Paired analysis of TCRα and TCRβ chains at the single-cell level in mice.
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.
St Jude Children’s Research Hospital, Memphis, Tennessee 38105-3678, USA.
Pradyot
P
Dash
Jennifer L
JL
McClaren
Thomas H
TH
Oguin
William
W
Rothwell
Brandon
B
Todd
Melissa Y
MY
Morris
Jared
J
Becksfort
Cory
C
Reynolds
Scott A
SA
Brown
Peter C
PC
Doherty
Paul G
PG
Thomas
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000276:immunology; D022801:Complementarity Determining Regions; D000939:Epitopes / Q000276:immunology; D005260:Female; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009975:Orthomyxoviridae / Q000276:immunology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014158:Transcription, Genetic
D000956:Antigens, Viral; D022801:Complementarity Determining Regions; D000939:Epitopes; D012333:RNA, Messenger; D016693:Receptors, Antigen, T-Cell, alpha-beta