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 single indicating cytotoxic attack from a T-cell are isolated and then analyzed by next-generation sequencing in order to identify the epitope sequences. Specifically disclosed is a method in which a library of candidate epitope-encoding nucleic acids are expressed in cells which feature a membrane-bound major histocompatibility complex (MHC) protein, said library produced by transfection of plasmids featuring both a nucleotide encoding the candidate epitope and a nucleotide encoding a FRET-based fluorescent protein cleaved by granzyme.
1. A method for determining epitopes recognized by cytotoxic T-cells, 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 a fluorescence-based signaling system in their cytosols that generates a fluorescent signal whenever a peptide linkage in the system is enzymatically cleaved by a granzyme A, B, H, K or M from granules of a cytotoxic T cell upon recognition of an expressed peptide in the context of an MHC protein on a reporter cell by such cytotoxic T cell;
contacting in a reaction mixture a mixed population of the reporter cells expressing different candidate epitope-encoding nucleic acids of the library with a sample comprising cytotoxic T-cells, wherein effector functions of cytotoxic I-cells recognizing reporter cells are activated so that granzyme A, H, K or M in granules of the Cytotoxic T-cells are delivered by, a granzyme-perforin pathway to recognized reporter cells and cause a fluorescent signal to be venerated by the reporter cells;
isolating intact reporter cells generating fluorescent 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.
2. The method of claim 1 wherein said sample is from an individual and said MHC proteins of said reporter cells are matched with MHC proteins expressed by the individual.
3. The method of claim 2 wherein said reporter cells comprise autologous cells of said individual genetically modified to express candidate epitopes of said library.
4. The method of claim 3 wherein said autologous cells are B cells of said individual.
5. The method of claim 4 wherein said autologous cells are stably transfected or transformed by a vector capable of expressing said candidate epitopes of said library.
6. The method of claim 3 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 granzyme A, B, H, K or M.
7. A method for determining epitopes recognized by cytotoxic T-cells, 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 a fluorescence-based signaling system that generates a fluorescent signal whenever a peptide linkage in the system is enzymatically cleaved by a granzyme A, B, H, K or M from granules of a granzyme-perforin pathway of a cytotoxic T cell upon recognition of an expressed peptide in the context of an MHC protein on a reporter cell by such cytotoxic T cell;
(b) contacting in a reaction mixture a mixed population of the reporter cells expressing different candidate epitope-encoding nucleic acids of the library with a sample comprising cytotoxic T-cells;
(c) isolating reporter cells generating a fluorescent signal indicating recognition by a cytotoxic T-cell;
(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 T-cell 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.
8. The method of claim 7 wherein said step of isolating includes sorting said reporter cells by a fluorescently activated cell sorter (FACS).