Age Dependent Influences on Human B Lymphocytes

虽然已知衰老对体液免疫有有害影响，但人们对其基础知之甚少。事实上，关于年轻人类骨髓中B淋巴细胞生成的正常过程仍然存在许多问题。该项目将利用最近的技术进步，使纯化人类造血干细胞成为可能，并通过多个步骤观察其分化以产生B淋巴细胞。这里提出的实验将提供关于老年人B淋巴细胞前体的第一个详细信息，因此是本计划项目的核心主题。淋巴细胞前体绝对数量和比例的年龄相关变化将通过多参数流式细胞术进行评估。单克隆抗体，以及可溶性基质细胞产物和标记细胞因子。这种高分辨率的人骨髓解剖将与Webb博士（项目IV）合作完成，他将评估一个重要转录因子的亚群。一个新的NOD/SCID移植模型将用于确定干细胞内在的年龄相关变化是否会影响它们产生B淋巴细胞的能力。将获得有关该系统中正常细胞因子需求的信息，并尝试通过注入重组因子来纠正任何分化缺陷。与Capra博士合作（项目II），我们将学习在环境条件受控的情况下，衰老如何影响成熟B细胞中免疫球蛋白Vh基因的利用和体细胞超突变。B淋巴细胞形成的早期步骤现在可以通过将人类干细胞放置在选定的小鼠基质细胞上来观察。这种令人兴奋的新方法将用于扩展嵌合小鼠模型的发现，克隆测定将允许识别受年龄影响的特定事件。我们的B淋巴生成的体内和体外研究将与Thompson博士（项目I）并行进行，他的重点是人类T淋巴细胞的发育。最后，淋巴细胞前体相对于微环境元素的取向将通过共聚焦显微镜确定，以了解衰老的其他后果。

While aging is known to have deleterious effects on humoral immunity the basis is poorly understood. Indeed, many questions remain about the normal process of B lymphocyte production within young human bone marrow. This project will exploit recent technological advances that now make it possible to purify human hematopoietic stem cells and observe their differentiation through multiple steps to yield B lymphocytes. Experiments proposed here will provide the first detailed information about B lymphocyte precursors in older humans and are thus central to the theme of this Program Project. Age-related changes in absolute numbers and proportions of lymphocyte precursors will be evaluated with multi-parameters flow cytometry. and monoclonal antibodies, as well as soluble stromal cell products and labeled cytokines. This high resolution dissection of human marrow will be done in collaboration with Dr. Webb (Project IV), who will evaluate subsets with respect to an important transcription factor. A new NOD/SCID transplantation model will be used to determine if intrinsic age-related changes ins tem cells influence their ability to give rise to B lymphocytes. Information will be obtained about normal cytokine requirements in this system and attempts made to correct any differentiation deficiencies by infusion of recombinant factors. In collaboration with Dr. Capra (Project II), we will learn how aging affects immunoglubin Vh gene utilization and somatic hypermutation in mature in mature B cells in a circumstance where environmental conditions are controlled. The early steps in B lymphocyte formation can now be observed by placing human stem cells on selected murine stromal cells. This exciting new approach will be used to extend findings made with the chimeric mouse model and clonal assays will permit identification of particular event that are influenced by age. Our in vivo and in vitro studies of B lymphopoiesis will be conducted in parallel and in collaboration of Dr. Thompson (Project I), whose focus is on human T lymphocyte development. Finally, the orientation of lymphocyte precursors relative to microenvironmental elements will be determined by confocal microscopy with a view to learning about other consequences of aging.