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.
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