Abstract HLA-DR alpha, beta 1 *1501 (and the closely related DR alpha, beta 1 *1502) heterodimers are the primary class II MHC restriction elements controlling T cell responses to a ragweed pollen allergen, Amb a 5 (M(r) = 5000). Using a novel, quantitative, competitive PCR (QC-PCR) assay and a TCR beta-gene internal standard (IS), we have examined the TCR beta-gene use in Amb a 5-specific T cells. Multiple TCR V beta (and V alpha) genes were found in polyclonal T cell lines from two unrelated subjects, and 30% of the cells were stained positive for V beta 5.2/5.3 as judged by a flow cytometry analysis. An Amb a 5-specific Th2 clone (AP1.2) was found to express V beta 5.2 (and V alpha 8) and to have a unique V-D-J junctional region sequence. To determine the relative frequency of clone AP1.2 beta-gene expression in the polyclonal T cell lines and in the PBMC using a QC-PCR assay, we first created a TCR IS by duplicating the J beta 1.5 gene segment of the AP1.2 beta-gene. A QC-PCR assay was performed using the modified AP1.2 beta-gene as a competitor and a V beta 5.2 or a D beta-specific primer, each paired with a C beta primer. Results showed the presence of 30% of AP1.2 beta transcripts in the polyclonal T cell lines, but no detectable amplified products were found in the total RNAs (200 ng) of autologous PBMC. These findings were confirmed by sequencing independent clones of PCR-amplified V beta 5.2-C beta fragments (from two unrelated subjects) from each cell line, which suggested that the frequency of the clone AP1.2 beta sequence is quite low in the PBMC but is dominant in polyclonal Amb a 5-specific cell lines.
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