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 β-chain mRNA of each of the 24 T-cell receptor β-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.