A rapid method for semiquantitative analysis of the human V beta-repertoire using TaqMan(R) PCR

Analysis of the V beta-repertoire of antigen-reactive T cell populations can be approached using either flow-cytometry or PCR-based techniques. While the former method requires a complete set of V beta-specific monoclonal antibodies (mAbs) and large cell numbers for analysis, the latter is both time-consuming and labour-intensive. To circumvent the drawbacks of both these methods we have employed the recently developed technique of TaqMan(R) PCR to analyse the V beta-usage of human T cell populations. TaqMan(R) PCR is based on the 5' --> 3' nuclease activity of Tag polymerase. During PCR amplification an internal oligonucleotide probe, that is labelled with a fluorescent reporter and a quencher dye, is cleaved by Tag polymerase. After cleavage, quenching of the reporter dye is lost and reporter fluorescence can be detected with a fluorescence plate reader. Using one C beta-specific fluorogenic probe and a panel of V beta-specific primers, we show that fluorescence-detected amplification of TCR beta cDNA is V beta-specific and linear within a 2-3-log range of template concentration. The sensitivity of TaqMan(R) PCR is comparable to conventional detection of PCR-products by agarose gel staining, while processing time is reduced. Furthermore, superantigen-induced skewing of the V beta-repertoire of human cells is readily detected with this method. Thus TaqMan(R) PCR is a reliable and fast method for semiquantitative analysis of the V beta-repertoire of human T cell populations.