Identification of antigen-specific TCR sequences based on biological and statistical enrichment in unselected individuals.

Neal P Smith,Bert Ruiter,Brinda Monian,Yamini V Virkud,James J Moon,Wayne G Shreffler,Ang A Tu,J Christopher Love

doi:10.1172/jci.insight.140028

Abstract

Recent advances in high-throughput T cell receptor (TCR) sequencing have allowed for new insights into the human TCR repertoire. However, methods for capturing antigen-specific repertoires remain an area of development. Here, we describe a potentially novel approach that utilizes both a biological and statistical enrichment to define putatively antigen-specific complementarity-determining region 3 (CDR3) repertoires in unselected individuals. The biological enrichment entailed FACS of in vitro antigen-activated memory CD4+ T cells, followed by TCRβ sequencing. The resulting TCRβ sequences were then filtered by selecting those that are statistically enriched when compared with their frequency in the autologous resting T cell compartment. Applying this method to define putatively peanut protein–specific repertoires in 27 peanut-allergic individuals resulted in a library of 7345 unique CDR3β amino acid sequences that had similar characteristics to other validated antigen-specific repertoires in terms of homology and diversity. In-depth analysis of these CDR3βs revealed 36 public sequences that demonstrated high levels of convergent recombination. In a network analysis, the public CDR3βs were shown to be core sequences with more edges than their private counterparts. This method has the potential to be applied to a wide range of T cell–mediated disorders and to yield new biomarkers and biological insights.

Highlights

T cells are defined by their antigen-specific T cell receptor (TCR), and the collection of all TCRs in a human, which is dynamic and comprises approximately 1010 unique TCRs at a given time, is known as their TCR repertoire [1]
T cells, and TCR CDR3β sequences were generated from both populations
To filter out sequences that were likely to be present in the CD154+ population due to bystander activation, we developed a statistical enrichment strategy to define ps-complementaritydetermining region 3 (CDR3)

Summary

Introduction

T cells are defined by their antigen-specific T cell receptor (TCR), and the collection of all TCRs in a human, which is dynamic and comprises approximately 1010 unique TCRs at a given time, is known as their TCR repertoire [1]. TCRs are dimeric proteins, comprising an α and β chain that are both generated through the process of genomic rearrangement of germline variable (V), diversity (D), and joining (J) genes concurrent with random nucleotide insertions and deletions in the VDJ junction This junction, known as the complementarity-determining region 3 (CDR3), interacts most closely with an epitope during antigen presentation, and is the primary focus of studies aiming to elucidate the mechanisms of TCR specificity [2]. The advantage of tetramer selection is the inherent functional validity it provides, as it labels cells specific for a single well-defined epitope It comes with a major drawback in the form of a limited scope of analysis. Most immune responses are polyantigenic, and current T cell epitope mapping information on most antigens is incomplete

Methods

Results

Conclusion