Project 2-Thymus

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The thymus has long been considered an enigmatic and mysterious organ; having suffered to some extent from a widespread notion, especially amongst those with a background in medical training, that it has only of eminent importance in early life. Indeed, this line of thinking originates from its critical role in establishing central immune tolerance in early life subsequently followed by a waning period of involution. However, an emerging body of evidence has suggested this “dogma” is, in effect, a “dog” in that many of the notions related to thymic function in humans are, in fact, limited in terms of their confidence as to physiological activities. Much of our understanding of the lymphatic system and its role in immunity has been based on studies of rodent models that appear to differ in considerable ways and cannot be extrapolated directly to humans. On top of this, there are clear voids in terms of the relationship between anatomy and function as well as the influences of age, sex, and race on thymic function. Hence, with this disconnect, and given the importance of the immune response to so many aspects of human disease, we believe it begs the notion of studying the anatomy of the human thymus, from normal organ donors (i.e., unaffected by thymic or immunological pathology), in an attempt to eventually impact our understanding of human disease. To be clear, in terms of targeted organs, given the role of the thymus in establishing central tolerance, there is perhaps no organ more relevant that should be subject to such anatomical investigation. Specifically, we believe a biomolecular mapping of the human thymus is essential to identify where and how human T cell selection occurs, T cell receptor (TCR) reactivities/binding avidities that pass selection or are subject to clonal deletion, and the specific regulatory elements governing T lymphocyte development prior to emigration to secondary lymphoid organs. To achieve this, we posit the following specific aims: AIM I: Establish systematic, anatomical best practices/benchmarks for collection, preservation, validation and standardization of QC protocols (SOPs) for acquiring and handling organ donor grade human thymus; AIM II: Reconstruct the three-dimensional (3D) human thymus; AIM III: Immunophenotype human thymus cells by fluorescence activated cell sorting (FACS). As the thymus is uniquely involved in T lymphocyte development, thymocyte and dendritic cell (DC) populations from dispersed thymus and peripheral blood mononuclear cells from the same patient will be defined by FACS and the immune repertoires compared. Isolated cell subsets will serve as the source for RNA expression profiling (AIM IVB) to guide the selection of markers used in AIMs II and IV; and AIM IV; ultimately, to determine the molecular composition and gene expression within the normal 3D human thymus.