Estimate of the total number of CD8+ clonal expansions in healthy adults using a new DNA heteroduplex-tracking assay for CDR3 repertoire analysis.
A T-cell receptor heteroduplex-tracking assay (TCR-HTA) was developed to analyze the sequence diversity of the TCR beta-chain mRNA of each of the 24 T-cell receptor beta-chain variable region (TRBV). TCR-HTA allowed an estimation of the number of expanded CD8 T-cell clones whose distinct CDR3 domain mRNA made up 2% or more of the transcript of each TRBV subfamily. An average of 40 CD8+ clonal expansions (range 34-49) was detected in three healthy adults. Correct sampling of the complex mRNA transcript populations was documented by the reproducible generation of TCR-HTA patterns using independently generated PCR amplicons. The CDR3 sequence of expanded T-cell clones could be rapidly determined by direct sequencing of DNA heteroduplex bands. CD4+ and CD8+ clonal expansions were found predominantly although not exclusively in CD45RO+ CD62L- effector/memory cells and the majority of expanded T-cell clones were stable over a period of at least 6 months. Fewer CD4+ than CD8+ clonal expansions were detected in peripheral blood cells. By providing a high-resolution method for the detection of clonally expanded T-cell clones and by simplifying the pattern generated using traditional DNA heteroduplex analysis, TCR-HTA is shown to be a sensitive method for assessing levels of oligoclonality and changes in TRBV repertoires.
Bernardin
Flavien
F
Department of Medicine, University of California, San Francisco, CA 94118, USA.
Doukhan
Laurence
L
Longone-Miller
Alcira
A
Champagne
Patrick
P
Sekaly
Rafick
R
Delwart
Eric
E
D000328:Adult; D001483:Base Sequence; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002999:Clone Cells; D022801:Complementarity Determining Regions / Q000145:classification / Q000235:genetics*; D017931:DNA Primers; D015342:DNA Probes; D004586:Electrophoresis; D019674:Genes, T-Cell Receptor beta; D014644:Genetic Variation; D020180:Heteroduplex Analysis / Q000379:methods*; D006801:Humans; D008213:Lymphocyte Activation; D018655:Lymphocyte Count; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA
D022801:Complementarity Determining Regions; D017931:DNA Primers; D015342:DNA Probes; D012333:RNA, Messenger; D016693:Receptors, Antigen, T-Cell, alpha-beta