Enforcement of γδ-lineage commitment by the pre-T-cell receptor in precursors with weak γδ-TCR signals

Developing thymocytes bifurcate from a bipotent precursor into alpha beta- or gamma delta-lineage T cells. Considering this common origin and the fact that the T-cell receptor (TCR) beta-, gamma-, and delta-chains simultaneously rearrange at the double negative (DN) stage of development, the possibility exists that a given DN cell can express and transmit signals through both the pre-TCR and gamma delta-TCR. Here, we tested this scenario by defining the differentiation outcomes and criteria for lineage choice when both TCR-beta and gamma delta-TCR are simultaneously expressed in Rag2(-/-) DN cells via retroviral transduction. Our results showed that Rag2(-/-) DN cells expressing both TCRs developed along the gamma delta-lineage, down-regulated CD24 expression, and up-regulated CD73 expression, showed a gamma delta-biased gene-expression profile, and produced IFN-gamma in response to stimulation. However, in the absence of Inhibitor of DNA-binding 3 expression and strong gamma delta-TCR ligand, gamma delta-expressing cells showed a lower propensity to differentiate along the gamma delta-lineage. Importantly, differentiation along the gamma delta-lineage was restored by preTCR coexpression, which induced greater down-regulation of CD24, higher levels of CD73, Nr4a2, and Rgs1, and recovery of functional competence to produce IFN-gamma. These results confirm a requirement for a strong gamma delta-TCR ligand engagement to promote maturation along the gamma delta T-cell lineage, whereas additional signals from the pre-TCR can serve to enforce a gamma delta-lineage choice in the case of weaker gamma delta-TCR signals. Taken together, these findings further cement the view that the cumulative signal strength sensed by developing DN cells serves to dictate its lineage choice.