T-cell receptor variable gene analysis of renal allograft-infiltrating cells in biopsy specimens using a nonradioisotopic micromethod

Background. A sensitive micromethod for T-cell receptor (TCR) analysis is needed for clonality analysis of renal allograft-infiltrating T cells (RAITs) obtained by needle biopsy. Methods. TCR cDNA was amplified by the anchored polymerase chain reaction and was hybridized with 28 different TCR beta variable (TCRBV) genes fixed on nylon membranes, and the percentage of each TCRBV gene was measured spectrophotometrically. Results. The specificity and linearity of the hybridization technique and the constancy of the TCRBV percentages over a wide range of sample amounts were demonstrated by control experiments. Analysis of RAITs of biopsy specimens from four patients showed broad or skewed TCRBV usage, indicating the presence of polyclonal and oligoclonal RAIT populations, respectively. In one patient who received OKT3 immunosuppressive treatment, the TCRBV skewness was dramatically reduced after the treatment. Conclusion. We have established a powerful method for analyzing BAIT clonality, which is especially useful for monitoring BAIT dynamics after immunosuppression therapy.