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

Abstract

The Eβ enhancer has been shown to be dispensable for germline transcription of nonrearranged TCRβ segments but appears to be required for TCRβ V to DJ rearrangement. Eβ dependency of the subsequent expression of VDJ-rearranged TCRβ genes in thymic subpopulations has so far not been analyzed. We generated transgenic mice, using a Vβ8.2Dβ1Jβ1.3-rearranged TCRβ bacterial artificial chromosome, which lacked Eβ, and monitored transgene expression by flow cytometry using Vβ-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 TCRβ gave rise to a pre-TCR, as suggested by its ability to suppress endogenous TCRβ rearrangement, to facilitate β-selection on a TCRβ-deficient background and to inhibit γδ T cell lineage development. The results suggest that the Vβ8.2 promoter is sufficient to drive expression of rearranged TCRβ VDJ genes Eβ independently in DN2/DN3 but not at later stages.