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Published May 16, 2023 | Published
Journal Article Open

Large libraries of single-chain trimer peptide-MHCs enable antigen-specific CD8+ T cell discovery and analysis


The discovery and characterization of antigen-specific CD8+ T cell clonotypes typically involves the labor-intensive synthesis and construction of peptide-MHC tetramers. We adapt single-chain trimer (SCT) technologies into a high throughput platform for pMHC library generation, showing that hundreds can be rapidly prepared across multiple Class I HLA alleles. We use this platform to explore the impact of peptide and SCT template mutations on protein expression yield, thermal stability, and functionality. SCT libraries were an efficient tool for identifying T cells recognizing commonly reported viral epitopes. We then construct SCT libraries to capture SARS-CoV-2 specific CD8+ T cells from COVID-19 participants and healthy donors. The immunogenicity of these epitopes is validated by functional assays of T cells with cloned TCRs captured using SCT libraries. These technologies should enable the rapid analyses of peptide-based T cell responses across several contexts, including autoimmunity, cancer, or infectious disease.

Additional Information

© The Author(s) 2023. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. We are grateful to all participants in this study and to the medical teams at Swedish Medical Center for their support. We thank the Northwest Genomic Center for help with sequencing services and the ISB-Swedish COVID-19 Biobanking Unit. We thank SJHC/SJCI COVID-19 clinical and research support teams. We thank Amazon Web Services for their support through cloud computing credits provided by the AWS Diagnostic Development Initiative (DDI). We acknowledge funding support from the Parker Institute for Cancer Immunotherapy (J.R.H. and P.D.G.), the Biomedical Advanced Research and Development Authority (HHSO10201600031C to J.R.H.), and the NIH through (1 R01 CA264090-01 JRH, PDG), (R21 AI154874 RKS), and (R01 AI121242 RKS). These authors contributed equally: William Chour, Jongchan Choi. Author contributions. W.C., J.C., and J.R.H. conceived this study. W.C., J.C., T.M.S., K.F., D.C.D., A.M.X., Y.S., A.H.C.N., and J.R.H. designed the experiments. W.C., J.C., J.X. M.E.C., D.C.D., Y.S., D.G.C., R.Z., D.Y., S.H., A.H.C.N., J.Z.B., R.A.E., and L.C.J. conducted the experiments. W.C., J.C., J.X., T.M.S., K.F., D.C.D., A.M.X., Y.S., D.G.C., R.Z., and D.Y. performed the analyses. S.P., G.L., K.M.M., R.K.S., J.K.L., J.D.G., P.D.G, and J.R.H provided resources. W.C., J.C., and J.R.H. wrote the paper. All authors were involved in editing and reviewing the paper. Data availability. The data generated in this study are provided in the main text, Supplementary Information, Supplementary Data Excel file, or available from the authors upon request. Raw images of the SDS-PAGE results from Figs. 1c and and2b2b may be found in Supplementary Figs. 1e and 2c, respectively. The processed sequencing data generated in this study have been deposited in the ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-11229. Code availability. The custom Python scripts used for data analysis are publicly available on GitHub. Competing interests. J.R.H. is a founder and board member of Isoplexis and PACT Pharma. P.D.G. is on the scientific advisory board of Celsius, Earli, Elpiscience, Immunoscape, Rapt, Metagenomi, and Nextech, was a scientific founder of Juno Therapeutics, and receives research support from Lonza. J.D.G. declared contracted research with Gilead, Lilly, and Regeneron and served on an advisory board for Gilead. The remaining authors declare no competing interests.

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