Methods of MHC class II epitope mapping, detection of autoimmune T cells and antigens, and autoimmune treatment

Abstract

The present invention provides of using multimeric MHC class II/peptide complexes. In one aspect, methods provided for identifying MHC class II-restricted immune epitopes of a predetermined polypeptide antigen. Methods for identifying an immunostimulatory epitope for a predetermined polypeptide antigen are provided. In a related aspect, methods for screening a therapeutic polypeptide agent for an MHC class II epitope are provided. In other aspects, methods for modulating T cells and for determining or monitoring an MHC class II-restricted immune status of a patient are also provided.

1 . A method for collecting T cells specific for an MHC class II epitope of a predetermined polypeptide antigen, comprising: 
 preparing a library of at least two peptide pools, each peptide pool comprising at least two peptides, the peptides comprising a fragment of the predetermined polypeptide antigen and sharing a region of contiguous amino acid sequence identity with at least one other peptide in the library;    forming pools of multimeric MHC class II/peptide complexes from the pools of peptides and soluble human MHC class II molecules;    contacting the pools of multimeric MHC class II/peptide complexes with human T cells;    identifying at least one pool of multimeric MHC class II/peptide complexes that binds to the T cells; and    collecting T cells that bind to the multimeric MHC class II/peptide complexes.    
     
     
         2 . The method of  claim 1 , wherein the identifying is by fluorescence activated cell sorting, T cell proliferation assay or cytokine secretion capture assay.  
     
     
         3 . The method of  claim 1 , wherein the collected T cells are cultured in vitro to increase the number of T cells.  
     
     
         4 . The method of  claim 3 , wherein the cultured T cells are administered to a human subject.  
     
     
         5 . The method of  claim 1 , further comprising contacting T cells with multimeric MHC class II/peptide complexes formed with individual peptides from the pool of multimeric MHC class II/peptide complexes that binds to the T cells to identify at least one epitope in the peptide pool.  
     
     
         6 . The method of  claim 1 , wherein at least some of the peptides comprise a candidate epitope of the predetermined polypeptide antigen, the candidate epitope predicted by a computer-implemented algorithm for candidate MHC class II epitope identification.  
     
     
         7 . The method of  claim 1 , wherein the soluble human MHC class II molecules further comprise a ligand, and the multimeric MHC class II complexes are formed by interaction of the ligand with a polyvalent binding partner.  
     
     
         8 . A method for identifying an MHC class II immunostimulatory epitope for a predetermined polypeptide antigen, comprising: 
 preparing pools of peptides, each peptide comprising a fragment of the predetermined polypeptide antigen and having a region of sequence identity with another peptide;    forming a first library of pools of multimeric MHC class II/peptide complexes from the pools of peptides and soluble MHC class II molecules;    contacting the first library with human T cells;    identifying at least one pool of multimeric MHC class II/peptide complexes in the first library that bind to the T cells;    forming a second library of multimeric MHC class II/peptide complexes from the individual peptides of the at least one pool of multimeric MHC class II/peptide complexes of the first library that binds to the T cells; and    identifying at least one epitope of the predetermined antigen.    
     
     
         9 . The method of  claim 8 , further comprising forming an immunogenic composition comprising the identified epitope.  
     
     
         10 . The method of  claim 8 , further comprising forming an immunogenic composition omitting the identified epitope.  
     
     
         11 . A method for identifying MHC class II epitopes of the proteome of an organism, comprising: 
 obtaining amino acid sequences for proteins of the organism;    analyzing the amino acid sequences with a computer-implemented algorithm for candidate MHC class II epitope identification to identify candidate epitopes;    preparing pools of peptides comprising the candidate epitopes;    forming a first library of pools of multimeric MHC class II/peptide complexes from the pools of peptides and soluble MHC class II molecules;    contacting the first library with T cells;    identifying at least one pool of multimeric MHC class II/peptide complexes in the first library that bind to the T cells; and    identifying at least one MHC class II epitope from the proteome of the organism.    
     
     
         12 . The method of  claim 11 , further comprising: 
 forming a second library of multimeric MHC class II/peptide complexes from the individual peptides of pools of multimeric MHC class II/peptide complexes of the first library that bind to the T cells.    
     
     
         13 . A method for screening a therapeutic polypeptide agent for an MHC class II epitope, comprising: 
 preparing pools of peptides, each peptide comprising a fragment of the polypeptide agent and having a region of sequence identity with another fragment of the polypeptide agent, each pool comprising at least two peptides;    forming a first library of pools of multimeric MHC class II/peptide complexes with the peptide pools and soluble human MHC class II molecules;    contacting the first library with human T cells;    determining whether the pools of multimeric MHC class II/peptide complexes bind to the T cells; and    determining whether the polypeptide agent has an MHC class II epitope.    
     
     
         14 . A method for modulating the state of T cells, comprising: 
 contacting a population of T cells with a multimeric MHC class II/peptide complex conjugated to a biologically active modulatory molecule, and    modulating the state of at least one T cell in the population.    
     
     
         15 . The method of  claim 14 , wherein the multimeric MHC class II/peptide complex confers epitope-specific binding and targeting of the biologically active modulatory molecule to the T cells.  
     
     
         16 . The method of  claim 14 , wherein the modulation of the statue of the T cell is apoptosis, anergy, activation, proliferation, or deviation towards alternative cytokine production, as compared with a T cell not contacted with the biologically active modulatory molecule.  
     
     
         17 . The method of  claim 14 , wherein the biologically active modulatory molecule is an antibody or a cytotoxin.  
     
     
         18 . The method of  claim 17 , wherein the antibody is anti-CD95 antibody, anti-CTLA4 antibody or anti-CD28 antibody.  
     
     
         19 . The method of  claim 14 , wherein the biologically active modulatory molecule is a member of the B7 family or CD95.  
     
     
         20 . The method of  claim 14 , wherein the biologically active modulatory molecule is coupled to substrate.  
     
     
         21 . The method of  claim 20 , wherein the multimeric complexes are bound to anti-MHC class II antibodies coupled to a microbe ad.  
     
     
         22 . The method of  claim 14 , wherein the T cells are human T cells.  
     
     
         23 . The method of  claim 14 , wherein the contacting is ex vivo.  
     
     
         24 . The method of  claim 14 , wherein the contacting is in vivo in a human subject.  
     
     
         25 . A method for monitoring an MHC class II-restricted immune status of a subject, comprising: 
 isolating T cells from the subject,    contacting the T cells with multimeric MHC class II/peptide complexes comprising soluble MHC class II molecules and peptides comprising at least one MHC class II epitope; and    determining whether the T cells bind to the multimeric MHC class II/peptide complexes.