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.

Germline T cell receptor exchange results in physiological T cell development and function.

T cell receptor (TCR) transgenic mice represent an invaluable tool to study antigen-specific immune responses. In the pre-existing models, a monoclonal TCR is driven by a non-physiologic promoter and randomly integrated into the genome. Here, we create a highly efficient methodology to develop T cell receptor exchange (TRex) mice, in which TCRs, specific to the self/tumor antigen mesothelin (Msln), are integrated into the Trac locus, with concomitant Msln disruption to circumvent T cell tolerance. We show that high affinity TRex thymocytes undergo all sequential stages of maturation, express the exogenous TCR at DN4, require MHC class I for positive selection and undergo negative selection only when both Msln alleles are present. By comparison of TCRs with the same specificity but varying affinity, we show that Trac targeting improves functional sensitivity of a lower affinity TCR and confers resistance to T cell functional loss. By generating P14 TRex mice with the same specificity as the widely used LCMV-P14 TCR transgenic mouse, we demonstrate increased avidity of Trac-targeted TCRs over transgenic TCRs, while preserving physiologic T cell development. Together, our results support that the TRex methodology is an advanced tool to study physiological antigen-specific T cell behavior.