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

描述（由申请人提供）：是否年龄相关的缺陷发生在造血祖T细胞以前不清楚。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。在体内评估衰老造血干细胞（HSC）和下游多能祖细胞（MPP）时，以及在体外使用表达Notch配体（OP9- DL1）支持T细胞分化的OP9基质细胞共培养时，T细胞发育缺陷明显。在当前的应用中，我们建议利用OP9-DL1细胞培养系统来阐明老年造血祖细胞中T谱系电位丧失的分子变化。我们假设衰老的干细胞和祖细胞中与年龄相关的T谱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，恢复这些分子的表达将恢复T谱系的潜力，以老年造血祖细胞。我们将确定在老年小鼠分离的HSC和下游MPP中表达改变的分子，并将其与年轻小鼠的HSC和MPP进行比较。这些分子的鉴定将首先使用基于DNA微阵列的分类纯化祖细胞的询问进行。我们将利用实时PCR技术确认造血祖细胞中已鉴定分子的表达改变，并在可能的情况下结合其他方法。我们将使用逆转录病毒基因转导HSC和MPP，以了解在老年祖细胞中表达减少的分子是否足以纠正OP9- DL1系统中老年HSC和MPP的缺陷T细胞发育。这些研究应该提供对与年龄相关的T祖细胞能力丧失的分子机制的见解。是否年龄相关的缺陷发生在造血祖细胞的T细胞以前不是很清楚。我们最近重新评估了来自老年小鼠的骨髓祖细胞对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。在目前的应用中，我们建议研究我们发现的T祖细胞年龄相关缺陷的分子基础。这些实验将允许对T细胞发育中与年龄相关的缺陷进行分子表征，并可能在临床环境（如衰老和骨髓移植）中增强T谱系重建的治疗性。

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.