Paired analysis of TCRα and TCRβ chains at the single-cell level in mice
Pradyot Dash, … , Peter C. Doherty, Paul G. Thomas
Pradyot Dash, … , Peter C. Doherty, Paul G. Thomas
Published December 6, 2010
Citation Information: J Clin Invest. 2011;121(1):288-295. https://doi.org/10.1172/JCI44752.
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Technical Advance Immunology

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

Abstract

Characterizing the TCRα and TCRβ 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 TCRα and TCRβ chain utilization is very limited for pathogen- and autoantigen-induced immunity. To address this problem, we have developed a multiplex nested RT-PCR method for the simultaneous amplification of transcripts encoding the TCRα and TCRβ chains from single cells. This multiplex method circumvented the lack of antibodies specific for variable regions of mouse TCRα chains and the need for prior knowledge of variable region usage in the TCRβ chain, resulting in a comprehensive, unbiased TCR repertoire analysis with paired coexpression of TCRα and TCRβ 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.

Authors

Pradyot Dash, Jennifer L. McClaren, Thomas H. Oguin III, William Rothwell, Brandon Todd, Melissa Y. Morris, Jared Becksfort, Cory Reynolds, Scott A. Brown, Peter C. Doherty, Paul G. Thomas

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Figure 1

Unbiased single-cell amplification of TCR CDR3α and CDR3β.

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Unbiased single-cell amplification of TCR CDR3α and CDR3β. (A) Schematic...
(A) Schematic diagram of the multiplex PCR method used to simultaneously amplify and sequence the TCR CDR3α and CDR3β regions. Following single-cell sorting of KbPB1703+CD8+ T cells into a PCR plate, the first round of PCR used a primer mixture of 23 TRAV and 19 TRBV forward and single TRAC and TRBC reverse primers. Subsequently, a nested PCR was performed for α and β in a separate plate using a corresponding internal primer mix (23 TRAV forward, single TRAC reverse, and 19 TRBV forward, single TRBC reverse, respectively). (B) Schematic representation of the TCR CDR3 region, showing the relative positions of the oligonucleotide primers. An agarose gel electrophoresis image of TCR segments containing CDR3α and CDR3β amplified from single KbPB1703+CD8+ T cells is also shown. L, 100-bp ladder lane. The α and β products were loaded alternately in each twin-lane (separated by vertical lines). Negative control PCR reactions (for contamination) without any cDNA are shown in the boxed region. (C) TRBV usage in the primary KbPB1703+CD8+ T cell response determined by multiplex RT-PCR and sequencing (n = 9 mice). (D) Correlation of the data in C to data acquired by costaining tetramer-specific KbPB1703+CD8+ T cells with a panel of anti-TRBV antibodies.