Intrathymic and Extrathymic Notch-Dependent T Lineage Checkpoints during Normal Development and after Bone Marrow Transplantation.

The essential role of Notch for T lineage commitment is well established, but it remains unclear where and on which progenitor subset(s) Notch signals act. Addressing these questions is critical to understand the regulation of lymphopoiesis in normal and lymphopenic settings, such as after bone marrow transplantation (BMT). In normal mice, a small number of progenitors settle in the thymus from the blood and expand to generate a pool of early T lineage progenitors (ETPs). At the population level, ETPs can give rise to T cells, NK cells, B cells, DCs and myeloid cells. We found that ETPs could be subdivided based on expression of the cytokine receptor Flt3, which is expressed on about 10% of ETPs and is inversely correlated with expression of Notch target genes. B lineage potential was restricted to ETPFlt3 positive cells. The Notch targets Hes-1, Hes-5, and Deltex1 were present at low levels in ETPFlt3 positive and high levels in ETPFlt3 negative cells. Induction of Notch signaling resulted in the rapid downregulation of surface Flt3 expression. In contrast, culture of ETPFlt3 negative cells in the absence of Notch ligands resulted in upregulation of Flt3. Although both ETP subsets were efficient T lineage progenitors, ETPFlt3 negative cells had a more rapid differentiation kinetics resembling DN2 thymocytes, consistent with a more advanced state of T lineage commitment relative to the Flt3 positive subset. In mice reconstituted with HSCs transduced with the pan-Notch inhibitor DNMAML1 (Maillard et al., Blood 2004), no ETPFlt3 negative and very few ETPFlt3 positive cells were observed, indicating that the generation of ETPs is Notch-dependent. These observations position the physiological Notch checkpoint either very early after thymic seeding and/or in a prethymic location.To further investigate if Notch-dependent prethymic T lineage commitment occurs in adult mice, we studied lymphoid reconstitution early after BMT. ETPs were absent in the thymus of recipients until ≥ 6 weeks post-BMT, despite the presence of donor-derived pre-T cells and double positive thymocytes as early as 2–3 weeks post-BMT. Instead, cells with pre-T cell characteristics were present in the spleen of BMT recipients in the first month post-BMT. These cells were absent when DNMAML1-transduced bone marrow was used as the source of HSCs, indicating that generation of extrathymic pre-T cells is Notch-dependent. Thus, extrathymic sites may be important for efficient lymphoid reconstitution after BMT. Altogether, our results indicate that both intrathymic and extrathymic Notch-dependent checkpoints regulate T lineage commitment during normal development and in the post-BMT setting.