T-CELL EPITOPE IDENTIFICATION
First Claim
1. 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 and generating a signal upon recognition thereof by a cytotoxic lymphocyte and activation of effector functions of the cytotoxic T-cell;
contacting the reporter cells containing the expressed library of candidate epitope-encoding nucleic acids with a sample comprising cytotoxic lymphocytes; and
analyzing the candidate epitope-encoding nucleic acids of reporter cells that generate a signal indicating recognition by a cytotoxic lymphocyte of the sample to identify sequences of the epitope-encoding nucleic acids.
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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 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.
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Citations
20 Claims
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1. A method for determining epitopes recognized by cytotoxic lymphocytes, the method comprising the steps of:
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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 and generating a signal upon recognition thereof by a cytotoxic lymphocyte and activation of effector functions of the cytotoxic T-cell; contacting the reporter cells containing the expressed library of candidate epitope-encoding nucleic acids with a sample comprising cytotoxic lymphocytes; and analyzing the candidate epitope-encoding nucleic acids of reporter cells that generate a signal indicating recognition by a cytotoxic lymphocyte of the sample to identify sequences of the epitope-encoding nucleic acids. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method for determining epitopes recognized by cytotoxic lymphocytes, the method comprising the steps of:
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(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 and generating a signal upon recognition thereof by a cytotoxic lympohcyte and activation of effector functions of the cytotoxic lymphocyte; (b) contacting the reporter cells containing the expressed library of candidate epitope-encoding nucleic acids with a sample comprising cytotoxic lymphocytes; (c) isolating reporter cells generating a 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) analyzing the candidate epitope-encoding nucleic acids of reporter cells generating a signal to identify sequences of the epitope-encoding nucleic acids. - View Dependent Claims (11, 12)
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13. A reporter cell for determining antigen-specific cytotoxic lymphocytes of an individual comprising:
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an autologous cell of the individual having MHC molecules capable of forming an MHC-epitope complex on a cell surface; an epitope displayed in an MHC-epitope complex on the cell surface, the epitope being expressed in the autologous cell by a transgene comprising a candidate epitope-encoding nucleic acid; and a signal generation product capable of generating a signal in response to action by an effector agent of a cytotoxic lymphocyte, the signal generation product being expressed in the autologous cell by a transgene comprising nucleic acids encoding the signal generation product. - View Dependent Claims (14)
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15. A method for testing the presence and/or level of cellular immunity in an individual to a predetermined antigen or class of antigens comprising the steps of:
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(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, the reported cells capable of generating a detectable signal in response to recognition by a cytotoxic T cell and activation of an effector response by cytotoxic T cell; (b) combining the reporter cells with a sample of cytotoxic T cells from the individual; (c) determining the presence and/or level of cellular immunity of the individual to the predetermined antigen or class of antigens by a number or frequency of reporter cells generating a signal. - View Dependent Claims (16, 17, 18, 19, 20)
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Specification