High-throughput and single-cell T cell receptor sequencing technologies

Abstract

T cells express T cell receptors (TCRs) composed of somatically recombined TCRα and TCRβ chains, which mediate recognition of major histocompatibility complex (MHC)-antigen complexes and drive the antigen-specific adaptive immune response to pathogens and cancer. The TCR repertoire in each individual is highly diverse, which allows for recognition of a wide array of foreign antigens, but also presents a challenge in analyzing this response using conventional methods. Recent studies have developed high-throughput sequencing technologies to identify TCR sequences, analyze their antigen specificities using experimental and computational tools, and pair TCRs with transcriptional and epigenetic cell state phenotypes in single cells. In this Review, we highlight these technological advances and describe how they have been applied to discover fundamental insights into T cell-mediated immunity.

Fig. 1 |. Characterization of T cell dynamics using multiomic TCR sequencing approaches.

a, TCR sequencing through bulk or single-cell methods enables profiling of TCR repertoire diversity and clonality. b, Paired TCR sequencing and profiling of gene or protein expression allows for joint analysis of T cell clonality and phenotype and enables the tracing of clones over time or throughout tissues. c, TCR sequencing combined with measuring chromatin accessibility reveals clonal epigenetic signatures and uncovers developmental trajectories that may be obscured at the level of gene or protein expression. d, TCR epitope discovery methods identify antigens driving the T cell response.

Fig. 2 |. Overview of single-cell TCR sequencing approaches.

a, Left, T cells are distributed among wells on a plate at a density of 10³–10⁵ cells per well in the pairSEQ method (top) or single T cells are sorted by FACS into individual wells (bottom). Middle, within each well, TCR transcripts are reverse transcribed, amplified and attached to well-specific barcodes. Right, after the amplified libraries are pooled and deep sequenced, TCRαβ pairings are computationally inferred by analyzing which TCRα and TCRβ sequences share the same set of well barcodes (top) or cellular barcodes (bottom). b, In emulsion-based methods, single T cells are encapsulated into droplets, where they undergo OE-PCR to enable linking of TCRα and TCRβ transcripts. Fused TCRαβ transcripts are then pooled, amplified and sequenced.

Fig. 3 |. Schematic of paired scRNA-seq and TCR sequencing methods.

a, Whole-transcriptome cDNA single-cell libraries barcoded at either the 3′ or 5′ end are subjected to gene expression profiling and targeted TCR capture with primers or hybridization probes. TCR and gene expression reads originating from the same cell can be linked on the basis of cellular barcodes. BC, barcode. b, Full-length whole-transcriptome cDNA libraries are generated from single cells by the SMART-seq or SMART-seq2 approach and sequenced. c, Computational methods for reconstructing TCR sequences from scRNA-seq libraries without targeted TCR amplification involve the alignment of reads to TCR gene reference databases or a ‘recombinome’ of possible V–J pairings. In a–c, colored lines indicate the different methods described.

Fig. 4 |. Overview of single-cell methods for linking TCR sequence to antigen specificity.

a,b, pMHC tetramers conjugated to a fluorophore (a) or magnetic nanoparticle (b) and a unique DNA barcode (DNA-BC) are incubated with a T cell population and single-cell sorted into wells (a) or encapsulated in droplets (b). In a, TCR transcripts and the pMHC DNA barcode (purple) are simultaneously captured and attached to well barcodes, allowing TCR sequences to be matched to pMHC specificity. In b, TCR transcripts are captured by cell-barcoded V region and C region primers containing the DNA barcode. Final sequenced TCR products contain both the cell barcode and pMHC DNA barcode, enabling pairing of TCRαβ sequences to antigen specificity.