Extrinsic and intrinsic regulation of differentiation and selection events during lymphocyte development

The development of lymphocytes is a precise, stepwise process that is subject to multiple extrinsic as well as intrinsic molecular mechanisms that guide cell differentiation and selection. Two essential regulators of lymphopoiesis are environmental signal mediators in the form of cytokines and transcription factors. Both influence proliferation, survival, differentiation and cell fate decisions.
In the present thesis, we investigated the role of the cytokines Flt3-ligand and IL-7 in lymphocyte development by the use of various mutant mouse strains. Our results unraveled crucial functions for both cytokines during lymphocyte differentiation. Flt3-ligand drives the proliferation of uncommitted progenitor populations such as CLP and EPLM, whereas IL-7 has a pro-survival effect on these cells. High levels of Flt3-ligand rescued the B-cell defect in IL-7-/- mice due to the tremendous expansion of the CLP and EPLM populations, surpassing the need for IL-7 as a survival factor. Thus, these observations clearly demonstrate that both, Flt3-ligand as well as IL-7, function in a permissive mode in the commitment process towards the B-cell lineage. Flt3 expression is suppressed upon commitment to the B-cell fate, but IL-7 has furthermore a proliferative function for committed CD19+ progenitor cells. Constitutive over-expression of both Flt3-ligand and IL-7 resulted in a lympho- and myelo-proliferative disease. The two cytokines had a synergistic effect on the development of B cells, resulting in the accumulation of progenitors also in peripheral lymphoid organs. Interestingly, even MPPs could be detected in lymph nodes of these mice and transplantation experiments confirmed the functionality of these progenitors, since they were capable of long-term multilineage reconstitution. Thus, Flt3-ligand and IL-7 act in concert during lymphocyte development. We further provided evidence that peripheral lymphoid organs have the capability to support extramedullary hematopoiesis in pathological situations.
In a second study we focused on the molecular and transcriptional regulation of the first essential checkpoint of the antigen-receptor rearrangement in T-cell development, called β-selection. Even though almost half of the cells are eliminated at this checkpoint, only little is known about the underlying molecular mechanisms. Improvement of the current staging of thymocyte development by the addition of CD27 downregulation as a marker for cells that failed productive β-chain rearrangement at the DN3 stage allowed us to investigate this process in more detail than previously possible. Transcriptional analysis revealed a specific expression of the transcription factor Duxbl in cells prior to β-selection. Transgenic expression of Duxbl blocked the development of pre-T cells in vitro and in vivo due to increased apoptosis and cell cycle arrest. Further studies revealed the involvement of the Oas/RNaseL apoptosis pathway in this Duxbl-mediated developmental arrest. Additional expression of the pro-survival factor Bcl2 partially rescued the block and in vitro knockdown experiments of Duxbl reduced apoptosis induction within the DN3 compartment. Thus, the specific expression of Duxbl in conjunction with the gain- and loss-of-function phenotypes of increased and reduced apoptosis, respectively, provide clear evidence for a key role of Duxbl in the elimination of DN3 cells that are not able to recombine a functional β-chain.
Overall, the results presented in this thesis, provide important new insights on the cell-extrinsic and -intrinsic regulation of differentiation and selection events in the development of lymphocytes. Furthermore, they highlight the validity of in vivo models for the investigation of immune cell development and function.