Linking the T cell receptor to the single cell transcriptome in antigen‐specific human T cells

Abstract

Heterogeneity of T cells is a hallmark of a successful adaptive immune response, harnessing the vast diversity of antigen-specific T cells into a coordinated evolution of effector and memory outcomes. The T cell receptor (TCR) repertoire is highly diverse to account for the highly heterogeneous antigenic world. During the response to a virus multiple individual clones of antigen specific CD8+ (Ag-specific) T cells can be identified against a single epitope and multiple epitopes are recognised. Advances in single-cell technologies have provided the potential to study Ag-specific T cell heterogeneity at both surface phenotype and transcriptome levels, thereby allowing investigation of the diversity within the same apparent sub-population. We propose a new method (VDJPuzzle) to reconstruct the native TCRαβ from single cell RNA-seq data of Ag-specific T cells and then to link these with the gene expression profile of individual cells. We applied this method using rare Ag-specific T cells isolated from peripheral blood of a subject who cleared hepatitis C virus infection. We successfully reconstructed productive TCRαβ in 56 of a total of 63 cells (89%), with double α and double β in 18, and 7% respectively, and double TCRαβ in 2 cells. The method was validated via standard single cell PCR sequencing of the TCR. We demonstrate that single-cell transcriptome analysis can successfully distinguish Ag-specific T cell populations sorted directly from resting memory cells in peripheral blood and sorted after ex vivo stimulation. This approach allows a detailed analysis of the TCR diversity and its relationship with the transcriptional profile of different clones.

Single-Cell RNA Sequencing Reveals Expanded Clones of Islet Antigen-Reactive CD4+ T Cells in Peripheral Blood of Subjects with Type 1 Diabetes

“…We tested this hypothesis by comparing individual cell and bulk profiles from BRI-4.13 (19), an islet antigen-reactive CD4+ T cell clone from an individual with T1D (Materials and Methods). Although single cell transcript profiling has been successfully used with several cell types (36), less is known (37) about the performance of single cell techniques with antigen-specific T cells, which contain very limited amounts of RNA. In our experiments, we detected non-linearity between individual cell and bulk profiles in expression of low abundance genes, indicating that genes expressed at low levels are less likely to be detected at the single cell level than in a bulk measurement (Figure 1A).…”

BASIC: BCR assembly from single cells

“…1A), which is generated through a series of somatic rearrangements and mutations that allow for more than 10 18 possible sequences (Davies et al , 1975; Edelman, 1959; Elhanati et al , 2015). Single-cell RNA-sequencing (scRNA-seq) offers a particularly powerful way to study adaptive immune cells, simultaneously allowing both for capture of quantitative gene expression as well as repertoire sequence information (Eltahla et al , 2016; Stubbington et al , 2016). By coupling gene expression with specific BCRs, exciting opportunities emerge in studying B-cell biology that are not approachable with bulk RNA-seq and repertoire studies, such as characterizing at single-cell resolution the transcriptional heterogeneity underlying BCR affinity maturation and receptor specificity.…”

Single-Cell Genomics: Approaches and Utility in Immunology

“…TraCeR [62] was the first tool for T cell receptor (TCR) reconstruction from scRNA-seq, followed quickly by VDJPuzzle [63]. Whereas, BASIC is currently the only algorithm designed specifically for B cells and the only approach that performs its own assembly of the V(D)J receptor sequence [64].…”

“…Already, this innovative combination showed that the T cell clonotypes responding to a murine Salmonella infection come from diverse T cell compartments and it identified a previously unappreciated repertoire diversity within HCV specific T cells before and after ex vivo cell culture [62,63]. Prior interrogation of both questions was impossible, and it will be very exciting to see what else this powerful new combination brings in the future.…”