A new high-throughput sequencing method for determining diversity and similarity of T cell receptor (TCR) α and β repertoires and identifying potential new invariant TCR α chains

BMC Immunology, Oct 2016

Background High-throughput sequencing of T cell receptor (TCR) genes is a powerful tool for analyses of antigen specificity, clonality and diversity of T lymphocytes. Here, we developed a new TCR repertoire analysis method using 454 DNA sequencing technology in combination with an adaptor-ligation mediated polymerase chain reaction (PCR). This method allows the amplification of all TCR genes without PCR bias. To compare gene usage, diversity and similarity of expressed TCR repertoires among individuals, we conducted next-generation sequencing (NGS) of TRA and TRB genes in peripheral blood mononuclear cells from 20 healthy human individuals. Results From a total of 267,037 sequence reads from 20 individuals, 149,216 unique sequence reads were identified. Preferential usage of several V and J genes were observed while some recombinations of TRAV with TRAJ appeared to be restricted. The extent of TCR diversity was not significantly different between TRA and TRB, while TRA repertoires were more similar between individuals than TRB repertoires were. The interindividual similarity of TRA depended largely on the frequent presence of shared TCRs among two or more individuals. A publicly available TRA had a near-germline TCR with a shorter CDR3. Notably, shared TRA sequences, especially those shared among a large number of individuals’, often contained TCRα related with invariant TCRα derived from invariant natural killer T cells and mucosal-associated invariant T cells. Conclusion These results suggest that retrieval of shared TCRs by NGS would be useful for the identification of potential new invariant TCRα chains. This NGS method will enable the comprehensive quantitative analysis of TCR repertoires at a clonal level.

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A new high-throughput sequencing method for determining diversity and similarity of T cell receptor (TCR) α and β repertoires and identifying potential new invariant TCR α chains

Kitaura et al. BMC Immunology A new high-throughput sequencing method for determining diversity and similarity of T cell receptor (TCR) α and β repertoires and identifying potential new invariant TCR α chains Kazutaka Kitaura 2 Tadasu Shini 0 1 Takaji Matsutani 2 Ryuji Suzuki 1 2 0 BITS. Co., Ltd , Tokyo , Japan 1 Department of Rheumatology and Clinical Immunology, Clinical Research Center for Rheumatology and Allergy, Sagamihara National Hospital, National Hospital Organization , Sagamihara , Japan 2 Repertoire Genesis Incorporation , 104 Saito-Bioincubator, 7-7-15, Saito-asagi, Ibaraki, Osaka 567-0085 , Japan Background: High-throughput sequencing of T cell receptor (TCR) genes is a powerful tool for analyses of antigen specificity, clonality and diversity of T lymphocytes. Here, we developed a new TCR repertoire analysis method using 454 DNA sequencing technology in combination with an adaptor-ligation mediated polymerase chain reaction (PCR). This method allows the amplification of all TCR genes without PCR bias. To compare gene usage, diversity and similarity of expressed TCR repertoires among individuals, we conducted next-generation sequencing (NGS) of TRA and TRB genes in peripheral blood mononuclear cells from 20 healthy human individuals. Results: From a total of 267,037 sequence reads from 20 individuals, 149,216 unique sequence reads were identified. Preferential usage of several V and J genes were observed while some recombinations of TRAV with TRAJ appeared to be restricted. The extent of TCR diversity was not significantly different between TRA and TRB, while TRA repertoires were more similar between individuals than TRB repertoires were. The interindividual similarity of TRA depended largely on the frequent presence of shared TCRs among two or more individuals. A publicly available TRA had a near-germline TCR with a shorter CDR3. Notably, shared TRA sequences, especially those shared among a large number of individuals', often contained TCRα related with invariant TCRα derived from invariant natural killer T cells and mucosal-associated invariant T cells. Conclusion: These results suggest that retrieval of shared TCRs by NGS would be useful for the identification of potential new invariant TCRα chains. This NGS method will enable the comprehensive quantitative analysis of TCR repertoires at a clonal level. T cell receptor; Repertoire; Next generation sequencing; Immune profiling; Invariant TCRα - Background The term T cell repertoire describes a collection of lymphocytes characterized by T cell receptor (TCR) expression, which plays a critical role in antigen recognition. Since alterations of the T cell repertoire provide a significant indication of immune status in physiological and disease conditions, T cell repertoire analyses have been conducted for the identification of antigen-specific T cells involved in the development of disease and for the diagnosis of T lymphocyte abnormalities. Comparison of variable-region usage by fluorescence-activated cell sorter analysis using a large panel of antibodies specific for TCR variable regions [1–4], polymerase chain reaction (PCR) with multiple primers [5] or PCR-based enzyme-linked immunosorbent assay [6, 7] have been widely used to detect changes in T cell repertoire. Length distribution analysis known as CDR3 spectratyping is based on the addition of non-template nucleotides in V-(D)-J region © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. and has been used to evaluate T cell clonality and diversity [8, 9]. To identify the antigen specificity of T cells further, PCR cloning of TCR clonotypes and subsequent sequence determination of the antigen recognition region, CDR3, have been required. These conventional approaches are commonly used but are time-consuming and a laborious way to study TCR repertoires. In recent years, advances in high-throughput sequencing technologies known as next-generation sequencing (NGS) have rapidly progressed and enabled large-scale analysis of sequence data [10, 11]. Although several NGS-based TCR repertoire analysis systems have been developed by other researches, many amplification techniques are based on multiple PCR with different primers specific for each variable region. Thus, bias during PCR amplification is unavoidable since bias is most commonly due to differential hybridization kinetics among variable region-specific primers to different target gen (...truncated)


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Kazutaka Kitaura, Tadasu Shini, Takaji Matsutani, Ryuji Suzuki. A new high-throughput sequencing method for determining diversity and similarity of T cell receptor (TCR) α and β repertoires and identifying potential new invariant TCR α chains, BMC Immunology, 2016, pp. 38, 17, DOI: 10.1186/s12865-016-0177-5