Single-cell immune receptor 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 technologies. However, these assays are more complicated to run and require expensive reagents and limited sequencing throughput compared to bulk immune receptor profiling methods. Here, we describe a single-cell immune profiling strategy using FACS-sorted cells in 96-well plates. The plate is pre-aliquoted with primers for both T-cell receptor (TCR) α/β or TCR γ/δ chains along with 30 crucial T-cell markers. We then perform multiplex RT-PCR amplification and next-generation sequencing (NGS) of the CDR3 regions to obtain adaptive immune receptor repertoire profile and immunophenotype information.
The resulting data provides counts of the most abundant clonotypes, along with chain pairing information for the α/β and γ/δ chains, together with the T-cell subtype information obtained from the gene-expression profiles. By analyzing TCR gene rearrangement at the single-cell level, researchers can better understand T-cell development, proliferation, and clonality, which are crucial for studying diseases such as cancer, immunodeficiency, and autoimmunity. Furthermore, single-cell TCR sequencing when combined with RNA sequencing datasets, facilitates the identification of γδ T cells. As such, this method could serve as a standardized tool for identifying potential γδ T cell-based cancer immunotherapies. The technology's cost-effectiveness and ability to analyze clonotypes and immunophenotypes of cells in a single assay could make it a valuable tool for unraveling the adaptive immune dynamics in many diseases.