Profiling the T-cell receptor beta-chain repertoire by massively parallel sequencing

Abstract

T-cell receptor (TCR) genomic loci undergo somatic V(D)J recombination, plus the addition/subtraction of nontemplated bases at recombination junctions, in order to generate the repertoire of structurally diverse T cells necessary for antigen recognition. TCR beta subunits can be unambiguously identified by their hypervariable CDR3 (Complement Determining Region 3) sequence. This is the site of V(D)J recombination encoding the principal site of antigen contact. The complexity and dynamics of the T-cell repertoire remain unknown because the potential repertoire size has made conventional sequence analysis intractable. Here, we use 59-RACE, Illumina sequencing, and a novel short read assembly strategy to sample CDR3 b diversity in human T lymphocytes from peripheral blood. Assembly of 40.5 million short reads identified 33,664 distinct TCR b clonotypes and provides precise measurements of CDR3 b length diversity, usage of nontemplated bases, sequence convergence, and preferences for TRBV (T-cell receptor beta variable gene) and TRBJ (T-cell receptor beta joining gene) gene usage and pairing. CDR3 length between conserved residues of TRBV and TRBJ ranged from 21 to 81 nucleotides (nt). TRBV gene usage ranged from 0.01% for TRBV17 to 24.6% for TRBV20-1. TRBJ gene usage ranged from 1.6% for TRBJ2-6 to 17.2% for TRBJ2-1. We identified 1573 examples of convergence where the same amino acid translation was specified by distinct CDR3 b nucleotide sequences. Direct sequence-based immunoprofiling will likely prove to be a useful tool for understanding repertoire dynamics in response to immune challenge, without a priori knowledge of antigen.

Reliable detection of subclonal single-nucleotide variants in tumour cell populations

“…It may be utilised for the analysis of pathogen populations, such as viruses or bacteria, for the assessment of T-cell diversity 22 , or for detecting rare somatic mutations associated with diseases, such as the Proteus syndrome 23 . Another application is the cost-effective pooled sequencing of multiple individuals.…”

Predicting the spectrum of TCR repertoire sharing with a data‐driven model of recombination

“…In this hypothesis, shared TCRs are simply those that have a higher‐than‐average generation probability and are thus more abundant in the unselected repertoire 13. The advent of high‐throughput sequencing of TCR repertoires14, 15, 16, 17 has largely confirmed this view through the analysis of shared TCR sequences between unrelated humans,18, 19, 20 monozygous human twins,21, 22 and mice 23. However, despite recent efforts to characterize the landscape of public TCRs,24 the contributions of V(D)J generation biases and convergent recombination relative to convergent selection remain to be elucidated and quantified.…”

High-throughput T cell receptor sequencing reveals distinct repertoires between tumor and adjacent non-tumor tissues in HBV-associated HCC

“…Notably, TRBV20-1 and TRBJ2-1 were also the most frequent gene segments in other studies of tumors and healthy subjects. 18,19 The usage patterns of Vb and Jb gene segments were similar between tumors and adjacent non-tumor tissues According to the heatmap of VJ gene segments (Fig. 2), the usage patterns of Vb and Jb gene segments were similar between tumors and adjacent non-tumor tissues, whereas some Vb and Jb gene segments exhibited significantly higher frequency in non-tumor tissues, such as TRBV20-1, TRBV9, TRBV10-3, TRBV5-4, TRBV10-2, and TRBJ2-7, TRBJ2-1, and TRBJ1-5.…”

“…The diversity of TCRs originates from the random combination of variable (V) and joining (J) gene segments in the a-chain and V, diversity (D) and J gene segments in the b-chain germline gene. Random insertions and deletions of non-template nucleotides at the junctions of V-(D)-J gene segments further increase the diversity of TCRs, and up to 10 18 TCR types have been reported. The specificity and diversity of TCRs predominantly depend on the complementarity determining region 3 (CDR3), which is encoded by V(D)J recombination and interacts with the peptide presented by the major histocompatibility complex (MHC).…”