Quantitative assessment of the human TCRBV repertoire by competitive PCR.
A novel quantitative protocol was developed for the measurement of the relative expression levels of the human TCRBV genes based on reverse transcription (RT) and subsequent competitive polymerase chain reaction (cPCR). Competitor DNA templates for the analysis of 24 different TCRBV families were generated by a simple and rapid one-step PCR procedure with a special PCR primer, which introduces a deletion in the constant region gene segment. A defined amount of TCRBV family-specific competitor DNA and the reverse transcribed cDNA of interest were amplified in the same tube with the same primer pair in a competitive way. The resulting fragments were separated on agarose gels and the densitometrical values were evaluated directly without the requirement for additional hybridization steps. For all of the 24 different TCRBV family-specific cPCRs equal amplification efficiencies were demonstrated by titration experiments for wild-type and competitor templates. For TCRBV repertoire studies, a short form of the cPCR assay was performed, requiring only one cPCR for quantitation of each TCRBV family. The exact initial amount of wild-type template in each cPCR was interpolated from TCRBV family-specific reference calibration curves. The RT-cPCR assay was applied for the quantitative assessment of the TCRBV repertoire of CD4+ and CD8+ T cell subsets in unstimulated PBMC and compared to flow cytometric analyses with a panel of monoclonal antibodies specific for TCRBV determinants. The RT-cPCR experiments revealed a differential expression of several BV families in either the CD4+ or the CD8+ fraction. The TCRBV family-specific cPCR assay presented here combines the simplicity and speed of conventional TCRBV family-specific PCR with the quantitative features of competitive PCR. TCRBV family-specific RT-cPCR has a broad application for all kinds of quantitative T cell repertoire studies and could be easily adapted for the usage with different BV-specific primer sets.
Uhrberg
M
M
Bone Marrow Donor Center, MED-Heinrich-Heine University, Düsseldorf, Germany.
Wernet
P
P
D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015496:CD4-Positive T-Lymphocytes / Q000737:chemistry; D018414:CD8-Positive T-Lymphocytes / Q000737:chemistry; D002138:Calibration; D004247:DNA / Q000032:analysis / Q000235:genetics; D017931:DNA Primers; D005434:Flow Cytometry; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D008969:Molecular Sequence Data; D010641:Phenotype; D016133:Polymerase Chain Reaction / Q000379:methods*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000032:analysis* / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016176:T-Lymphocyte Subsets; D013698:Templates, Genetic; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D016693:Receptors, Antigen, T-Cell, alpha-beta; D004247:DNA