Massively Scalable Single-cell Multiomic Profiling of T Cell Repertoire with REFLEX

Research Square 2025

P. Skene, M. Hart, Z. Thomson, Saransh N. Kaul, Saskia Ilkisin, M. Landreau, Tyanna J. Stuckey, Peter J. Wittig et al.

Abstract

Single-cell profiling of T cell state with immune repertoire is critical for understanding heterogenous T cell phenotypes and responses to antigen, however, existing technologies struggle to generate this information at sufficient throughput to match biological complexity. We present “REFLEX”, a novel single-cell method enabling highly scalable, cost-efficient, multiomic profiling with paired-chain TCR sequencing. REFLEX utilizes in-situ reverse transcription with integrated sample multiplexing barcodes in a way that merges seamlessly with the commonly used 10x FLEX platform to allow capture of TCR sequences at unprecedented scale and depth. We profile >2 million cells from CMV-peptide-pulsed T cell expansions, capturing TCR sequences and rich multiomic information from 1.4M T cells, identifying many putative novel CMV reactive clonotypes and illustrating the scale and transformative impact on our understanding of T cell mediated adaptive immunity achievable with REFLEX.

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