The Lymphoblastic Leukemia Gene 1 (Lyl1) Regulates Lymphoid Specification and Maintenance of Early T Lineage Progenitors

Abstract 548

Long-term thymopoiesis crucially depends on the recruitment and expansion of bone marrow derived progenitor cells. Therefore, a tight regulation of the thymus-settling progenitor cells is required to maintain T cell lineage homeostasis. Lyl1, a transcription factor involved in homeostatic control of immature hematopoietic cells, remains expressed in early T-lineage progenitors (ETPs) until T cell lineage commitment.

Here we demonstrate a critical requirement for Lyl1 in lymphoid priming of bone marrow (BM) progenitors and in the maintenance of ETPs. Lyl1 deficient hematopoiesis was unable to generate sufficient numbers of lymphoid-primed progenitor populations such as LMPPs, CLPs and in particular ETPs. We found a significant (p>0.001) 4-fold reduction of LMPPs in the BM and a 20-fold reduction (p<0.001) of ETPs in the thymus of Lyl1 KO mice compared to wildtype controls. Transplantation assays revealed a selective defect of Lyl1^−/− LMPPs for thymic engraftment and T lineage development. Intra-thymic injections of Lyl1^−/− LMPPs demonstrated, in absence of homing requirements, a cell autonomous defect of Lyl1^−/− progenitors in their ability to undergo the ETP to DN2 transition and to expand in response to Notch. Injection of Lyl1^+/+ LMPPs resulted in a 5-fold higher recovery of total donor cells compared to injections of Lyl1^−/− LMPPs (p<0.001). 40% of the donor cells derived after intra-thymic injection of Lyl1^+/+ LMPPs were committed to the T cell lineage (DN3, DP, SP), whereas T lineage commitment after injection of Lyl1^−/− cells was only seen in 20% of the recovered cells (p<0.01). By absolute numbers, wildtype LMPPs had generated more DN3 (8-fold), more DP (21-fold) and more SP (9-fold) T cell lineage committed thymocytes, and fewer Lyl1^+/+ cells remained in the c-kit positive “ETP/LMPP-like” stage (-0.5-fold).

In contrast, reintroduction of Lyl1 cDNA into Lyl1−/− BM progenitors using retroviral vectors restored the thymic progenitor pool and enhanced T cell lineage output. At 12 weeks after transplantation of MIG-Lyl1 transduced cells we observed a significant expansion of transfected (GFP^pos) cells in the peripheral blood solely attributable to expansion of normal, mature and poly-clonal T cells (p<0.001). The thymuses of MIG-Lyl1 transplanted recipients showed significantly greater overall cellularity (p<0.01) attributable to a significantly higher proportion of GFP^pos thymocytes (p<0.01) compared to the MIG-GFP transplanted control group.

To gain a more detailed understanding of the underlying molecular mechanisms of Lyl1-mediated T-lymphoid specification, we performed global gene expression profiling of wildtype and Lyl1^−/− LMPPs as well as whole genome ChIP-seqencing in HPC-7 cells after pull-down with anti-Lyl1 antibodies. Here, we identified the lymphoid-promoting factor Gfi1 as a critical transcriptional target of Lyl1-mediated T lymphopoiesis. We found that Gfi1 expression was decreased in Lyl1^−/− LMPPs and ETPs by 2- and 7.5- fold respectively. ChIP assays using ckit^pos BM cells from Lyl1^+/+ and Lyl1^−/− mice revealed a strong enrichment of Lyl1 at a known enhancer region of the Gfi1 locus located 35kb upstream of Gfi1 (p<0.001). Binding of Lyl1 activated the Gfi1 35kb enhancer element in transactivation assays (p<0.001). Finally, intra-thymic injection of Lyl1−/− progenitors after retroviral transduction with Gfi1 cDNA allowed lymphoid development and enhanced the T cell lineage output compared to GFP-transduced controls (p=0.08).

Collectively, our data provide evidence that pro-T cell expansion in the thymus is regulated through intrinsic control of thymus progenitor cells that employ a transcriptional program already established in hematopoietic stem and progenitor cells. We identify Lyl1 as a critical component of this regulatory network, which is vital for the maintenance of T cell lineage homeostasis. Finally, identification of important downstream mediators of Lyl1 function not only illuminates the molecular mechanisms underlying early T-cell development, but also suggests previously unrecognized pathways likely to play a role in Lyl1-mediated development of leukemia and lymphoma.