Paired analysis of TCRα and TCRβ chains at the single-cell level in mice

被引:158
作者
Dash, Pradyot
McClaren, Jennifer L.
Oguin, Thomas H., III
Rothwell, William
Todd, Brandon
Morris, Melissa Y.
Becksfort, Jared
Reynolds, Cory
Brown, Scott A.
Doherty, Peter C. [2 ]
Thomas, Paul G. [1 ]
机构
[1] St Jude Childrens Res Hosp, Dept Immunol, Memphis, TN 38105 USA
[2] Univ Melbourne, Dept Microbiol & Immunol, Melbourne, Vic, Australia
关键词
T-CELL; INFLUENZA-VIRUS; REPERTOIRE DIVERSITY; PREMATURE TERMINATION; ALLELIC EXCLUSION; RECALL RESPONSES; PCR ANALYSIS; IN-VIVO; AUTOIMMUNITY; INFECTION;
D O I
10.1172/JCI44752
中图分类号
R-3 [医学研究方法]; R3 [基础医学];
学科分类号
1001 ;
摘要
Characterizing the TCR alpha and TCR beta chains expressed by T cells responding to a given pathogen or underlying autoimmunity helps in the development of vaccines and immunotherapies, respectively However, our understanding of complementary TCR alpha and TCR beta chain utilization is very limited for pathogen- and auto-antigen-induced immunity To address this problem, we have developed a multiplex nested RT-PCR method for the simultaneous amplification of transcripts encoding the TCR alpha and TCR beta chains from single cells This multiplex method circumvented the lack of antibodies specific for variable regions of mouse TCR alpha chains and the need for prior knowledge of variable region usage in the TCR beta chain, resulting in a comprehensive, unbiased TCR repertoire analysis with paired coexpression of TCR alpha and TCR beta chains with single-cell resolution Using CD8(+) CTLs specific for an influenza epitope recovered directly from the pneumonic lungs of mice, this technique determined that 25% of such effectors expressed a dominant, nonproductively rearranged TCRa transcript T cells with these out-of-frame TCRa mRNAs also expressed an alternate, in-frame TCRa, whereas approximately 10% of T cells had 2 productive TCRa transcripts The proportion of cells with biallelic transcription increased over the course of a response, a finding that has implications for immune memory and autoimmunity This technique may have broad applications in mouse models of human disease
引用
收藏
页码:288 / 295
页数:8
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