HLA-DRα,β1*1501 (and the closely related DRα,β1*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 β-gene internal standard (IS), we have examined the TCR β-gene use in Amb a 5-specific T cells. Multiple TCR Vβ (and Vα) genes were found in polyclonal T cell lines from two unrelated subjects, and 30% of the cells were stained positive for Vβ5.2/5.3 as judged by a flow cytometry analysis. An Amb a 5-specific Th2 clone (AP1.2) was found to express Vβ5.2 (and Vα8) and to have a unique V- D-J junctional region sequence. To determine the relative frequency of clone AP1.2 β-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 Jβ1.5 gene segment of the AP1.2 β-gene. A QC-PCR assay was performed using the modified AP1.2 β-gene as a competitor and a Vβ5.2 or a Dβ-specific primer, each paired with a Cβ primer. Results showed the presence of 30% of AP1.2 β 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 Vβ5.2-Cβ fragments (from two unrelated subjects) from each cell line, which suggested that the frequency of the clone AP1.2 β sequence is quite low in the PBMC but is dominant in polyclonal Amb a 5-specific cell lines.
|Number of pages||6|
|Journal||Journal of Immunology|
|Publication status||Published - May 31 1995|
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