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Abstract

Development of αβ and γδ T cells depends on productive rearrangement of the appropriate TCR genes and their subsequent expression as proteins. TCRβ and TCRγδ proteins first appear in DN3 and DN4 thymocytes, respectively. So far, it is not clear whether this is due to a delayed expression of TCRγδ proteins or to a more rapid progression to DN4 of thymocytes expressing TCRγδ. The answer to this question bears on the distinction between instructive and stochastic models of αβ/γδ 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 TCRβ transgene was expressed in nearly all DN3 and DN4 cells, accelerated DN3 to DN4 transition, and strongly diminished the number of cells that express TCRγδ proteins. In contrast, TCRγδ 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 TCRβ proteins. The TCRβ transgene partially inhibited endogenous TCRγ rearrangements, whereas the TCRγδ transgenes did not inhibit endogenous TCRβ rearrangements. Second, we analyzed frequencies of productive TCRβ and TCRγδ V(D)J junctions in DN3 and DN4 subsets. Most importantly, frequencies of productive TCRγδ rearrangements (Vδ5, Vγ1.1, and Vγ2) appeared unselected in DN3. The results suggest a late and restricted expression of the corresponding γδTCR, severely limiting their putative instructional opportunities in αβ/γδ divergence.