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Finding TCR epitopes in peptide haystacks
Identification of the peptide-MHC ligands recognized by a specific T cell receptor (TCR) remains a formidable bottleneck for T cell biology. Lee and Meyerson developed an epitope discovery platform based on introduction of DNA-encoded peptide pools into immortalized antigen-presenting cells (APCs) engineered to express defined class I or II MHC molecules. When αβ T cells of interest were activated by a cognate peptide-MHC complex, secreted cytokines were captured by anti-cytokine antibodies tethered to the APC surface. Positive selection for cytokine-displaying APCs followed by next-generation DNA sequencing revealed the peptide epitope seen by the TCR. This approach enabled a proof-of-principle experiment that defined several previously unknown CD4+ and CD8+ T cell epitopes from cytomegalovirus recognized by “public” TCRs shared among multiple CMV seropositive donors.
Abstract
A major limitation to understanding the associations of human leukocyte antigen (HLA) and CD8+ and CD4+ T cell receptor (TCR) genes with disease pathophysiology is the technological barrier of identifying which HLA molecules, epitopes, and TCRs form functional complexes. Here, we present a high-throughput epitope identification system that combines capture of T cell–secreted cytokines by barcoded antigen-presenting cells (APCs), cell sorting, and next-generation sequencing to identify class I– and class II–restricted epitopes starting from highly complex peptide-encoding oligonucleotide pools. We engineered APCs to express anti-cytokine antibodies, a library of DNA-encoded peptides, and multiple HLA class I or II molecules. We demonstrate that these engineered APCs link T cell activation–dependent cytokines with the DNA that encodes the presented peptide. We validated this technology by showing that we could select known targets of viral epitope–, neoepitope-, and autoimmune epitope–specific TCRs, starting from mixtures of peptide-encoding oligonucleotides. Then, starting from 10 TCRβ sequences that are found commonly in humans but lack known targets, we identified seven CD8+ or CD4+ TCR-targeted epitopes encoded by the human cytomegalovirus (CMV) genome. These included known epitopes, as well as a class I and a class II CMV epitope that have not been previously described. Thus, our cytokine capture–based assay makes use of a signal secreted by both CD8+ and CD4+ T cells and allows pooled screening of thousands of encoded peptides to enable epitope discovery for orphan TCRs. Our technology may enable identification of HLA-epitope-TCR complexes relevant to disease control, etiology, or treatment.
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