A DOMINANT T-CELL RECEPTOR BETA-CHAIN IN RESPONSE TO A SHORT RAGWEED ALLERGEN, AMB-A-5

HLA-DR alpha,beta(1)1501 (and the closely related DR alpha,beta(1)*15 02) heterodimers are the primary class II MHC restriction elements con trolling T cell responses to a ragweed pollen allergen, Amb a 5 (M(r) = 5000). Using a novel, quantitative, competitive PCR (QC-PCR) assay a nd a TCR beta-gene internal standard (IS), we have examined the TCR be ta-gene use in Amb a 5-specific T cells. Multiple TCR V beta (and V al pha) genes were found in polyclonal T cell lines from two unrelated su bjects, 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 fre quency of clone AP1.2 beta-gene expression in the polyclonal T cell li nes 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 compet itor 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 transc ripts in the polyclonal T cell lines, but no detectable amplified prod ucts were found in the total RNAs (200 ng) of autologous PBMC. These f indings were confirmed by sequencing independent clones of PCR-amplifi ed 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.