Hematopoietic stem and progenitor recovery in sex steroid ablation-mediated immune regeneration
thesisposted on 06.02.2017, 05:56 by Khong, Danika Mei Po
Lifelong hematopoiesis is supported by hematopoietic stem cells (HSCs) differentiating within the bone marrow (BM) down all blood cell lineages including erythrocytes, platelets, myeloid and lymphoid cells. Although most blood cells are generated entirely within the BM, T cells complete their development in the thymus, a primary lymphoid organ within the mediastinal cavity. Together, B and T cells represent the lymphoid arm of the immune system and are vital in the adaptive protection against foreign pathogens. While hematopoiesis is continuous for the lifespan of the organism, it is currently well established that, paradoxical to its fundamental importance for establishing and maintaining good health, the adaptive immune system degenerates very early with age; a phenomenon temporally linked to sex steroid exposure. Beginning from birth, but accelerated at the onset of puberty, both the primary lymphoid organs – the BM and thymus, gradually deteriorate, resulting in a decline in the generation of new naïve B and T cells. In healthy adults this does not pose a major threat, however in patients who are immunocompromised following cytoreductive treatments, such as chemo- or radiation- therapy or chronic infections best exemplified by acquired immunodeficiency syndrome (AIDS), immune recovery is considerably delayed leaving these individuals susceptible to opportunistic infections and malignant relapses. As a corollary to the hypothesized underlying cause of immune atrophy, our laboratory and others have previously demonstrated that removing the negative effects of sex steroids by either surgical or chemical (reversible) castration (sex steroid ablation; SSA) facilitates significant immune regeneration in aged mice and can enhance lymphoid recovery post HSC transplantation (HSCT) or treatment with chemotherapy. 3 This thesis aimed to elucidate the mechanisms underlying SSA-mediated lymphoid recovery by comprehensively assessing the impact of SSA on (1) the function of hematopoietic stem and progenitor cells in the BM, (2) the ability of the supporting BM stromal microenvironment to support hematopoiesis, and (3) the function of the earliest T-lineage progenitors in the thymus. Consistent with previous reports, we observed an age-associated accumulation of HSCs that demonstrated inferior self-renewal capacity and lymphoid differentiation potential when compared to young HSCs. We further demonstrated both a numerical and functional enhancement of the primitive long-term HSC (LT-HSC) population following SSA. Not only did these LT-HSCs show enhanced self-renewal, they were also more efficient at differentiating into downstream lymphoid cells with SSA. Detailed molecular analysis revealed important cell intrinsic changes pertaining to quiescence, self-renewal, lymphoid differentiation and DNA replication processes occurring within these primitive HSCs, collectively suggesting their role in establishing SSA-mediated immune regeneration. Since HSCs rely heavily on their stromal cell-based microenvironmental “niches” for signals governing their differentiation and survival, the effects of SSA on the the endosteal and vascular (central marrow) compartments were profiled. Interestingly, a population of osteoblasts (OBLs) with high Runx2 expression was revealed within the vascular niche of the BM that numerically increased with age; this correlated with an accumulation of LT- HSCs seen with age in the BM. These Runx2 –rich OBLs also expressed hematopoietic supporting molecules, albeit to a lesser degree than the canonical LT-HSC-associated endosteal osteoblasts. These age-related changes were not observed within the endosteal osteoblasts compartment however. While SSA was unable to reverse the age-associated increase in the vascular OBL population, there was increased expression of genes linked to promoting HSC quiescence, self-renewal, lymphoid differentiation and cell adhesion. This 4 clearly indicates the important synergisms between the hematopoietic and stromal compartments of the bone marrow – with both contributing to the SSA-induced rejuvenation of the blood system. With this evidence suggesting that enhanced HSC and BM niche function led to downstream improvements in lymphopoiesis, the final section of this thesis focused on the effects of SSA on the lymphoid progenitors: their ability to seed the thymus and subsequently differentiate into T cells. While there has been considerable insight into the developmental hierarchy of HSCs and the downstream progenitors within the BM, little is known about the identity of the thymic-bound progenitor that egresses from the BM. Increases in the CD27+CD62L+ lymphoid primed multipotent progenitors (LMPP) and common lymphoid progenitors (CLPs) within the BM, and the early thymic progenitor (ETP) and CLP-2 within the thymus were evident following SSA. The earliest T cell progenitor within the thymus is the ETP, which has proficient T differentiation potential. However the earliest cellular increase in the thymus post SSA was by the CLP2 population at day 2. The CLP and its downstream CLP-2 produced in the BM, are by default a B cell progenitors, yet have a very efficient capacity for thymic entry and the propensity to form T cells once influenced by Notch signaling within the thymus. Several thymic homing molecules, CCR-7, CCR-9 and PSGL-1 have been identified on various circulating thymic progenitors (CTPs), however the expression of these molecules was unaltered following SSA. This would suggest the increased ability of these BM derived CLP-2s to migrate into the aged regenerating thymus, is possibly due to a noncanonical “stress” or “damage” associated pathway. The data are thus consistent with the downstream improvements observed in lymphopoiesis being the result of an increased supply of lymphoid progenitors from the BM, subsequently entering the thymus coupled with an enhanced ability of the intrathymic microenvironment in supporting thymopoiesis, rather than an enhanced intrinsic ability of these progenitors to differentiate into T cells, on a per cell level. Collectively, the work presented here provides a tantalizing glimpse into the mechanisms underlying SSA-mediated lymphoid regeneration, and hence a means of improving transplant outcomes, perhaps by either conditioning donor HSCs or the recipient BM niche, or by targeted acceleration of the regeneration process to reduce the time required for immune recovery.