Analyzing the CDR3 Repertoire with respect to TCR-Beta Chain V-D-J and V-J Rearrangements in Peripheral T Cells using HTS.

Long Ma,Aihua Peng,Yuehong Li,Liwen Yang,Teng Zhang,Suhong Sun,Bin Shi Bin Shi,Xiaoyan He,Xinsheng Yao,Rui Ma

doi:10.1038/srep29544

Long Ma, Aihua Peng + Show 8 more

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https://doi.org/10.1038/srep29544

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Abstract

V-D-J rearrangement of the TCR—beta chain follows the 12/23 rule and the beyond 12/23 restriction. Currently, the proportion and characteristics of TCR—beta chain V—J rearrangement is unclear. We used high-throughput sequencing to compare and analyze TCR—beta chain V-J rearrangement and V-D-J rearrangement in the CDR3 repertoires of T cells from the PBMCs of six volunteers and six BALB/c mice. The results showed that the percentage of V-J rearrangement of the volunteers was approximately 0.7%, whereas that of the mice was 2.2%. The clonality of mice V-J rearrangement was significantly reduced compared with the V-D-J rearrangement, whereas the clonality of human V-J rearrangement was slightly reduced compared with the V-D-J rearrangement. V-J rearrangement in CDR3 involved the significant usage of N, S, F and L, whereas V-D-J rearrangement in CDR3 involved the significant usage of R and G. The levels of V deletion and J deletion in V-J rearrangement were significantly reduced compared with V-D-J rearrangement. TRBD and TRBJ usage in V-J rearrangement differed from that of V-D-J rearrangement, including dominant usage of TRBV and TRBJ and their pairing. Taken together, these results provide new ideas and technology for studies of V-D-J rearrangement and V-J rearrangement in the CDR3 repertoire.

Highlights

The alpha-beta and gamma-delta TCRs of humans and mice are formed by germ line genetic rearrangement of the variable (V), diversity (D), joining (J), and constant (C) regions
In 2000, Bassing CH et al.[4] used embryonic stem cells containing only the TCR beta gene locus modified by TRBD1 and TRBJ1 fragments to make a BALB/c mouse model (BALB/c mice with no TRBD2 and TRBJ2 cluster: TRBJ1 M3 allele) and found that TRBV mainly rearranged with TRBD1 but not with TRBJ1; the additional B 23/12 restriction played an important role in the regulation of V gene rearrangement
In a mechanistic study of the beyond 12/23 (B 12/23) restriction, Tillman RE et al.[7,8] confirmed that in the case of non-lymphocyte (CHO cell line) expressing recombinant activated gene protein (RAG) 1 and 2, TRBV 23-RSS exhibited more rearrangement with TRBD 12-RSS compared with TRBJ 12-RSS rearrangement, and this bias may correlate with preferential combining of 5′TRBD 12-RSS and RAG

Summary

Introduction

The alpha-beta and gamma-delta TCRs of humans and mice are formed by germ line genetic rearrangement of the variable (V), diversity (D), joining (J), and constant (C) regions. Previous studies have examined the 12/23 rule and the beyond 12/23 restriction (B 23/12 restriction) of TCR beta chain rearrangement. Using 5′TRBJ1.2 12-RSS to replace TRBD1 12-RSS, TRBV directly rearranged with the modified TRBJ1.2. In a mechanistic study of the B 12/23 restriction, Tillman RE et al.[7,8] confirmed that in the case of non-lymphocyte (CHO cell line) expressing recombinant activated gene protein (RAG) 1 and 2, TRBV 23-RSS exhibited more rearrangement with TRBD 12-RSS compared with TRBJ 12-RSS rearrangement (following B 12/23 restriction), and this bias may correlate with preferential combining of 5′TRBD 12-RSS and RAG. Regarding the efficiency of the B 12/23 restriction, Jung, D CHO and 3T3 cell (plasmid transfection experiments in vitro) models revealed that TRBV 23-RSS mainly rearranges with TRBD 12-RSS and rarely with TRBJ 12-RSS. In 2014, Peepei Liu et al.[16] used high-throughput sequencing (HTS) to detect the alpha-beta TCR complementarity determining region 3 (CDR3) repertoire in a large number of CDR3 sequences from 3 volunteers and found that approximately 2% of sequences exhibited a fusion rearrangement of TRBD genes in the TCR beta chain (or TRBD tandem rearrangement)

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