High-resolution analysis of the human T-cell receptor repertoire.

Eliana Ruggiero,Manfred Schmidt,Anna Paruzynski,Raffaele Fronza,Sergij Goerdt,Ali Nowrouzi,Christina Lulay,Anne Arens,Jan P Nicolay,Peter H Krammer,Gökçe Ürenden,Christof Von Kalle,Hanno Glimm,Sven Schneider

doi:10.1038/ncomms9081

Abstract

Unbiased dissection of T-cell receptor (TCR) repertoire diversity at the nucleotide level could provide important insights into human immunity. Here we show that TCR ligation-anchored-magnetically captured PCR (TCR-LA-MC PCR) identifies TCR α- and β-chain diversity without sequence-associated or quantitative restrictions in healthy and diseased conditions. TCR-LA-MC PCR identifies convergent recombination events, classifies different stages of cutaneous T-cell lymphoma in vivo and demonstrates TCR reactivation after in vitro cytomegalovirus stimulation. TCR-LA-MC PCR allows ultra-deep data access to both physiological TCR diversity and mechanisms influencing clonality in all clinical settings with restricted or distorted TCR repertoires.

Highlights

Unbiased dissection of T-cell receptor (TCR) repertoire diversity at the nucleotide level could provide important insights into human immunity
The naive TCR repertoire is assembled in the thymus and formed by somatic rearrangements of non-contiguous genes belonging to the variable (V), diversity (D; only for the b- and d-chain) and joining (J) families
We show the identification of the events leading to the generation of the TCR diversity in healthy donors (HDs), discrimination of different stages of cutaneous T-cell lymphoma in vivo and monitoring of the reactivation of specific TCR clones after in vitro stimulation with peptide pools of two immunodominant cytomegalovirus (CMV) antigens, providing high-resolution characterization of TCR clonality repertoires

Summary

Introduction

Unbiased dissection of T-cell receptor (TCR) repertoire diversity at the nucleotide level could provide important insights into human immunity. Correspondence and requests for materials should be addressed to C.v.K. The naive TCR repertoire is assembled in the thymus and formed by somatic rearrangements of non-contiguous genes belonging to the variable (V), diversity (D; only for the b- and d-chain) and joining (J) families (combinatorial diversity). To obtain the highest possible resolution at high specificity, we have made use of previously established PCR-based methods: ligationanchored (LA) PCR10,11 and non-restrictive linear amplificationmediated PCR12–14, and developed TCR-LA-MC PCR as a new sequencing technology for the immunogenetic characterization of normal and reduced clonality in the a- and b-chain TCR repertoire. We show the identification of the events leading to the generation of the TCR diversity in healthy donors (HDs), discrimination of different stages of cutaneous T-cell lymphoma in vivo and monitoring of the reactivation of specific TCR clones after in vitro stimulation with peptide pools of two immunodominant cytomegalovirus (CMV) antigens, providing high-resolution characterization of TCR clonality repertoires

Methods

Results

Conclusion