Methods and compositions for identifying epitopes

A system for detection of granzyme B activity in an antigen presenting cell, comprising:

• a) a fusion polypeptide comprising a CRE recombinase functionally linked to a plasma membrane attachment peptide, wherein the CRE recombinase and membrane attachment peptide are separated by a GzB cleavage site;
• b) a reporter of CRE activity comprising a nucleic acid sequence encoding GFP and RFP in head-to-head orientation flanked by LoxP sites;
• and/or
• c) a nucleic acid sequence encoding a candidate antigen in expressible form, located proximally to a CRE activated primer recognition sequence comprising an inactive primer flanked by LoxP sites, wherein CRE induced rearrangement of the LoxP sites produces a functional primer recognition sequence.

A method for identifying an antigen that is recognized by a cytotoxic T cell and/or NK cell, comprising:

• a) contacting an antigen presenting cell (APC) or a library of APC of any one of with one or more cytotoxic T cells (CTLs) and/or NK cells under conditions appropriate for antigen recognition, wherein the APC comprises:
• i. an exogenous nucleic acid encoding one or more candidate antigens, wherein the one or more candidate antigens are expressed and presented with MHC class I or MHC class II molecules;
• ii. a molecular reporter of Granzyme B (GzB) activity;
• and
• iii. an exogenous inhibitor of caspase-activated deoxyribonuclease (CAD)-mediated DNA degradation, a CAD knockout, or a caspase knockout;
• b) identifying APC(s) that express recognized antigen by assaying for granzyme B activity in the APC(s), wherein increased granzyme B activity, as compared to an appropriate control, indicates the APC expresses antigen that was recognized by the cytotoxic T cell and/or NK cell;
• and
• c) isolating nucleic acid that encodes the recognized antigen from the APC identified in step b), optionally further comprising sequencing the nucleic acid isolated in step c).