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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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 3

Biallelic expression of TCRα in KbPB1703+CD8+ T cells.

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Biallelic expression of TCRα in KbPB1703+CD8+ T cells. (A) Split RT-...
(A) Split RT-PCR of single cells shows both mono- (m) and biallelic (b) TCRα expression. The cDNA from a single cell was split 3 ways, and 2 rounds of PCR were used to amplify the CDR3α, as shown by agarose gel electrophoresis (vertical lines separate triplet-lanes). Some cells contained transcript from both alleles, others from a single allele. Most cells with nonproductive transcripts (bold) also had in-frame transcripts. (B) Schematic representation of the PCR sequencing-cloning-sequencing method used to identify the alternate allele. The PCR products (from single cells) that showed either an out-of-frame, in-frame, or unreadable overlap Tcra transcript sequence pairing with the identical CDR3β were cloned using TA cloning, and multiple products were sequenced. (C) The percentage of cells that had 2 transcripts (dual–in-frame and in-frame/out-of-frame) was 35%. In addition, approximately 42% and 23% of all cells analyzed had a monoallelic productive or nonproductive transcript, respectively (data derived from 3 mice and 240 split reactions). Values are mean ± SEM. (D) Comparing nonbiased amplification of TCRα by TRAV primers. Relative frequencies of in-frame and out-of-frame TCRαs paired with the same TCRβ showed that the varying efficiency of amplification was clone specific, rather than TRAV specific (data derived from 78 sequences from 5 mice). (E) KbPB1703+CD8+ T cells were analyzed on days 7, 8, 9, and 10 after primary virus challenge (672 cells from 16 individual mice) for the proportion of the total response represented by out-of-frame cells, showing a significant change (P = 0.0215) between day 7 and day 10.