Comparative Ligand Mapping from MHC Positive Cells

Abstract

A method of comparative ligand mapping to identify peptide ligands presented by MHC positive cells that distinguish an infected/transfected cell from an uninfected/non-transfected cell is disclosed.

What is claimed is: 
     
         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:
 culturing a non-transfected cell line and a transfected cell line, each containing a construct that encodes an individual soluble class I MHC molecule, and 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, wherein the cell lines are cultured 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, and wherein the transfected cell line is produced by transfecting a non-transfected cell line with at least one of a gene from a microorganism and a tumor gene;   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;   identifying at least one individual, endogenously loaded peptide ligand wherein:
 (a) the peptide ligand is 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; or 
 (b) the peptide ligand is 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; and 
   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.   
     
     
         2 . The method of  claim 1 , wherein the identified 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. 
     
     
         3 . The method of  claim 1 , wherein the identified peptide ligand is obtained from a protein encoded by the non-transfected cell line. 
     
     
         4 . The method of  claim 1 , wherein 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. 
     
     
         5 . The method of  claim 1 , wherein the non-transfected cell line is class I MHC negative. 
     
     
         6 . The method of  claim 1 , wherein the non-transfected cell line expresses endogenous class I MHC molecules. 
     
     
         7 . 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. 
     
     
         8 . The method of  claim 1 , further comprising the step of transfecting a portion of the non-transfected cell line with at least one gene from a microorganism and/or a tumor gene, thereby providing the transfected cell line. 
     
     
         9 . The method of  claim 1 , wherein the cell lines containing the construct that encodes the individual soluble class I MHC molecule are 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 . A method for identifying at least one peptide ligand for a class I MHC molecule, wherein the at least one peptide ligand distinguishes an infected cell from an uninfected cell, the method comprising the steps of:
 separately culturing an uninfected cell line and an infected cell line, each containing a construct that encodes a soluble class I MHC molecule, under conditions that allow for expression and secretion of recombinant soluble class I MHC molecules that have endogenously produced and loaded peptide ligands bound thereto;   isolating the peptide ligands from the soluble class I MHC molecules secreted from the infected and uninfected cell lines;   comparing the peptide ligands isolated from the infected cell line with the peptide ligands isolated from the uninfected cell line to identify at least one peptide ligand that distinguishes infected cells from uninfected cells; and   identifying a source protein from which the at least one peptide ligand is obtained.   
     
     
         11 . The method of  claim 10 , wherein the infected cell line is infected with a microorganism, and wherein the at least one peptide ligand identified as distinguishing infected cells from uninfected cells is presented by the class I MHC molecules on the infected cell line but not on the uninfected cell line. 
     
     
         12 . The method of  claim 11 , wherein the at least one peptide ligand is obtained from a protein encoded by the microorganism. 
     
     
         13 . The method of  claim 10 , wherein the uninfected and infected cell lines do not express endogenous class I MHC molecules. 
     
     
         14 . The method of  claim 10 , wherein the uninfected cell line expresses endogenous class I MHC molecules. 
     
     
         15 . The method of  claim 10 , wherein the at least one peptide ligand is obtained from a protein encoded by the uninfected cell line. 
     
     
         16 . The method of  claim 10 , wherein the at least one peptide ligand is presented by the class I MHC molecules on the uninfected cell line but not on the infected cell line. 
     
     
         17 . The method of  claim 10 , further comprising the step of infecting a portion of the uninfected cell line with a microorganism, thereby providing the infected cell line. 
     
     
         18 . The method of  claim 10 , wherein 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. 
     
     
         19 . The method of  claim 10 , wherein the cell lines containing the construct that encodes the individual soluble class I MHC molecule are 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.