Tracking T cell clonotypes in complex T lymphocyte populations by real-time quantitative PCR using fluorogenic complementarity-determining region-3-specific probes.
The T cell receptor (TCR) alpha and beta chains are encoded by a series of stochastic rearrangements between variable (V), diversity (D) for TCR beta chain only, and joining (J) gene segments, creating hypervariable complementarity-determining region 3 (CDR3) regions that contact the peptide/MHC complex and confer specificity. In the present paper, we applied the recently developed real-time quantitative RT-PCR technique to the detection of rearranged TCR beta chain mRNA transcripts. We designed BV- and BJ-specific primers together with TaqMan probes specific for the CDR3 regions of the clones of interest. As an external reference, we used plasmids containing the entire TCR beta chains, making it possible to normalize the number of specific rearranged BV-J mRNA copies among the total number of TCR beta chains. Here, we present data validating this fluorogenic PCR-based method for the quantification of several TCR clonotypes characteristic of the CD4 T cell response to hen egg white lysozyme (HEL) in mice of the H-2d haplotype. This accurate and sensitive procedure permits the precise determination of T cell clone frequencies ranging from 10(-2) to less than 10(-5) in normal biological samples; it may provide an alternative approach when frequencies are too low to be assessed by flow cytometry.
Gallard
Alexandra
A
Institut National de la Santé et de la Recherche Médicale U.563, Centre de Physiopathologie de Toulouse Purpan, Institut Claude de Préval, Hôpital Purpan, Place du Dr Baylac, 31059 Toulouse Cedex, France.
Foucras
Gilles
G
Coureau
Christiane
C
Guéry
Jean-Charles
JC
D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000145:classification / Q000276:immunology*; D022801:Complementarity Determining Regions / Q000235:genetics*; D017931:DNA Primers; D015342:DNA Probes; D005260:Female; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D009113:Muramidase / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012333:RNA, Messenger; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D012235:Rhodamines
C114557:5-carboxytetramethylrhodamine succinimidyl ester; D022801:Complementarity Determining Regions; D017931:DNA Primers; D015342:DNA Probes; D005452:Fluoresceins; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D016693:Receptors, Antigen, T-Cell, alpha-beta; D012235:Rhodamines; C024098:6-carboxyfluorescein; C421864:hen egg lysozyme; D009113:Muramidase