Molecular mechanisms that control the loss of T progenitor competence in aging

描述（由申请人提供）：以前对T细胞的造血祖细胞是否发生年龄相关缺陷没有很好的了解。我们最近评估了老年小鼠骨髓祖细胞对T细胞谱系的贡献能力，并确定衰老显著影响造血祖细胞的T细胞潜能。当在体内评估老化的造血干细胞（HSC）和下游多能祖细胞（MPP）时，以及在体外使用与经工程化以表达支持T细胞分化的Notch配体（0 P9-DL 1）的0 P9基质细胞的细胞共培养物评估老化的造血干细胞（HSC）和下游多能祖细胞（MPP）时，T细胞发育的缺陷是明显的。在本申请中，我们提出利用OP 9-DL 1细胞培养系统来阐明衰老造血祖细胞中T谱系潜能丧失的分子变化。我们推测，衰老干细胞和祖细胞中与年龄相关的T细胞系能力丧失是由基因表达的特定分子变化引起的。我们进一步假设这些分子表达的恢复将恢复衰老造血祖细胞的T谱系潜能。我们将鉴定与年轻小鼠的HSC和MPP相比，从老年小鼠分离的HSC和下游MPP中表达改变的分子。这些分子的鉴定最初将使用基于DNA微阵列的分选纯化的祖细胞的询问来进行。我们将使用实时PCR，在可能的情况下结合其他方法，通过造血祖细胞确认已鉴定分子的表达改变。我们将使用HSC和MPP的逆转录病毒基因转导来询问在老年祖细胞中表现出表达降低的分子的强制表达是否足以纠正OP 9-DL 1系统中来自老年HSC和MPP的缺陷性T细胞发育。这些研究应该提供深入了解与年龄相关的T祖细胞能力丧失的分子机制。与公共卫生相关的T细胞造血祖细胞是否发生年龄相关的缺陷以前并不清楚。我们最近重新评估了老年小鼠骨髓祖细胞对T细胞谱系的贡献能力，并确定衰老显著影响造血祖细胞的T细胞潜能。在当前的应用中，我们建议研究我们已经发现的T祖细胞潜能中与年龄相关的缺陷的分子基础。这些实验将允许T细胞发育中年龄相关缺陷的分子表征，并且可以实现在临床环境（如衰老和骨髓移植）中T谱系重建的治疗增强。

DESCRIPTION (provided by applicant): Whether age-related defects occur in hematopoietic progenitors for T cells was previously not well understood. We recently assessed the ability of bone marrow progenitors from aged mice to contribute to the T cell lineage, and established that aging significantly impacts the T cell potential of hematopoietic progenitors. Defects in T cell development were clearly evident when aged hematopoietic stem cells (HSC) and downstream multipotent progenitors (MPP) were assessed in vivo, and also in vitro using cell co-culture with OP9 stromal cells engineered to express Notch ligands (OP9- DL1) that support T cell differentiation. In the current application, we propose to exploit the OP9-DL1 cell culture system to elucidate the molecular changes underlying the loss of T lineage potential in aged hematopoietic progenitors. We hypothesize that the age-associated loss of T lineage competence in aged stem and progenitor cells is caused by specific molecular changes in gene expression. We further hypothesize that restoration of expression of these molecules will restore T lineage potential to aged hematopoietic progenitors. We will identify molecules whose expression is altered in HSC and downstream MPP isolated from aged mice, as compared to HSC and MPP from young mice. Identification of such molecules will be initially performed using DNA microarray based interrogation of sort-purified progenitors. We will confirm altered expression of identified molecules by hematopoietic progenitors using real-time PCR, coupled with additional approaches when possible. We will use retroviral gene transduction of HSC and MPP to ask whether forced expression of molecules showing reduced expression in aged progenitors is sufficient to correct defective T cell development from aged HSC and MPP in the OP9- DL1 system. These studies should provide insight into molecular mechanisms underlying the age-associated loss of T progenitor competence. PUBLIC HEALTH RELEVANCE Whether age-related defects occur in hematopoietic progenitors for T cells was previously not well understood. We recently reassessed the ability of bone marrow progenitors from aged mice to contribute to the T cell lineage, and established that aging significantly impacts the T cell potential of hematopoietic progenitors. In the current application, we propose to investigate the molecular basis of the age-related defects in T progenitor potential that we have discovered. These experiments will allow a molecular characterization of age-related defects in T cell development, and may enable therapeutic enhancement of T lineage reconstitution in clinical settings such as aging and bone marrow transplantation.