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

CD8 + cytotoxic T cell responses against viral infection represent a major element of the adaptive immune response. We describe the development of a peptide antigen - major histompatibility complex (pMHC) library representing the full SARS-CoV-2 viral proteome, and comprised of 634 pMHC multimers representing the A*02.01, A*24.02, and B*07.02 HLA alleles, as well as specific antigens associated with the cytomegalovirus (CMV). These libraries were used to capture non-expanded CD8 + T cells from blood samples collected from 64 infected individuals, and then analyzed using single cell RNA-seq. The discovery and characterization of antigen-specific CD8 ⁺ T cell clonotypes typically involves the labor-intensive synthesis and construction of peptide-MHC tetramers. We adapted 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 used 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 constructed SCT libraries designed to capture SARS-CoV-2 specific CD8 ⁺ T cells from COVID-19 participants and healthy donors. The immunogenicity of these epitopes was 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.