841 A novel 96-well plate-based FACS-sorted single-cell approach allows the identification of immune receptor chain pairing & immunophenotyping of T or B cells

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Background Single-cell adaptive immune receptor (AIR) profiling is a revolutionary approach that allows investigators to combine clonotype repertoire identification with paired-chain information and the phenotype of cells (e.g., cell subtype). Single-cell immune receptor profiling can be performed using a medium-throughput approach (1,000–5,000 cells) using microwell arrays or droplet microfluidics platforms. However, these assays are more complicated to run, require expensive reagents, and are difficult to scale for a large number of samples. Methods In this study, we describe a cost-effective single-cell immune profiling method for identifying T-cell receptor (TCR) α/β, TCR γ/δ, or B-cell receptor (BCR) IGH/IGK/IGL chain-pairing information, high-abundant clonotype counts, along with gene expression profile of the cells. After FACS sorting T or B cells in a 96-well plate, the primers for immune receptor profiling and 30 crucial T- and B-cell markers are added with the buffer. This is followed by multiplex RT-PCR amplification and sequencing of the CDR3 regions or full-length receptor regions. The assay contains UMIs to allow unbiased, quantitative amplification of clonotypes. Immune receptor profiling data is analyzed with the MiXCR software pipeline and T- and B-cell marker gene expression profiling data is analyzed with the Salmon/Alevin aligner tool. Results We obtained the sequence of each clonotype along with the chain pairing information for these α/β, γ/δ or IGH/IGK/IGL chains, together with the T- and B-cell subtype information using gene-expression profiles. The assay is optimized for low crossover between wells and the UMI-based uniform clonotype detection allows accurate identification of sorted T- and B-cells, e.g. antigen-activated T-cells labeled with specific MHC-peptide antigens. Conclusions This first-of-its-kind, 96-well plate-based single-cell AIR profiling assay combines TCR/BCR chain sequencing paired-chain information and the phenotype of cells (e.g., cell subtype) in a single multiplex assay without the need for any specialized instrument such as microwell arrays or droplet microfluidics-based single-cell platforms. This allows cost-effective single-cell profiling without the need for expensive reagents or chips. The single-cell TCR/BCR gene arrangement information could be used to develop T-cell-based cancer therapies and synthesize antibodies with the help of BCR IGH/IGK/IGL chain pair information which could be an effective immunotherapy strategy for cancer and autoimmune diseases.

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