Sequence analysis of T-cell receptor TCR V regions. Expression of TCRβ variable (BV) genes was analysed by reverse transcription polymerase chain reaction (RT-PCR). Total RNA was isolated using Trizol (Life Technologies, Gaithersburg, MD, USA). Two micrograms of RNA were used for cDNA synthesis using random primers in a total reaction volume of 50 µl. One microlitres of cDNA were subjected to PCR using forward primers specific for different Vβ gene families. As a reverse primer, an oligonucleotide specific for Cβ1 was used. The primers used for amplification of TCR Vβ gene families were as follows: BV1, GCA AAA GGA AAC ATT CTT GAA CG; BV2, AGA GTC TCA TGC TGA TGG C; BV3, AGG ACG GGA GAG AAA GTT TTT C; BV4, GGC CAC TAT GAG AGT GGA TTT GTC; BV5, CCC TAA CTA TAG CTC TGA GCT G; BV6, GGG GCA GGG CCC AGA GTT TCT AAT; BV7, CTT AAA CCT TCA CCT ACA CGC; BV8, CTT TAA CAA CAA CGT TCC G; BV9, CAG TTC CAA ATC GCT TCT CAC; BV10, GGA TTG TGT TCC TAT AAA AGC; BV11, CCA CTA TTC CTA TGG AGT TAA TTC C; BV12, GTC ACC AGA CTG AGA ACC ACC GCT A; BV13, GCA TGA CAC TGC AGT GTG CCC; BV14, ACC CAA GAT ACC TCA TCA CAG; BV15, TCT CAG ACT AAG GGT CAT GAT AGA; BV16, CTG TTA CAT TTT GTG AAA GAG TC; BV17, CTC ACA GAT AGT AAA TGA CTT TC; BV18, AGC CCA ATG AAA GGA CAC AGT CAT; BV19, ACC CCC GAA AAA GGA CAT ACT TTT; BV20, GAG GGA ACA TCA AAC CCC AAC CTA; BV21, TCC AGC CTG CAG AGC TTG GGG GAC T; BV22, AAG TGA TCT TGC GCT GTG TCC CCA; BV23, GCA GGG TCC AGG TCA GGA CCC CCA; BV24, CCC AGT TTG GAA AGC CAG TGA CCC; BV25, ATG TCT TTG ATG AAA CAG GTA; BV26, TCC AGT ACC AAA ACA TTG CAG; CB1, GTG TTT GAG CCA TCA GAA GC. The nomenclature is according to Arden et al (1995). PCR was carried out with Taq polymerase (Life Technologies, Gaithersburg, MD, USA), according to standard procedures, in 35 cycles consisting of 30 s at 95°C, 30 s at 55°C and 1 min at 72°C. PCR products were separated on a 1·5% agarose gel and analysed by ethidium bromide staining. The expressed Vα genes were analysed in a similar fashion. The primers used were the following: AV1, CAG CTT CTC CTC AAG TAC; AV2, TGG AAG GTT TAC AGC ACA; AV3, TCC ACC TAA TTT TAA TAC GT; AV4, TGC CTT GTA ACC ACT CCA; AV5, CAC CCT TAA CCA GAG TTT G; AV6, AAC CTT GTC ATC TCC GCT; AV7, ACC CAC ATT TCT GTC TTA CA; AV8, TTA TTA TAG ACA TTC GTT CAA AT; AV9, GAG ACA CAT CTC TAG AGA G; AV10, CCT CCT GGT GAC AGT AGT TAC; AV11, GTG TCC AAT GCT TAC AAC TT; AV12, CAG TTC CTT CAA CTT CAC C; AV13, CTG TCG CTA CGG AAC GCT; AV14, GAG AAT CGT TTC TCT GTG AA; AV15, CCA GTT GCT GAC GTA TAT TTT; AV16, CCT ATT CAG TCT CTG GAA AC; AV17, TAC GTC CAG ATG TGA GTG; AV18, CCA AAA CAC CCG AGG CCT; AV19, ACA ATA AAC ATA CAG GAA AAG; AV20, CAA GGA TAC AAG ACA AAA GTT; AV21, CTG AAG GTC CTA CAT TCC; AV22, ACA TAC CGT AAA GAA ACC ACT; AV23, GCT ATT TAC AAC CTC CAG; AV24, CCC CTT CAG CAA CTT AAG; AV25, GCT GGT GAA TTG ACC TCA; AV26, ACT GCC AAG TTG GAT GAG; AV27, TGA TAC CAA AGC CCG TCT; AV28, GGA AAA GAA AGC TCC CAC; AV29, GAA AAA ATA TCT GCT TCA TTT A; AV30, CTA AAA GCC ACA TTA ACA AAG; AV31, CAC GGG GTA CCC TAC C; AV32, AGG AGA GGA CTT CAC CAC; CaCONF, CAG CAT TAT TCC AGA AGA CAC; CaCONR, CCT CAG CTG GAC CAC AGC. For determination of the nucleotide sequence of the TCR α and β chain variable regions, specific PCR products were isolated with the Qiaquick gel extraction kit (Qiagen, Hilden, Germany). Cycle sequence reactions were carried out with the Cβ− and Cα-specific primers using the big dye terminator cycle sequencing ready reaction kit [Applied Biosystems (ABI), Warrington, UK]. Reaction mixtures were analysed on an ABI Prism 3100 genetic analyser.