Regulation of the lymphoid cell fate by Hoxa9

项目摘要 骨髓中稳态造血对于维持血细胞生成和免疫功能至关重要。 能力贯穿一生。随着年龄的增长，骨髓造血干细胞（HSC） 室经历细胞内在和外在的变化，使造血输出偏向 髓系造血干细胞亚群优势的偏倚和造血输出量的变化有助于 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 应答因此，研究重点是了解支持无偏见，平衡 支持淋巴样分化能力的HSC库具有提供新的治疗方法的潜力。 恢复HSC发育轨迹以恢复老年人免疫功能的途径。同源异型盒 转录因子Hoxa 9在原始HSC中表达，并在谱系定型时失活。小鼠 hoxa 9的种系缺失是可行的但显示可移植的多谱系缺陷， 证实了细胞固有缺陷。竞争性移植实验显示， 再生能力和体外研究显示对早期作用细胞因子的增殖反应较差。骨 在骨髓中，hoxa 9-/-小鼠表现出减少的细胞构成，受损的淋巴启动，淋巴-骨髓细胞减少， 祖细胞、普通淋巴祖细胞和B细胞前体。Hoxa 9-/-小鼠的胸腺也较小， 早期胸腺祖细胞减少。有趣的是，流式细胞术分析的长期再增殖干 在年轻的成年hoxa 9-/-小鼠中的LT-HSC显示LT-HSC频率增加 表达高水平的CD 150，其在功能上与髓系偏斜相关。这是对比 通过显著降低hoxa 9-/-骨髓中CD 150 lo LT-HSC的频率，其具有平衡的淋巴细胞增殖， 骨髓潜能随着淋巴细胞生成的减少，沿着骨髓造血细胞数量的增加， 让人想起衰老。Hoxa 9靶基因与淋巴细胞和B细胞年龄相关性改变的关系 骨髓中的基因是p16 ink 4a。细胞周期蛋白依赖性激酶抑制剂p16 ink 4a几乎检测不到 HSC和B细胞前体来自年轻小鼠，但随着年龄的增长，这两个亚群都增加。实时荧光定量PCR 来自年轻hoxa 9-/-小鼠的Sca 1+造血祖细胞显示， p16ink4a.为了确定p16 ink 4a的过度表达是否是淋巴/B细胞的分子基础， 在hoxa 9-/-小鼠中缺乏时，我们产生了hoxa 9-/-p16 ink 4a-/-小鼠。重要的是，p16 ink 4a的缺失恢复了 骨髓和胸腺细胞结构，并挽救了淋巴，B和T细胞前体的缺陷， hoxa 9-/-小鼠。这些新的实验发现支持了Hoxa 9抑制p16 ink 4a的假说， 是淋巴细胞生成的关键目前，关于Hoxa 9在以下方面的作用的信息很少： 调节淋巴造血。本提案中详细介绍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.