Tu156 - A High-throughput Solution for Building Tcr-antigen Maps at Single Cell Resolution

Bruce Adams; Payam Shahi; Daniel Reyes; Nima Mousavi; Sreenath Krishnan; Nandhini Ramen; FuNien Tsai; Poornasree Kumar; Peter Finnegan; Sunjay Fernandes; Gunmeet Bali; Jessica Terry

Abstract Text: Understanding T-cell receptor (TCR) antigen recognition is essential to understanding the adaptive immune response to disease. However, building a TCR-antigen map is a challenge due to the formidable diversity of TCRs and antigens. To address this we developed Barcode Enabled Antigen Mapping (BEAM), a technology fully integrated with the 10x Genomics Immune Profiling Solution enabling simultaneous profiling of peptide-MHC or antigen binding to CD8+ T or B-cells, along with paired receptor sequences and gene expression. Thereby presenting a comprehensive picture of the immune-receptor, its target, and phenotypic cell state.

We demonstrate assay performance with a series of spike-in experiments with SARS CoV2 peptide-stimulated T-cells. The background was a complex mixture of 40% CMV-reactive T-cells, 10% Flu-reactive T-cells and HLA-matched human PBMCs. Cells were labeled with a panel of five viral pathogen peptide-loaded multimers along with a scrambled control peptide. The use of a negative control allows us to evaluate the specificity of binding and report antigen specificity scores for each cell.

In each experiment we profiled ~10,000 antigen-enriched T-cells and were able to identify a rare cluster of SARS CoV2 reactive CD8+ T-cell clonotypes at 1% spike-in. While the experimental design screened a mixture of 5 peptides, we observed highly-specific binding of each peptide to its target receptors. We further tested the robustness of our protocol on a variety of samples, including mice splenocytes and dissociated tumor cells.

We envisage single cell, high-throughput, and multimodal experiments such as these will underpin the rapidly evolving therapeutic and diagnostic landscape of tomorrow.