T-CELL EPITOPE IDENTIFICATION
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.