Regulation of the lymphoid cell fate by Hoxa9

项目总结 骨髓中稳定的造血对于维持血细胞的生成和免疫至关重要 终生的能力。随着年龄的增长，骨髓造血干细胞(HSC) 隔室经历了细胞内在和外在的变化，使造血输出偏向于 髓系血统。HSC亚群优势的偏向和造血量的变化有助于 老年人的免疫功能障碍，包括感染倾向增加和疫苗接种减少 回应。因此，研究的重点是了解支持无偏见、平衡的 支持淋巴系分化能力的HSC池有可能提供新的治疗方法 恢复HSC发育轨迹以恢复老年人免疫功能的途径。同源异形盒 转录因子Hoxa9在原始的HSCs中表达，并在谱系承诺时失活。老鼠 用生殖系缺失hoxa9是可行的，但表现出可移植的多系缺陷， 确认了细胞的固有缺陷。竞争性移植实验显示长期受损 再生能力和体外研究表明，对早期作用的细胞因子的增殖反应很差。在骨子里 骨髓，hoxa9-/-小鼠表现出细胞减少，淋巴启动功能受损，淋巴髓细胞减少 祖细胞、共同淋巴祖细胞和B细胞前体细胞。Hoxa9-/-小鼠的胸腺也更小， 早期胸腺祖细胞的减少。有趣的是，长期再生干细胞的流式细胞术分析 年轻成年Hoxa9-/-小鼠的细胞室(LT-HSC)显示LT-HSC的频率增加 表达高水平的CD150，这在功能上与髓系扭曲有关。这与之形成对比 通过显著降低CD150lo LT-HSC在HOXA9/-骨髓中的频率，使其具有平衡的淋巴细胞和 髓系潜能。骨髓偏向的HSCs数量增加而淋巴系生成减少 让人回想起衰老。与年龄相关的淋巴细胞和B细胞改变相关的Hoxa9靶基因 骨髓中的Genesis是p16INK4a。细胞周期蛋白依赖的激酶抑制物p16INK4a在 HSC和B细胞前体来自幼鼠，但这两个亚群都随着年龄的增长而增加。实时定量聚合酶链式反应检测LIN- 年轻hoxa9-/-小鼠的SCA1+造血祖细胞的mRNA转录增加 P16INK4a。确定p16INK4a的过度表达是否是淋巴/B细胞的分子基础 在hoxa9-/-小鼠中，我们产生了hoxa9-/-p16INK4a-/-小鼠。重要的是，恢复了p16INK4a的缺失 骨髓和胸腺细胞和挽救淋巴、B和T细胞前体细胞缺陷 HOXA9-/-小鼠。这些新的实验发现支持Hoxa9抑制p16INK4a的假设 对淋巴生成至关重要。目前，关于Hoxa9在人类免疫缺陷中的作用的信息很少。 淋巴-造血的调节。这项建议中详述的研究旨在填补这一空白 知识。

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-hematopoiesis. The studies detailed in this proposal are aimed at filling this gap in the knowledge.