A quantitative real time PCR method to analyze T cell receptor Vbeta subgroup expansion by staphylococcal superantigens.
BACKGROUND
Staphylococcal enterotoxins (SEs), SE-like (SEl) toxins, and toxic shock syndrome toxin-1 (TSST-1), produced by Staphylococcus aureus, belong to the subgroup of microbial superantigens (SAgs). SAgs induce clonal proliferation of T cells bearing specific variable regions of the T cell receptor beta chain (Vbeta). Quantitative real time PCR (qRT-PCR) has become widely accepted for rapid and reproducible mRNA quantification. Although the quantification of Vbeta subgroups using qRT-PCR has been reported, qRT-PCR using both primers annealing to selected Vbeta nucleotide sequences and SYBR Green I reporter has not been applied to assess Vbeta-dependent expansion of T cells by SAgs.


METHODS
Human peripheral blood mononuclear cells were stimulated with various SAgs or a monoclonal antibody specific to human CD3. Highly specific expansion of Vbeta subgroups was assessed by qRT-PCR using SYBR Green I reporter and primers corresponding to selected Vbeta nucleotide sequences.


RESULTS
qRT-PCR specificities were confirmed by sequencing amplified PCR products and melting curve analysis. To assess qRT-PCR efficiencies, standard curves were generated for each primer set. The average slope and R2 of standard curves were -3.3764 +/- 0.0245 and 0.99856 +/- 0.000478, respectively, demonstrating that the qRT-PCR established in this study is highly efficient. With some exceptions, SAg Vbeta specificities observed in this study were similar to those reported in previous studies.


CONCLUSIONS
The qRT-PCR method established in this study produced an accurate and reproducible assessment of Vbeta-dependent expansion of human T cells by staphylococcal SAgs. This method could be a useful tool in the characterization T cell proliferation by newly discovered SAg and in the investigation of biological effects of SAgs linked to pathogenesis.
Seo
Keun Seok
KS
Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, ID 83844, USA. baram717@uidaho.edu
Park
Joo Youn
JY
Terman
David S
DS
Bohach
Gregory A
GA
D000942:Antigens, Bacterial / Q000276:immunology; D004768:Enterotoxins / Q000276:immunology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics / Q000276:immunology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013211:Staphylococcus aureus / Q000235:genetics / Q000276:immunology*; D018089:Superantigens / Q000276:immunology*; D016176:T-Lymphocyte Subsets / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology
D000942:Antigens, Bacterial; D004768:Enterotoxins; D016693:Receptors, Antigen, T-Cell, alpha-beta; D018089:Superantigens