Rapid selection and identification of functional CD8+ T cell epitopes from large peptide-coding libraries

Cytotoxic CD8 + T cells recognize and eliminate infected or malignant cells that present peptide epitopes derived from intracellularly processed antigens on their surface. However, comprehensive profiling of specific major histocompatibility complex (MHC)-bound peptide epitopes that are naturally processed and capable of eliciting a functional T cell response has been challenging. Here, we report a method for deep and unbiased T cell epitope profiling, using in vitro co-culture of CD8 + T cells together with target cells transduced with high-complexity, epitope-encoding minigene libraries. Target cells that are subject to cytotoxic attack from T cells in co-culture are isolated prior to apoptosis by fluorescence-activated cell sorting, and characterized by sequencing the encoded minigenes. We then validate this highly parallelized method using known murine T cell receptor/peptide-MHC pairs and diverse minigene-encoded epitope libraries. Our data thus suggest that this epitope profiling method allows unambiguous and sensitive identification of naturally processed and MHC-presented peptide epitopes. High-throughput assays for TCR specificity are a bottleneck in understanding T cell immunity and harnessing it for medicine. Here the authors develop a functional screening method to identify T cell specificity in the natural context of peptide-MHC presentation, enabling detection of physiologically relevant T cell antigens from large libraries of peptide-coding sequences.