Hematopoiesis is the process of differentiation from hematopoietic stem cells (HSC) into mature blood cells. Once a multi-potent stem cell has been influenced by intrinsic (transcription factors) or extrinsic (cytokines and micro-environmental) factors, it undergoes a loss of lineage potential and become committed to a cell lineage or lineages. Differentiation from multipotent HSC into functional T, B, or Natural Killer (NK) cells consists of passage through intermediate progenitor stages prior to becoming a mature lymphoid cell.
This thesis examines the process of lymphoid differentiation using several different aproaches. The majority of my research has focused on the identification of a functionally and molecularly distinct stage of early human hematopoietic commitment. This stage of commitment corresponds to the loss of erythroid and megakaryocytic potential while maintaining the capacity to generate all cells of the lymphoid lineage and the monocytic/macrophage cells of the myeloid lineage, going against the model of hematopoiesis that the earliest lineage decision bifurcates lymphoid potential and myeloid-erythroid potential. These progenitors are distinctive from all previously identified human lymphoid-committed progenitors, in that this population lacks the cell surface antigen CD10 and therefore does not have a bias toward B cell potential and minimal T and NK cell potential. These cells are functionally analogous to a population of cells described in mouse, the Lymphoid-primed Multi-Potent Progenitor (LMPP), and both the mouse LMPP and our novel human LMPP are strong candidates to be thymic seeding cells.
In the second part of my work, we applied the identification of the human LMPP progenitor population in normal BM to the clinically relevant question how the lack of IL2RG signaling affects the regulation of the lymphoid commitment. In humans with X-linked Severe Combined Immune Deficiency (X-SCID) a null mutation of the IL2RG gene results in an inability to differentiate or function in response to multiple lymphoid cytokines resulting in an absence of peripheral T and NK cells, and present but functionally impaired B cells. In contrast, mice lacking IL2RG signaling are fully unable to develop B cells, with deficits in the early stage of B cell commitment. The in-progress studies described in this thesis demonstrate that the earliest stages of B lymphoid development are unaffected by defects in IL2RG signaling in these human patients. These results demonstrate the critical differences between lymphopoiesis in mice and humans, and the importance of overcoming experimental hurdles in order to directly investigate human lymphoid commitment.
The third part was of my work was done in collaboration with others, examinig factors that influenced thymic entry in a murine transplant model. Differentiation of progenitors into T lymphoid cells is a multi-step process in which T cell precursors receive critical extrinsic signals in the thymus to undergo the beginning stages of thymocyte development. In comparison to adults, neonatal mice have stronger thymocyte proliferation and vascular endothelial growth factor (VEGF) dependent angiogenesis in the thymus. Neonatal mice undergo more rapid thymic reconstitution than adults post-transplantation, even when BM engraftment is minimal and in the absence of pre-transplantation radiation. We show that inhibition of VEGF prior to transplantation prevents rapid thymic reconstitution in neonates, but has no effect on thymic reconstitution in adults. These data suggest that the early radiation-independent thymic reconstitution in the neonatal host is mediated by VEGF, and reveals a novel pathway that might be targeted to improve immune reconstitution post-transplantation.
To summarize, the body of this thesis examines the stages of lymphoid differentiation in several contexts: normal human hematopoiesis, dysfunctional human hematopoiesis, and murine thymic reconstitution post-HSCT. Taken together, description of a human LMPP progenitor phenotype opens up the possibility for future study molecular regulation of the first stages of lymphoid commitment in normal and aberrant human hematopoiesis.