Visualizing epithelial network connectivity in thymus biology, aging, and regeneration

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Project summary. T lymphocytes are cells of the immune system that, like all blood cells, are routinely lost throughout life and must be replenished. The thymus is the primary site for T lymphocyte production. However, it contains no lymphoid stem cells, and instead depends on continuous importation of stem-like cells that circulate in the blood. Microenvironmental conditions unique to the thymus then instruct these stem-like cells to proliferate and differentiate into T lineage cells that recognize foreign substances but are tolerant to self. The durable thymic microenvironment is established by its stromal cells, consisting primarily of epithelial cells (thymic epithelial cells, TEC). TEC form a pervasive reticular matrix (network) upon which lymphocyte development depends, and competition for the TEC network limits lymphoid cellularity in the thymus, and thus T cell output. For reasons that are still not understood, the thymus exhibits premature and accelerated age-related atrophy, a disorder that is primarily a consequence of changes in TEC. Importantly, the thymus retains latent potential for short-lived regeneration, which can be induced by stimuli such as surgical castration. The mechanisms of this process are not well known, but are believed to require proliferation of TEC cells themselves, or expansion of a TEC precursor population. We have generated a large temporo-spatial database of global stromal gene transcription during induced regrowth. Multiple independent data mining approaches have consistently indicated that genes associated with projection, extension, and outgrowth of cellular processes, such as those that characterize the TEC network, are dynamically regulated during regeneration. To begin to evaluate the role of TEC projections in lymphoid development, thymic aging, and thymic regeneration, we have adopted contemporary neuroscience imaging technologies (Rosa26[Brainbow 2.1], a.k.a. Confetti) in thick organ slices to visualize individual TEC and the TEC network. We find that the morphology of individual TEC, particularly those in the cortex (cTEC), is vastly different than what is suggested by conventional immuno-staining of thin sections, with overall globular rather than radially aligned reticular shapes, each possessing elaborate processes that envelop large numbers of lymphocytes. In the `aged' thymus (from 6 month-old Confetti mice), these processes become both shorter and thinner, and thus provide a diminished surface for lymphoid contact that explains reduced cellularity with age. We predict that rather than TEC proliferation, surgical castration will induce the (transient) lengthening and strengthening of these processes, thus increasing lymphocyte production. Testing this hypothesis is a key objective in this project, but in order to accomplish this, we are first obligated to establish a baseline in the healthy state for TEC and the TEC network. The proposed project relies heavily on established and emerging neuroscience paradigms, and is consistent with the stated mission of the NIH to “to seek fundamental knowledge about the nature and behavior of living systems, and the application of that knowledge to enhance health…and reduce illness.”