Abstract
Analysis of T-cell receptor beta chain (TCR-β) rearrangement is essential to investigate T-cell responses in human autoimmune diseases, infection and cancer. Since the TCR-β locus contains 55 variable (V) region gene segments, multiple assays have been necessary to determine TCR-β rearrangements of individual T cells. We established a seminested rtPCR method for single T-cell analysis with two sets of degenerate primers covering 76 and 24% of the TCR-Vβ genes, respectively. The specificity of the approach was validated by screening cDNAs obtained from T-cell clones (TCC) with defined TCR-β rearrangement. We applied the method successfully to profile TCR-β rearrangement of single T cells sorted from body fluids or dissected tissue. Concomitant analysis of other gene transcripts allowed determining phenotype and function of TCR-β-defined single T cells. Our fast, cost-efficient and high throughput approach will facilitate studies on T-cell responses in human diseases.
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