The TCRbeta enhancer is dispensable for the expression of rearranged TCRbeta 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.

Delayed and restricted expression limits putative instructional opportunities of Vgamma1.1/Vgamma2 gammadelta TCR in alphabeta/gammadelta lineage choice in the thymus.

Development of alphabeta and gammadelta T cells depends on productive rearrangement of the appropriate TCR genes and their subsequent expression as proteins. TCRbeta and TCRgammadelta proteins first appear in DN3 and DN4 thymocytes, respectively. So far, it is not clear whether this is due to a delayed expression of TCRgammadelta proteins or to a more rapid progression to DN4 of thymocytes expressing TCRgammadelta. The answer to this question bears on the distinction between instructive and stochastic models of alphabeta/gammadelta lineage decision. To study this question, we first monitored initial TCR protein expression in wild-type and TCR transgenic mice in reaggregate thymic organ cultures. A TCRbeta transgene was expressed in nearly all DN3 and DN4 cells, accelerated DN3 to DN4 transition, and strongly diminished the number of cells that express TCRgammadelta proteins. In contrast, TCRgammadelta transgenes were expressed only in a fraction of DN4 cells, did not accelerate DN3 to DN4 transition, and did not reduce the number of DN4 cells expressing TCRbeta proteins. The TCRbeta transgene partially inhibited endogenous TCRgamma rearrangements, whereas the TCRgammadelta transgenes did not inhibit endogenous TCRbeta rearrangements. Second, we analyzed frequencies of productive TCRbeta and TCRgammadelta V(D)J junctions in DN3 and DN4 subsets. Most importantly, frequencies of productive TCRgammadelta rearrangements (Vdelta5, Vgamma1.1, and Vgamma2) appeared unselected in DN3. The results suggest a late and restricted expression of the corresponding gammadeltaTCR, severely limiting their putative instructional opportunities in alphabeta/gammadelta divergence.