The TCRβ Enhancer Is Dispensable for the Expression of Rearranged TCRβ Genes in Thymic DN2/DN3 Populations but Not at Later Stages

The Ebeta enhancer has been shown to be dispensable for germline transcription of nonrearranged TCRbeta segments but appears to be required for TCRbeta V to DJ rearrangement. Ebeta dependency of the subsequent expression of VDJ-rearranged TCRbeta genes in thymic subpopulations has so far not been analyzed. We generated transgenic mice, using a Vbeta8.2Dbeta1Jbeta1.3-rearranged TCRbeta bacterial artificial chromosome, which lacked Ebeta, and monitored transgene expression by flow cytometry using Vbeta-specific mAbs and an IRES-eGFP reporter. Transgene expression was found in double negative (DN)2 and DN3 but not at later stages of thymopoesis. There was no toxicity associated with the transgene given that apoptosis in DN3, DN4 was not increased, and the number of DN4 cells generated from DN3 cells in reaggregate thymic organ cultures was not diminished. The transgenic TCRbeta gave rise to a pre-TCR, as suggested by its ability to suppress endogenous TCRbeta rearrangement, to facilitate beta-selection on a TCRbeta-deficient background and to inhibit gammadelta T cell lineage development. The results suggest that the Vbeta8.2 promoter is sufficient to drive expression of rearranged TCRbeta VDJ genes Ebeta independently in DN2/DN3 but not at later stages.