T-cell epitope identification

Abstract

The present invention is a method for determining the identity of the epitopes recognized by T-cells. The method consists of expressing an encoded library of candidate epitope sequences in a recipient reporter cell capable of providing a detectable signal upon cytotoxic attack from a single cognate T-cell followed by contacting the reporter cells with T-cells of interest. The reporter cells with a signal indicating cytotoxic attack from a T-cell are isolated and then analyzed by next-generation sequencing in order to identify the epitope sequences.

1 - 20 . (canceled) 
     
     
         21 . A method for determining epitopes recognized by cytotoxic lymphocytes, the method comprising the steps of:
 expressing a library of candidate epitope-encoding nucleic acids in reporter cells capable of presenting expressed peptides of such candidate epitope-encoding nucleic acids in the context of a membrane-bound major histocompatibility complex (MHC) protein wherein the reporter cells are modified to carry an optically-based signaling system that generates an optical signal whenever a peptide linkage in the system is enzymatically cleaved by a serine protease from granules of a cytotoxic lymphocyte upon recognition of an expressed peptide in the context of an MHC protein on a reporter cell by such cytotoxic lymphocyte;   contacting in a reaction mixture a mixed population of reporter cells expressing different candidate epitope-encoding nucleic acids of the library with a sample comprising cytotoxic lymphocytes, wherein effector functions of cytotoxic lymphocytes recognizing reporter cells are activated so that serine proteases in granules of the cytotoxic lymphocytes are delivered by a granzyme-perforin pathway to recognized reporter cells and cause an optical signal to be generated by the reporter cells;   isolating intact reporter cells generating optical signals;   extracting candidate epitope-encoding nucleic acids from the isolated intact reporter cells; and   sequencing the candidate epitope-encoding nucleic acids of the reporter cells to identify the epitope-encoding nucleic acids.   
     
     
         22 . The method of  claim 21  wherein said sample is from an individual and said MHC proteins of said reporter cells are matched with MHC proteins expressed by the individual. 
     
     
         23 . The method of  claim 22  wherein said reporter cells comprise autologous cells of said individual genetically modified to express candidate epitopes of said library. 
     
     
         24 . The method of  claim 23  wherein said autologous cells are B cells of said individual. 
     
     
         25 . The method of  claim 24  wherein said autologous cells are stably transfected or transformed by a vector capable of expressing said candidate epitopes of said library. 
     
     
         26 . The method of  claim 23  wherein said reporter cells are genetically modified to express a FRET-based fluorescent protein signaling system that generates a fluorescent signal whenever enzymatically cleaved by one of said serine proteases. 
     
     
         27 . The method of  claim 21  wherein said serine protease is a granzyme. 
     
     
         28 . The method of  claim 27  wherein said granzyme is a granzyme A, B, H, K or M. 
     
     
         29 . The method of  claim 21  wherein said cytotoxic lymphocytes are cytotoxic T cells or natural killer cells. 
     
     
         30 . A method for determining epitopes recognized by cytotoxic lymphocytes, the method comprising the steps of:
 (a) expressing a library of candidate epitope-encoding nucleic acids or an enriched library of candidate epitope-encoding nucleic acids in reporter cells capable of presenting expressed peptides of such candidate epitope-encoding nucleic acids in the context of a membrane-bound major histocompatibility complex (MHC) protein wherein the reporter cells are modified to carry an optically-based signaling system that generates an optical signal whenever a peptide linkage in the system is enzymatically cleaved by a serine protease from granules of a granzyme-perforin pathway of a cytotoxic lymphocyte upon recognition of an expressed peptide in the context of an MHC protein on a reporter cell by such cytotoxic lymphocyte;   (b) contacting in a reaction mixture a mixed population of reporter cells expressing different candidate epitope-encoding nucleic acids of the library with a sample comprising cytotoxic lymphocytes;   (c) isolating reporter cells generating an optical signal indicating recognition by a cytotoxic lymphocyte;   (d) extracting candidate epitope-encoding nucleic acids from the isolated reporter cells and generating therefrom an enriched library of candidate epitope-encoding nucleic acids;   (e) repeating steps (a)-(d) with the enriched library of candidate epitope-encoding nucleic acids until a frequency of reporter cells generating the signal indicating recognition by a cytotoxic lymphocyte is greater than or equal to a predetermined value;   (f) sequencing the candidate epitope-encoding nucleic acids of reporter cells generating said signal to identify sequences of the epitope-encoding nucleic acids.   
     
     
         31 . The method of  claim 30  wherein said optical signal indicating recognition by a cytotoxic lymphocyte is a fluorescent signal and said step of isolating includes sorting said reporter cells by a fluorescently activated cell sorter (FACS). 
     
     
         32 . The method of  claim 30  wherein said serine protease is a granzyme. 
     
     
         33 . The method of  claim 32  wherein said granzyme is a granzyme A, B, H, K or M. 
     
     
         34 . The method of  claim 32  wherein said cytotoxic lymphocytes are cytotoxic T cells or natural killer cells. 
     
     
         35 . A method for testing the presence and level of cellular immunity in an individual to a predetermined antigen or class of antigens comprising the steps of:
 (a) providing reporter cells expressing a library of epitopes derived from the predetermined antigen or class of antigens, the reporter cells being MHC matched to the individual, and the reporter cells each having an optically-based signaling system that generates an optical signal whenever a peptide linkage in the system is enzymatically cleaved by a serine protease from granules of a granzyme-perforin pathway of a cytotoxic lymphocyte activated upon recognition of an expressed peptide in the context of an MHC protein on the reporter cell by such cytotoxic lymphocyte;   (b) combining the reporter cells with a sample of cytotoxic lymphocytes from the individual;   (c) determining a level of cellular immunity of the individual to the predetermined antigen or class of antigens by a number or frequency of reporter cells generating an optical signal.   
     
     
         36 . The method of  claim 35  wherein said optical signal of said reporter cell is generated by a FRET-based fluorescent protein signaling system. 
     
     
         37 . The method of  claim 35  wherein said predetermined antigen or class of antigens are proteins from an infectious agent. 
     
     
         38 . The method of  claim 37  wherein said infectious agent is a virus, a bacteria, a fungus, or a protozoa.