non-transfected cell line, the portion of the non-transfected cell line is transfected with a gene from HIV. 
     
     
         15 . The method of  claim 1 , wherein the transfected cell is further defined as a tumorigenic cell, and the non-transfected cell is further defined as a non-tumorigenic cell, whereby the step of transfecting a portion of the non-tumorigenic cell line with at least one of a gene from a microorganism and a tumor gene, thereby provides a transformed, tumorigenic cell line. 
     
     
         16 . 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 gene from a microorganism or tumor gene with which the transfected cell line is transfected but is encoded by the non-transfected cell line.   
     
     
         17 . The method of  claim 16 , wherein the transfected cell is further defined as a tumorigenic cell, and the non-transfected cell is further defined as a non-tumorigenic cell, whereby the step of transfecting a portion of the non-tumorigenic cell line with at least one of a gene from a microorganism and a tumor gene, thereby provides a transformed, tumorigenic cell line. 
     
     
         18 . A method for identifying at least one individual, endogenously loaded peptide ligand for an individual class I MHC molecule that distinguishes a transfected cell from a non-transfected cell, comprising the steps of:
 providing a non-transfected cell line containing a construct that encodes an individual soluble class I MHC molecule, the non-transfected cell line being able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of class I MHC molecules;   transfecting a portion of the non-transfected cell line with at least one of a gene from a microorganism and a tumor gene, thereby providing a transfected cell line;   culturing the non-transfected cell line and the transfected 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 non-transfected cell line and the transfected cell line;   separating the endogenously loaded peptide ligands from the individual soluble class I MHC molecules from the non-transfected cell line and separating the endogenously loaded peptide ligands from the individual soluble class I MHC molecules from the transfected cell line;   isolating the endogenously loaded peptide ligands from the non-transfected cell line and the endogenously loaded peptide ligands from the transfected cell line;   comparing the endogenously loaded peptide ligands isolated from the non-transfected cell line to the endogenously loaded peptide ligands isolated from the transfected cell line; and   identifying at least one individual, endogenously loaded peptide ligand presented by the individual soluble class I MHC molecule on the non-transfected cell line that is not presented by the individual soluble class I MHC molecule on the transfected cell line.   
     
     
         19 . The method of  claim 18  wherein, in the step of providing a non-transfected 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. 
     
     
         20 . The method of  claim 18 , wherein the non-transfected cell line is class I MHC negative. 
     
     
         21 . The method of  claim 18 , wherein the non-transfected cell line expresses endogenous class I MHC molecules. 
     
     
         22 . The method of  claim 18  wherein, in the step of providing a non-transfected cell line containing a construct that encodes an individual soluble class I MHC molecule, the non-transfected 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 a non-transfected cell line. 
 
     
     
         23 . The method of  claim 22  wherein, in the step of providing a non-transfected 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. 
     
     
         24 . The method of  claim 23 , wherein the tag is selected from the group consisting of a HIS tail and a FLAG tail. 
     
     
         25 . The method of  claim 23 , 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. 
     
     
         26 . The method of  claim 24  wherein the tag is encoded by the expression vector into which the PCR product is cloned. 
     
     
         27 . The method of  claim 18 , 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 non-transfected cell line and not presented by the individual soluble class I MHC molecule on the transfected cell line is obtained. 
     
     
         28 . The method of  claim 18  wherein, in the step of transfecting a portion of the non-transfected cell line, the portion of the non-transfected cell line is transfected with a gene from HIV. 
     
     
         29 . The method of  claim 18 , wherein the transfected cell is further defined as a tumorigenic cell, and the non-transfected cell is further defined as a non-tumorigenic cell, whereby the step of transfecting a portion of the non-tumorigenic cell line with at least one of a gene from a microorganism and a tumor gene, thereby provides a transformed, tumorigenic cell line. 
     
     
         30 . The method of  claim 18 , further comprising the step of:
 determining the source protein from which the at least one endogenously loaded peptide ligand is obtained.   
     
     
         31 . The method of  claim 30 , wherein the transfected cell is further defined as a tumorigenic cell, and the non-transfected cell is further defined as a non-tumorigenic cell, whereby the step of transfecting a portion of the non-tumorigenic cell line with at least one of a gene from a microorganism and a tumor gene, thereby provides a transformed, tumorigenic cell line.