An automated method for the analysis of T-cell receptor repertoires - Rapid RT-PCR fragment length analysis of the T-cell receptor beta chain complementarity-determining region 3

The examination of T-cell receptor (TCR) repertoires has an important role in the study of lymphoproliferative disorders and autoimmune diseases. Anal ysis of the complementarity-determining region 3 (CDR3) of the TCR beta cha in is used to assess the clonality of T-cell populations. We developed a ra pid fluorescence-based method for CDR3 length analysis of expressed TCR gen e families. TCR beta chain complementary DNA is amplified by a nested polym erase chain reaction with V beta family-specific oligonucleotide primers an d a fluorochrome-labeled C beta pr-inter: The polymerase chain reaction pro ducts were analyzed on a compact automated DNA sequencing system (OpenGene system Visible Genetics, Toronto, Ontario). To demonstrate the usefulness o f our technique, we examined the CDR3 length distribution of peripheral blo od T cells from a healthy subject, intestinal T cells from a patient with u lcerative colitis, and the T-cell leukemia cell line Jurkat. The analysis r evealed polyclonal, oligoclonal, and monoclonal CDR3 distributions, respect ively, for the 3 T-cell populations. Our new method shows virtually identic al CDR3 length patterns compared with the traditional radioisotope-based me thod The new technique offers the convenience of rapid throughput, nonradio active labeling, and quality data analysis.