Research ResourcesT CELLS

Antigen identification for HLA class I– and HLA class II–restricted T cell receptors using cytokine-capturing antigen-presenting cells

See allHide authors and affiliations

Science Immunology  22 Jan 2021:
Vol. 6, Issue 55, eabf4001
DOI: 10.1126/sciimmunol.abf4001

You are currently viewing the abstract.

View Full Text

Log in to view the full text

Log in through your institution

Log in through your institution

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.


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.

View Full Text

Stay Connected to Science Immunology