An integrated reagent and multimodal analysis workflow to enrich and characterize peptide-specific CD8+T cells

Abstract Immune responses to disease are complex and the only way to understand such a response is to consider the behavior of multiple different cell types across both the innate and adaptive branches of the immune system. We developed Barcode Enabled Antigen Mapping (BEAM) technology to enable easy enrichment of antigen-specific cells. This technology is fully integrated with the 10x Genomics Chromium Single Cell Immune Profiling Solution to allow multimodal evaluation of immune cells including full-length, paired sequences of T and B cell receptor genes while simultaneously screening their specificity for a set of genes. Our antigen-specific TCR workflow (BEAM-T) identifies antigen-specific CD8+T cells. We demonstrate the assay’s performance with spike-in experiments using various percentages of HLA-A*02:01-restricted peptide-stimulated T cells (targeted mixture being 40% T cells with specificity for cytomegalovirus peptide, 10% T cells with specificity for flu peptide and 0–5% anti-SARS-CoV-2 T cells) into a background of human PBMCs. Cells were labeled with a panel of five viral pathogen peptide-loaded multimers, plus a HLA-A*02:01 negative control peptide to assess antigen specificity and were then sorted for CD8+PE+ T cells. We demonstrated that the major VDJ-T clonotypes identified for the SARS-CoV2 cells were retained, albeit with roughly proportionally fewer cells, as the percentage of spiked cells decreased. We further feature the robustness of the protocol with various sample types and models including H2Kb-restricted OT-1 transgenic mouse splenocytes, as well as healthy human donor PBMCs and dissociated tumor cells to demonstrate the utility of an integrated reagent-to-data analysis offering across a range of samples.