The influence of the thymus on the immune defense system will be studied using a quantitative balance-sheet approach to the intrathymic pathways of T cell development. The investigation will use both the intact mouse thymus, and culture models of the intra-thymic environment consisting of lymphoid elements incubated on monolayers of epithelial and other non-lymphoid cells. Thymocyte subsets from the cortex and the medulla will be isolated on the basis of surface markers using a range of monoclonal reagents and fluorescence activated cell sorting. The earliest cells of the pathway, intrathymic stem cells and precursor cells, will be separated from the blast population on the basis of markers, thymus reconstitution potential and generative ability in vitro. Factors controlling the arrival of new stem cells into the adult thymus will be determined. The cells at the end of the pathway, the immediate post-thymus migrants, will be isolated from the spleen or lymph nodes by cell sorting on the basis of surface fluorescence a few hours after intrathymic labeling. Precursor-product relationships between thymocyte subsets will be established by using surface markers, by using localized application of isotopically labeled DNA precursors and surface fluorescein tracers, and by using short-term culture. The basis for the lack of function in cortical thymocytes will be explored using a variety of stimuli which might lead to activation. Their interaction with epithelial components via surface protein or carbohydrate determinants will be investigated. The total functional potential of the thymocyte subset will be assessed in a cell-by-cell way using new, high cloning efficiency limit-dilution microculture systems, with either a lectin-mediated non-specific readout, or with a specific readout to measure the frequency of cytotoxic precursors of a given H-2 restriction pattern. The latter approach will require development of culture conditions which maintain precise immunological specificity, and the basis of cytotoxic T cell specificity degradation will be explored. Possible changes in H-2 restriction repertoire during intrathymic development, from the earliest to the latest cells in the sequence, will be followed. Culture of isolated subjects of early thymocytes on non-lymphoid monolayers differing in H-2 status will be used in an attempt to mimic any thymus influence on the H-2 restriction repertoire.
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