Th179 - Single-cell TCR 𝛼/𝛽 and TCR 𝛾/𝛿 Immune Receptor Analysis and Immunotyping in a 96-well Plate

Tianbing Liu – Cellecta, Inc.; Mikhail Makhanov – Cellecta, Inc.; Dongfang Hu – Cellecta, Inc.; Lester Kobzik – lkobzik@cellecta.com; Khadija Ghias – Cellecta, Inc.; Paul Diehl – Cellecta, Inc.

Abstract Text: This study describes an easy-to-run, low-throughput, single-cell immune profiling methodology by FACS sorting single cells in a 96-well plate to assess the clonotype repertoire diversity, paired-chain information, and cell phenotypes using a multiplex RT-PCR-based assay without any specialized equipment. This is an alternative cost-effective and convenient workflow to conventional microwell arrays or droplet microfluidics-based single-cell workflows. The 96-well plate is pre-loaded with primers for T-cell receptor (TCR) 𝛼/𝛽 or 𝛾/𝛿, alongside 30 key T-cell markers. T cells from PBMCs are sorted in a single cell per well format. We conduct RT-PCR library prep using the DriverMap™ AIR Profiling Assay and sequence the CDR3 region. The results show clonotype frequencies, chain pairing details for 𝛼/𝛽 and 𝛾/𝛿 chains, and T-cell subtype classifications based on gene-expression profiling. This approach facilitates in-depth analysis of TCR gene rearrangements at the single-cell level, enhancing the understanding of T cell development, proliferation, and clonality, essential in studying cancer, immunodeficiencies, and autoimmune disorders. Additionally, we can effectively identify and characterize γδ T cells by integrating single-cell TCR sequencing with RNA sequencing data to potentially develop γδ cancer immunotherapies. In summary, this method provides a cost-effective and convenient workflow to analyze paired clonotypes and immunophenotypes in a single 96-well plate assay.