Re-stimulation in non-polarizing conditions of TCRα transgenic cultures in vitro shows progressive focusing of favored TCRβ usage correlating with adoption of a Th2 cell phenotype. CFA/peptide primed draining lymph nodes were used to establish T cell lines in vitro from TCRα transgenic lines 20 and 34 with an elongated Valpha CDR3 region, TCRα transgenic line 30 with a short Valpha CDR3 region, and littermate controls. Every 10 days, lines were re-stimulated with peptide and APC. (A) Analysis of the TCRβ chain repertoire of TCRα chain transgenic lines 20, 34 and littermate control T cell lines at successive re-stimulations. Sequence analysis of the repertoire of TCRβ CDR3 regions for TCRα chain transgenic and littermate control T cell lines at the re-stimulation numbers shown. The percent of each T cell line TCRβ repertoire attributed to each unique TCRβ chain sequence identified is shown. (B) cDNA from TCRα chain transgenic line 20 and littermate control T cell lines at re-stimulation numbers 2, 4 and 6 was used to spectratype Vβ gene families TRBV2, TRBV13-1, TRBV16 and TRBV31. (C) IFNγ, IL-4, -5, -13 were measured at each re-stimulation of TCRα chain transgenic lines with an elongated CDR3 (line 20) (black bars) (n = 3), transgene negative littermate control lines (white bars) (n = 5) and TCRα chain transgenic lines with a short CDR3 (line 30) (gray bars) (n = 3). (D) Relative expression level of GATA-3 was determined by real-time PCR for TCRα chain transgenic lines with an elongated CDR3 (line 20) (black bars) (n = 3) compared to littermate control lines (n = 5). Error bars indicate SE. This experiment was repeated on three separate occasions. APC, antigen presenting cell.