Adoptive immune therapies based on the transfer of antigen-specific T cells have been used successfully to treat various cancers and viral infections, but improved techniques are needed to identify optimally protective human T cell receptors (TCRs). Here we present a high-throughput approach to the identification of natively paired human TCRα and TCRβ (TCRα:β) genes encoding heterodimeric TCRs that recognize specific peptide antigens bound to major histocompatibility complex molecules (pMHCs). We first captured and cloned TCRα:β genes from individual cells, ensuring fidelity using a suppression PCR.
View Article and Find Full Text PDFNew approaches in high-throughput analysis of immune receptor repertoires are enabling major advances in immunology and for the discovery of precision immunotherapeutics. Commensurate with growth of the field, there has been an increased need for the establishment of techniques for quality control of immune receptor data. Our laboratory has standardized the use of multiple quality control techniques in immunoglobulin (IG) and T-cell receptor (TR) sequencing experiments to ensure quality control throughout diverse experimental conditions.
View Article and Find Full Text PDFFunctional analyses of the T cell receptor (TCR) landscape can reveal critical information about protection from disease and molecular responses to vaccines. However, it has proven difficult to combine advanced next-generation sequencing technologies with methods to decode the peptide-major histocompatibility complex (pMHC) specificity of individual TCRs. We developed a new high-throughput approach to enable repertoire-scale functional evaluations of natively paired TCRs.
View Article and Find Full Text PDFUnderstanding mechanisms of protective antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We report a monoclonal antibody, 910-30, targeting the SARS-CoV-2 receptor-binding site for ACE2 as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. Sequence and structural analyses of 910-30 and related antibodies explore how class recognition features correlate with SARS-CoV-2 neutralization.
View Article and Find Full Text PDFBiotin-labeled molecular probes, comprising specific regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. Here, we design constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions include full-length spike ectodomain as well as various subregions, and we also design mutants that eliminate recognition of the angiotensin-converting enzyme 2 (ACE2) receptor.
View Article and Find Full Text PDFEmulsion-based techniques have dramatically advanced our understanding of single-cell biology and complex single-cell features over the past two decades. Most approaches for precise single cell isolation rely on microfluidics, which has proven highly effective but requires substantial investment in equipment and expertise that can be difficult to access for researchers that specialize in other areas of bioengineering and molecular biotechnology. Inspired by the robust droplet generation technologies in modern flow cytometry instrumentation, here we established a new platform for high-throughput isolation of single cells within droplets of tunable sizes by combining flow focusing with ultrasonic vibration for rapid and effective droplet formation.
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