PROJECT SUMMARY Steady-state hematopoiesis in bone marrow is critical for maintenance of blood cell genesis and immune competence throughout life. Concomitant with aging, the bone marrow hematopoietic stem cell (HSC) compartment undergoes cell intrinsic and extrinsic changes that biases hematopoietic output toward the myeloid lineage. The bias in HSC-subset predominance and change in hematopoietic output contributes to immune dysfunction in the elderly including an increased propensity to infection and lessened vaccine responses. Thus, studies focused on understanding essential regulators that support a non-biased, balanced HSC pool that supports lymphoid differentiation capability has the potential to provide novel therapeutic avenues to rejuvenate HSC developmental trajectories to restore immune function in the aged. The homeobox transcription factor Hoxa9 is expressed in primitive HSCs and is inactivated upon lineage commitment. Mice with germline deletion of hoxa9 are viable but display multilineage deficiencies that are transplantable, confirming a cell intrinsic defect. Competitive transplantation experiments revealed impaired long-term repopulating ability and in vitro studies showed poor proliferative responses to early-acting cytokines. In bone marrow, hoxa9-/- mice exhibit reduced cellularity, impaired lymphoid priming, reductions in lympho-myeloid progenitors, common lymphoid progenitors, and B cell precursors. Hoxa9-/- mice also have smaller thymi, and reductions in early thymic progenitors. Interestingly, flow cytometric analysis of the long-term repopulating stem cell compartment (LT-HSC) in young adult hoxa9-/- mice revealed increased frequencies of LT-HSCs expressing high levels of the CD150, which is functionally associated with myeloid skewing. This is contrasted by significant decreases in frequencies of CD150lo LT-HSC in hoxa9-/- marrow, which have balanced lympho- myeloid potential. Increased numbers of myeloid-biased HSCs along with diminished lymphopoiesis is reminiscent of aging. A Hoxa9 target gene implicated in age-associated alterations in lymphopoiesis and B cell genesis in the marrow is p16ink4a. The cyclin dependent kinase inhibitor p16ink4a is virtually undetectable in HSC and B cell precursors from young mice but increases in both subsets with age. Realtime PCR of Lin- Sca1+ hematopoietic progenitors from young hoxa9-/- mice revealed increased mRNA transcripts for p16ink4a. To determine if over expression of p16ink4a was the molecular basis of the lymphoid/B cell deficiency in hoxa9-/- mice, we generated hoxa9-/-p16ink4a-/- mice. Importantly, deletion of p16ink4a restored bone marrow and thymus cellularity and rescued the deficiencies in lymphoid, B and T cell precursors in hoxa9-/- mice. These novel experimental findings support the hypothesis that Hoxa9 suppression of p16ink4a is critical for lymphopoiesis. At present, there is a paucity of information regarding the role of Hoxa9 in regulation of lympho-hematopoi...